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Sample records for understanding protein folding

  1. Understanding ensemble protein folding at atomic detail

    International Nuclear Information System (INIS)

    Wallin, Stefan; Shakhnovich, Eugene I

    2008-01-01

    Although far from routine, simulating the folding of specific short protein chains on the computer, at a detailed atomic level, is starting to become a reality. This remarkable progress, which has been made over the last decade or so, allows a fundamental aspect of the protein folding process to be addressed, namely its statistical nature. In order to make quantitative comparisons with experimental kinetic data a complete ensemble view of folding must be achieved, with key observables averaged over the large number of microscopically different folding trajectories available to a protein chain. Here we review recent advances in atomic-level protein folding simulations and the new insight provided by them into the protein folding process. An important element in understanding ensemble folding kinetics are methods for analyzing many separate folding trajectories, and we discuss techniques developed to condense the large amount of information contained in an ensemble of trajectories into a manageable picture of the folding process. (topical review)

  2. Kinetics and Thermodynamics of Membrane Protein Folding

    Directory of Open Access Journals (Sweden)

    Ernesto A. Roman

    2014-03-01

    Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

  3. Physics of protein folding

    Science.gov (United States)

    Finkelstein, A. V.; Galzitskaya, O. V.

    2004-04-01

    Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.

  4. PREFACE Protein folding: lessons learned and new frontiers Protein folding: lessons learned and new frontiers

    Science.gov (United States)

    Pappu, Rohit V.; Nussinov, Ruth

    2009-03-01

    In appropriate physiological milieux proteins spontaneously fold into their functional three-dimensional structures. The amino acid sequences of functional proteins contain all the information necessary to specify the folds. This remarkable observation has spawned research aimed at answering two major questions. (1) Of all the conceivable structures that a protein can adopt, why is the ensemble of native-like structures the most favorable? (2) What are the paths by which proteins manage to robustly and reproducibly fold into their native structures? Anfinsen's thermodynamic hypothesis has guided the pursuit of answers to the first question whereas Levinthal's paradox has influenced the development of models for protein folding dynamics. Decades of work have led to significant advances in the folding problem. Mean-field models have been developed to capture our current, coarse grain understanding of the driving forces for protein folding. These models are being used to predict three-dimensional protein structures from sequence and stability profiles as a function of thermodynamic and chemical perturbations. Impressive strides have also been made in the field of protein design, also known as the inverse folding problem, thereby testing our understanding of the determinants of the fold specificities of different sequences. Early work on protein folding pathways focused on the specific sequence of events that could lead to a simplification of the search process. However, unifying principles proved to be elusive. Proteins that show reversible two-state folding-unfolding transitions turned out to be a gift of natural selection. Focusing on these simple systems helped researchers to uncover general principles regarding the origins of cooperativity in protein folding thermodynamics and kinetics. On the theoretical front, concepts borrowed from polymer physics and the physics of spin glasses led to the development of a framework based on energy landscape theories. These

  5. Glycoprotein folding and quality-control mechanisms in protein-folding diseases

    Directory of Open Access Journals (Sweden)

    Sean P. Ferris

    2014-03-01

    Full Text Available Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER. A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.

  6. Solitons and protein folding: An In Silico experiment

    Energy Technology Data Exchange (ETDEWEB)

    Ilieva, N., E-mail: nevena.ilieva@parallel.bas.bg [Institute of Information and Communication Technologies, Bulgarian Aacademy of Sciences, Sofia (Bulgaria); Dai, J., E-mail: daijing491@gmail.com [School of Physics, Beijing Institute of Technology, Beijing (China); Sieradzan, A., E-mail: adams86@wp.pl [Faculty of Chemistry, University of Gdańsk, Gdańsk (Poland); Niemi, A., E-mail: Antti.Niemi@physics.uu.se [Department of Physics and Astronomy, Uppsala University, Uppsala (Sweden); LMPT–CNRS, Université de Tours, Tours (France)

    2015-10-28

    Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

  7. Solitons and protein folding: An In Silico experiment

    International Nuclear Information System (INIS)

    Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.

    2015-01-01

    Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics

  8. Energetic frustrations in protein folding at residue resolution: a homologous simulation study of Im9 proteins.

    Directory of Open Access Journals (Sweden)

    Yunxiang Sun

    Full Text Available Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.

  9. Experimental investigation of protein folding and misfolding.

    Science.gov (United States)

    Dobson, Christopher M

    2004-09-01

    Newly synthesised proteins need to fold, often to intricate and close-packed structures, in order to function. The underlying mechanism by which this complex process takes place both in vitro and in vivo is now becoming understood, at least in general terms, as a result of the application of a wide range of biophysical and computational methods used in combination with the techniques of biochemistry and protein engineering. It is increasingly apparent, however, that folding is not only crucial for generating biological activity, but that it is also coupled to a wide range of processes within the cell, ranging from the trafficking of proteins to specific organelles to the regulation of cell growth and differentiation. Not surprisingly, therefore, the failure of proteins to fold appropriately, or to remain correctly folded, is associated with a large number of cellular malfunctions that give rise to disease. Misfolding, and its consequences such as aggregation, can be investigated by extending the types of techniques used to study the normal folding process. Application of these techniques is enabling the development of a unified description of the interconversion and regulation of the different conformational states available to proteins in living systems. Such a description proves a generic basis for understanding the fundamental links between protein misfolding and its associated clinical disorders, such as Alzheimer's disease and Type II diabetes, and for exploring novel therapeutic strategies directed at their prevention and treatment on a rational basis.

  10. BiP clustering facilitates protein folding in the endoplasmic reticulum.

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    Marc Griesemer

    2014-07-01

    Full Text Available The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER: translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'. Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

  11. Folding propensity of intrinsically disordered proteins by osmotic stress

    International Nuclear Information System (INIS)

    Mansouri, Amanda L.; Grese, Laura N.; Rowe, Erica L.

    2016-01-01

    Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR). Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain a-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. In conclusion, by focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.

  12. Teaching computers to fold proteins

    DEFF Research Database (Denmark)

    Winther, Ole; Krogh, Anders Stærmose

    2004-01-01

    A new general algorithm for optimization of potential functions for protein folding is introduced. It is based upon gradient optimization of the thermodynamic stability of native folds of a training set of proteins with known structure. The iterative update rule contains two thermodynamic averages...

  13. Current Understanding and Future Directions for Vocal Fold Mechanobiology

    Science.gov (United States)

    Li, Nicole Y.K.; Heris, Hossein K.; Mongeau, Luc

    2013-01-01

    The vocal folds, which are located in the larynx, are the main organ of voice production for human communication. The vocal folds are under continuous biomechanical stress similar to other mechanically active organs, such as the heart, lungs, tendons and muscles. During speech and singing, the vocal folds oscillate at frequencies ranging from 20 Hz to 3 kHz with amplitudes of a few millimeters. The biomechanical stress associated with accumulated phonation is believed to alter vocal fold cell activity and tissue structure in many ways. Excessive phonatory stress can damage tissue structure and induce a cell-mediated inflammatory response, resulting in a pathological vocal fold lesion. On the other hand, phonatory stress is one major factor in the maturation of the vocal folds into a specialized tri-layer structure. One specific form of vocal fold oscillation, which involves low impact and large amplitude excursion, is prescribed therapeutically for patients with mild vocal fold injuries. Although biomechanical forces affect vocal fold physiology and pathology, there is little understanding of how mechanical forces regulate these processes at the cellular and molecular level. Research into vocal fold mechanobiology has burgeoned over the past several years. Vocal fold bioreactors are being developed in several laboratories to provide a biomimic environment that allows the systematic manipulation of physical and biological factors on the cells of interest in vitro. Computer models have been used to simulate the integrated response of cells and proteins as a function of phonation stress. The purpose of this paper is to review current research on the mechanobiology of the vocal folds as it relates to growth, pathogenesis and treatment as well as to propose specific research directions that will advance our understanding of this subject. PMID:24812638

  14. Protein folding and protein metallocluster studies using synchrotron small angler X-ray scattering

    International Nuclear Information System (INIS)

    Eliezer, D.

    1994-06-01

    Proteins, biological macromolecules composed of amino-acid building blocks, possess unique three dimensional shapes or conformations which are intimately related to their biological function. All of the information necessary to determine this conformation is stored in a protein's amino acid sequence. The problem of understanding the process by which nature maps protein amino-acid sequences to three-dimensional conformations is known as the protein folding problem, and is one of the central unsolved problems in biophysics today. The possible applications of a solution are broad, ranging from the elucidation of thousands of protein structures to the rational modification and design of protein-based drugs. The scattering of X-rays by matter has long been useful as a tool for the characterization of physical properties of materials, including biological samples. The high photon flux available at synchrotron X-ray sources allows for the measurement of scattering cross-sections of dilute and/or disordered samples. Such measurements do not yield the detailed geometrical information available from crystalline samples, but do allow for lower resolution studies of dynamical processes not observable in the crystalline state. The main focus of the work described here has been the study of the protein folding process using time-resolved small-angle x-ray scattering measurements. The original intention was to observe the decrease in overall size which must accompany the folding of a protein from an extended conformation to its compact native state. Although this process proved too fast for the current time-resolution of the technique, upper bounds were set on the probable compaction times of several small proteins. In addition, an interesting and unexpected process was detected, in which the folding protein passes through an intermediate state which shows a tendency to associate. This state is proposed to be a kinetic molten globule folding intermediate

  15. An overlapping region between the two terminal folding units of the outer surface protein A (OspA) controls its folding behavior.

    Science.gov (United States)

    Makabe, Koki; Nakamura, Takashi; Dhar, Debanjan; Ikura, Teikichi; Koide, Shohei; Kuwajima, Kunihiro

    2018-04-27

    Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. Copyright © 2018. Published by Elsevier Ltd.

  16. Improving Protein Fold Recognition by Deep Learning Networks

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    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-12-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

  17. Improving Protein Fold Recognition by Deep Learning Networks.

    Science.gov (United States)

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-12-04

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

  18. Protein folding and misfolding shining light by infrared spectroscopy

    CERN Document Server

    Fabian, Heinz

    2012-01-01

    Infrared spectroscopy is a new and innovative technology to study protein folding/misfolding events in the broad arsenal of techniques conventionally used in this field. The progress in understanding protein folding and misfolding is primarily due to the development of biophysical methods which permit to probe conformational changes with high kinetic and structural resolution. The most commonly used approaches rely on rapid mixing methods to initiate the folding event via a sudden change in solvent conditions. Traditionally, techniques such as fluorescence, circular dichroism or visible absorption are applied to probe the process. In contrast to these techniques, infrared spectroscopy came into play only very recently, and the progress made in this field up to date which now permits to probe folding events over the time scale from picoseconds to minutes has not yet been discussed in a book. The aim of this book is to provide an overview of the developments as seen by some of the main contributors to the field...

  19. Nucleation phenomena in protein folding: the modulating role of protein sequence

    International Nuclear Information System (INIS)

    Travasso, Rui D M; FaIsca, Patricia F N; Gama, Margarida M Telo da

    2007-01-01

    For the vast majority of naturally occurring, small, single-domain proteins, folding is often described as a two-state process that lacks detectable intermediates. This observation has often been rationalized on the basis of a nucleation mechanism for protein folding whose basic premise is the idea that, after completion of a specific set of contacts forming the so-called folding nucleus, the native state is achieved promptly. Here we propose a methodology to identify folding nuclei in small lattice polymers and apply it to the study of protein molecules with a chain length of N = 48. To investigate the extent to which protein topology is a robust determinant of the nucleation mechanism, we compare the nucleation scenario of a native-centric model with that of a sequence-specific model sharing the same native fold. To evaluate the impact of the sequence's finer details in the nucleation mechanism, we consider the folding of two non-homologous sequences. We conclude that, in a sequence-specific model, the folding nucleus is, to some extent, formed by the most stable contacts in the protein and that the less stable linkages in the folding nucleus are solely determined by the fold's topology. We have also found that, independently of the protein sequence, the folding nucleus performs the same 'topological' function. This unifying feature of the nucleation mechanism results from the residues forming the folding nucleus being distributed along the protein chain in a similar and well-defined manner that is determined by the fold's topological features

  20. Solvent Effects on Protein Folding/Unfolding

    Science.gov (United States)

    García, A. E.; Hillson, N.; Onuchic, J. N.

    Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

  1. Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

    Science.gov (United States)

    Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

    2017-12-01

    Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent

  2. Detecting protein folding by thermal fluctuations of microcantilevers.

    Directory of Open Access Journals (Sweden)

    Romina Muñoz

    Full Text Available The accurate characterization of proteins in both their native and denatured states is essential to effectively understand protein function, folding and stability. As a proof of concept, a micro rheological method is applied, based on the characterization of thermal fluctuations of a micro cantilever immersed in a bovine serum albumin solution, to assess changes in the viscosity associated with modifications in the protein's structure under the denaturant effect of urea. Through modeling the power spectrum density of the cantilever's fluctuations over a broad frequency band, it is possible to implement a fitting procedure to accurately determine the viscosity of the fluid, even at low volumes. Increases in viscosity during the denaturant process are identified using the assumption that the protein is a hard sphere, with a hydrodynamic radius that increases during unfolding. This is modeled accordingly through the Einstein-Batchelor formula. The Einstein-Batchelor formula estimates are verified through dynamic light scattering, which measures the hydrodynamic radius of proteins. Thus, this methodology is proven to be suitable for the study of protein folding in samples of small size at vanishing shear stresses.

  3. General Protein Data Bank-Based Collective Variables for Protein Folding.

    Science.gov (United States)

    Ardevol, Albert; Palazzesi, Ferruccio; Tribello, Gareth A; Parrinello, Michele

    2016-01-12

    New, automated forms of data analysis are required to understand the high-dimensional trajectories that are obtained from molecular dynamics simulations on proteins. Dimensionality reduction algorithms are particularly appealing in this regard as they allow one to construct unbiased, low-dimensional representations of the trajectory using only the information encoded in the trajectory. The downside of this approach is that a different set of coordinates are required for each different chemical system under study precisely because the coordinates are constructed using information from the trajectory. In this paper, we show how one can resolve this problem by using the sketch-map algorithm that we recently proposed to construct a low-dimensional representation of the structures contained in the protein data bank. We show that the resulting coordinates are as useful for analyzing trajectory data as coordinates constructed using landmark configurations taken from the trajectory and that these coordinates can thus be used for understanding protein folding across a range of systems.

  4. CASP10-BCL::Fold efficiently samples topologies of large proteins.

    Science.gov (United States)

    Heinze, Sten; Putnam, Daniel K; Fischer, Axel W; Kohlmann, Tim; Weiner, Brian E; Meiler, Jens

    2015-03-01

    During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein. © 2015 Wiley Periodicals, Inc.

  5. Frustration in Condensed Matter and Protein Folding

    Science.gov (United States)

    Li, Z.; Tanner, S.; Conroy, B.; Owens, F.; Tran, M. M.; Boekema, C.

    2014-03-01

    By means of computer modeling, we are studying frustration in condensed matter and protein folding, including the influence of temperature and Thomson-figure formation. Frustration is due to competing interactions in a disordered state. The key issue is how the particles interact to reach the lowest frustration. The relaxation for frustration is mostly a power function (randomly assigned pattern) or an exponential function (regular patterns like Thomson figures). For the atomic Thomson model, frustration is predicted to decrease with the formation of Thomson figures at zero kelvin. We attempt to apply our frustration modeling to protein folding and dynamics. We investigate the homogeneous protein frustration that would cause the speed of the protein folding to increase. Increase of protein frustration (where frustration and hydrophobicity interplay with protein folding) may lead to a protein mutation. Research is supported by WiSE@SJSU and AFC San Jose.

  6. Self-organized critical model for protein folding

    Science.gov (United States)

    Moret, M. A.

    2011-09-01

    The major factor that drives a protein toward collapse and folding is the hydrophobic effect. At the folding process a hydrophobic core is shielded by the solvent-accessible surface area of the protein. We study the fractal behavior of 5526 protein structures present in the Brookhaven Protein Data Bank. Power laws of protein mass, volume and solvent-accessible surface area are measured independently. The present findings indicate that self-organized criticality is an alternative explanation for the protein folding. Also we note that the protein packing is an independent and constant value because the self-similar behavior of the volumes and protein masses have the same fractal dimension. This power law guarantees that a protein is a complex system. From the analyzed data, q-Gaussian distributions seem to fit well this class of systems.

  7. In vitro folding of inclusion body proteins.

    Science.gov (United States)

    Rudolph, R; Lilie, H

    1996-01-01

    Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

  8. High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

    Science.gov (United States)

    Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

    2016-11-23

    Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

  9. Heterochiral Knottin Protein: Folding and Solution Structure.

    Science.gov (United States)

    Mong, Surin K; Cochran, Frank V; Yu, Hongtao; Graziano, Zachary; Lin, Yu-Shan; Cochran, Jennifer R; Pentelute, Bradley L

    2017-10-31

    Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.

  10. Exploring the Evolutionary Accident Hypothesis: Are Extant Protein Folds the Fittest or the Luckiest?

    Science.gov (United States)

    Shannon, G.; Wei, C.; Pohorille, A.

    2017-01-01

    Considering the range of functions proteins perform, it is surprising they fold into a relatively small set of structures or "folds" that facilitate such function. One explanation is that only a minority were fit enough to emerge from Darwinian selection during the early evolution of life. Alternatively, perhaps only a fraction of all possible folds were trialed. Understanding proto-catalyst selection will aid understanding of the origins and early evolution of life. To investigate which explanation is correct, we study a protein evolved in vitro to bind ATP by Jack Szostak (Fig. 1). This protein adopts a fold which is absent from nature. We are testing whether this fold would have possessed the capability to evolve that would have been essential to survive natural selection on early Earth. Folds that couldn't improve their fitness and evolve to perform new functions would have been replaced by rivals that could. To determine whether the fold is evolvable, we are attempting to change the function of the protein by rationally redesigning to bind GTP. Two design strategies in the region of the nucleobase have been implemented to provide hydrogen bonding partners for the ligand i) an insertion ii) a MET to ASN mutation. Redesigns are being studied computationally at Ames Research Center including free energy of binding calculations. Binding affinities of promising redesigns are to be validated by experimental collaborators at ForteBio using Super Streptavidin Biosensors. If the fold is found to be non-evolvable, this may suggest that many structures were trialed, but the majority were pruned on the basis of their evolvability. Alternatively, if the fold is demonstrated to be evolvable, it would be difficult to explain its absence from nature without considering the possibility that the fold simply wasn't sampled on early Earth. This would not only further our understanding of the origins of life on Earth but also suggest a common phe-nomenon of proto

  11. Evolution of a protein folding nucleus.

    Science.gov (United States)

    Xia, Xue; Longo, Liam M; Sutherland, Mason A; Blaber, Michael

    2016-07-01

    The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric β-trefoil protein by ϕ-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the β-trefoil. The results show structural acquisition of the FN during gene fusion events, incorporating novel turn structure created by gene fusion. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. FN plasticity by way of circular permutation is made possible by the intrinsic C3 cyclic symmetry of the β-trefoil architecture, identifying a possible selective advantage that helps explain the prevalence of cyclic structural symmetry in the proteome. © 2015 The Protein Society.

  12. SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition.

    Science.gov (United States)

    Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina

    2007-05-22

    Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at http://svm-fold.c2b2.columbia.edu. Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach

  13. Fluorescence of Alexa fluor dye tracks protein folding.

    Directory of Open Access Journals (Sweden)

    Simon Lindhoud

    Full Text Available Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488, which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

  14. Protein folding and the organization of the protein topology universe

    DEFF Research Database (Denmark)

    Lindorff-Larsen,, Kresten; Røgen, Peter; Paci, Emanuele

    2005-01-01

    residues and, in addition, that the topology of the transition state is closer to that of the native state than to that of any other fold in the protein universe. Here, we review the evidence for these conclusions and suggest a molecular mechanism that rationalizes these findings by presenting a view...... of protein folds that is based on the topological features of the polypeptide backbone, rather than the conventional view that depends on the arrangement of different types of secondary-structure elements. By linking the folding process to the organization of the protein structure universe, we propose...

  15. Protein solubility and folding enhancement by interaction with RNA.

    Directory of Open Access Journals (Sweden)

    Seong Il Choi

    Full Text Available While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.

  16. Impact of hydrodynamic interactions on protein folding rates depends on temperature

    Science.gov (United States)

    Zegarra, Fabio C.; Homouz, Dirar; Eliaz, Yossi; Gasic, Andrei G.; Cheung, Margaret S.

    2018-03-01

    We investigated the impact of hydrodynamic interactions (HI) on protein folding using a coarse-grained model. The extent of the impact of hydrodynamic interactions, whether it accelerates, retards, or has no effect on protein folding, has been controversial. Together with a theoretical framework of the energy landscape theory (ELT) for protein folding that describes the dynamics of the collective motion with a single reaction coordinate across a folding barrier, we compared the kinetic effects of HI on the folding rates of two protein models that use a chain of single beads with distinctive topologies: a 64-residue α /β chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue β -barrel α -spectrin Src-homology 3 domain (SH3) protein. When comparing the protein folding kinetics simulated with Brownian dynamics in the presence of HI to that in the absence of HI, we find that the effect of HI on protein folding appears to have a "crossover" behavior about the folding temperature. This means that at a temperature greater than the folding temperature, the enhanced friction from the hydrodynamic solvents between the beads in an unfolded configuration results in lowered folding rate; conversely, at a temperature lower than the folding temperature, HI accelerates folding by the backflow of solvent toward the folded configuration of a protein. Additionally, the extent of acceleration depends on the topology of a protein: for a protein like CI2, where its folding nucleus is rather diffuse in a transition state, HI channels the formation of contacts by favoring a major folding pathway in a complex free energy landscape, thus accelerating folding. For a protein like SH3, where its folding nucleus is already specific and less diffuse, HI matters less at a temperature lower than the folding temperature. Our findings provide further theoretical insight to protein folding kinetic experiments and simulations.

  17. Microwave-enhanced folding and denaturation of globular proteins

    DEFF Research Database (Denmark)

    Bohr, Henrik; Bohr, Jakob

    2000-01-01

    It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from...... its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves...

  18. Ligand-promoted protein folding by biased kinetic partitioning.

    Science.gov (United States)

    Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

    2017-04-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

  19. Why and how does native topology dictate the folding speed of a protein?

    Science.gov (United States)

    Rustad, Mark; Ghosh, Kingshuk

    2012-11-01

    Since the pioneering work of Plaxco, Simons, and Baker, it is now well known that the rates of protein folding strongly correlate with the average sequence separation (absolute contact order (ACO)) of native contacts. In spite of multitude of papers, our understanding to the basis of the relation between folding speed and ACO is still lacking. We model the transition state as a Gaussian polymer chain decorated with weak springs between native contacts while the unfolded state is modeled as a Gaussian chain only. Using these hamiltonians, our perturbative calculation explicitly shows folding speed and ACO are linearly related when only the first order term in the series is considered. However, to the second order, we notice the existence of two new topological metrics, termed COC1 and COC2 (COC stands for contact order correction). These additional correction terms are needed to properly account for the entropy loss due to overlapping (nested or linked) loops that are not well described by simple addition of entropies in ACO. COC1 and COC2 are related to fluctuations and correlations among different sequence separations. The new metric combining ACO, COC1, and COC2 improves folding speed dependence on native topology when applied to three different databases: (i) two-state proteins with only α/β and β proteins, (ii) two-state proteins (α/β, β and purely helical proteins all combined), and (iii) master set (multi-state and two-state) folding proteins. Furthermore, the first principle calculation provides us direct physical insights to the meaning of the fit parameters. The coefficient of ACO, for example, is related to the average strength of the contacts, while the constant term is related to the protein folding speed limit. With the new scaling law, our estimate of the folding speed limit is in close agreement with the widely accepted value of 1 μs observed in proteins and RNA. Analyzing an exhaustive set (7367) of monomeric proteins from protein data bank

  20. Intermediates and the folding of proteins L and G

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Scott; Head-Gordon, Teresa

    2003-07-01

    We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G that are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted {beta}-1 and {beta}-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third {beta}-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.

  1. Transiently disordered tails accelerate folding of globular proteins.

    Science.gov (United States)

    Mallik, Saurav; Ray, Tanaya; Kundu, Sudip

    2017-07-01

    Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. © 2017 Federation of European Biochemical Societies.

  2. Folding of multidomain proteins: biophysical consequences of tethering even in apparently independent folding.

    Science.gov (United States)

    Arviv, Oshrit; Levy, Yaakov

    2012-12-01

    Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering-induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse-grained and atomistic molecular dynamics simulations of two two-domain constructs from the immunoglobulin-like β-sandwich fold. Each of these was experimentally shown to behave as the "sum of its parts," that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two-domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Copyright © 2012 Wiley Periodicals, Inc.

  3. How Many Protein Sequences Fold to a Given Structure? A Coevolutionary Analysis.

    Science.gov (United States)

    Tian, Pengfei; Best, Robert B

    2017-10-17

    Quantifying the relationship between protein sequence and structure is key to understanding the protein universe. A fundamental measure of this relationship is the total number of amino acid sequences that can fold to a target protein structure, known as the "sequence capacity," which has been suggested as a proxy for how designable a given protein fold is. Although sequence capacity has been extensively studied using lattice models and theory, numerical estimates for real protein structures are currently lacking. In this work, we have quantitatively estimated the sequence capacity of 10 proteins with a variety of different structures using a statistical model based on residue-residue co-evolution to capture the variation of sequences from the same protein family. Remarkably, we find that even for the smallest protein folds, such as the WW domain, the number of foldable sequences is extremely large, exceeding the Avogadro constant. In agreement with earlier theoretical work, the calculated sequence capacity is positively correlated with the size of the protein, or better, the density of contacts. This allows the absolute sequence capacity of a given protein to be approximately predicted from its structure. On the other hand, the relative sequence capacity, i.e., normalized by the total number of possible sequences, is an extremely tiny number and is strongly anti-correlated with the protein length. Thus, although there may be more foldable sequences for larger proteins, it will be much harder to find them. Lastly, we have correlated the evolutionary age of proteins in the CATH database with their sequence capacity as predicted by our model. The results suggest a trade-off between the opposing requirements of high designability and the likelihood of a novel fold emerging by chance. Published by Elsevier Inc.

  4. Autonomously folding protein fragments reveal differences in the energy landscapes of homologous RNases H.

    Directory of Open Access Journals (Sweden)

    Laura E Rosen

    Full Text Available An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I(core and I(core+1 hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH fragments have very similar stabilities, suggesting that the presence of additional residues in the I(core+1 fragment does not affect the folding or structure as compared to I(core. NMR experiments provide additional evidence that only the I(core intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I(core+1 intermediate. We determine that the ttRNH I(core+1 intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which I(core is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.

  5. Roles of beta-turns in protein folding: from peptide models to protein engineering.

    Science.gov (United States)

    Marcelino, Anna Marie C; Gierasch, Lila M

    2008-05-01

    Reverse turns are a major class of protein secondary structure; they represent sites of chain reversal and thus sites where the globular character of a protein is created. It has been speculated for many years that turns may nucleate the formation of structure in protein folding, as their propensity to occur will favor the approximation of their flanking regions and their general tendency to be hydrophilic will favor their disposition at the solvent-accessible surface. Reverse turns are local features, and it is therefore not surprising that their structural properties have been extensively studied using peptide models. In this article, we review research on peptide models of turns to test the hypothesis that the propensities of turns to form in short peptides will relate to the roles of corresponding sequences in protein folding. Turns with significant stability as isolated entities should actively promote the folding of a protein, and by contrast, turn sequences that merely allow the chain to adopt conformations required for chain reversal are predicted to be passive in the folding mechanism. We discuss results of protein engineering studies of the roles of turn residues in folding mechanisms. Factors that correlate with the importance of turns in folding indeed include their intrinsic stability, as well as their topological context and their participation in hydrophobic networks within the protein's structure.

  6. A multi-directional rapidly exploring random graph (mRRG) for protein folding

    KAUST Repository

    Nath, Shuvra Kanti; Thomas, Shawna; Ekenna, Chinwe; Amato, Nancy M.

    2012-01-01

    Modeling large-scale protein motions, such as those involved in folding and binding interactions, is crucial to better understanding not only how proteins move and interact with other molecules but also how proteins misfold, thus causing many devastating diseases. Robotic motion planning algorithms, such as Rapidly Exploring Random Trees (RRTs), have been successful in simulating protein folding pathways. Here, we propose a new multi-directional Rapidly Exploring Random Graph (mRRG) specifically tailored for proteins. Unlike traditional RRGs which only expand a parent conformation in a single direction, our strategy expands the parent conformation in multiple directions to generate new samples. Resulting samples are connected to the parent conformation and its nearest neighbors. By leveraging multiple directions, mRRG can model the protein motion landscape with reduced computational time compared to several other robotics-based methods for small to moderate-sized proteins. Our results on several proteins agree with experimental hydrogen out-exchange, pulse-labeling, and F-value analysis. We also show that mRRG covers the conformation space better as compared to the other computation methods. Copyright © 2012 ACM.

  7. Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

    Science.gov (United States)

    Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting

    2017-07-01

    To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project

  8. Examining a Thermodynamic Order Parameter of Protein Folding.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2018-05-08

    Dimensionality reduction with a suitable choice of order parameters or reaction coordinates is commonly used for analyzing high-dimensional time-series data generated by atomistic biomolecular simulations. So far, geometric order parameters, such as the root mean square deviation, fraction of native amino acid contacts, and collective coordinates that best characterize rare or large conformational transitions, have been prevailing in protein folding studies. Here, we show that the solvent-averaged effective energy, which is a thermodynamic quantity but unambiguously defined for individual protein conformations, serves as a good order parameter of protein folding. This is illustrated through the application to the folding-unfolding simulation trajectory of villin headpiece subdomain. We rationalize the suitability of the effective energy as an order parameter by the funneledness of the underlying protein free energy landscape. We also demonstrate that an improved conformational space discretization is achieved by incorporating the effective energy. The most distinctive feature of this thermodynamic order parameter is that it works in pointing to near-native folded structures even when the knowledge of the native structure is lacking, and the use of the effective energy will also find applications in combination with methods of protein structure prediction.

  9. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

    Science.gov (United States)

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).

  10. Cotranslational protein folding reveals the selective use of ...

    Indian Academy of Sciences (India)

    to fold properly by decelerating the translation rate at these sites. Thus the cotranslational protein folding is believed to be true for many proteins and is an important selection factor for the selective codon usage to optimize proper gene expres- sion and function (Komar 2009). A web server CS and S has been created by ...

  11. Water dynamics clue to key residues in protein folding

    International Nuclear Information System (INIS)

    Gao, Meng; Zhu, Huaiqiu; Yao, Xin-Qiu; She, Zhen-Su

    2010-01-01

    A computational method independent of experimental protein structure information is proposed to recognize key residues in protein folding, from the study of hydration water dynamics. Based on all-atom molecular dynamics simulation, two key residues are recognized with distinct water dynamical behavior in a folding process of the Trp-cage protein. The identified key residues are shown to play an essential role in both 3D structure and hydrophobic-induced collapse. With observations on hydration water dynamics around key residues, a dynamical pathway of folding can be interpreted.

  12. Nanoscale Dewetting Transition in Protein Complex Folding

    Science.gov (United States)

    Hua, Lan; Huang, Xuhui; Liu, Pu; Zhou, Ruhong; Berne, Bruce J.

    2011-01-01

    In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding. PMID:17608515

  13. Protein Folding: Search for Basic Physical Models

    Directory of Open Access Journals (Sweden)

    Ivan Y. Torshin

    2003-01-01

    Full Text Available How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins, the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces. Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

  14. Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

    Science.gov (United States)

    Maity, Hiranmay; Reddy, Govardhan

    2018-04-01

    Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

  15. There and back again: Two views on the protein folding puzzle.

    Science.gov (United States)

    Finkelstein, Alexei V; Badretdin, Azat J; Galzitskaya, Oxana V; Ivankov, Dmitry N; Bogatyreva, Natalya S; Garbuzynskiy, Sergiy O

    2017-07-01

    The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the

  16. Aggregation of natively folded proteins: a theoretical approach

    International Nuclear Information System (INIS)

    Trovato, Antonio; Maritan, Amos; Seno, Flavio

    2007-01-01

    The reliable identification of β-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. While the list of aggregation related diseases is growing, it has also been shown that many proteins that are normally well behaved can be induced to aggregate in vitro. This fact suggests the existence of a unified framework that could explain both folding and aggregation. By assuming this universal behaviour, we have recently introduced an algorithm (PASTA: prediction of amyloid structure aggregation), which is based on a sequence-specific energy function derived from the propensity of two residue types to be found paired in neighbouring strands within β-sheets in globular proteins. The algorithm is able to predict the most aggregation-prone portions of several proteins initially unfolded, in excellent agreement with experimental results. Here, we apply the method to a set of proteins which are known to aggregate, but which are natively folded. The quality of the prediction is again very high, corroborating the hypothesis that the amyloid structure is stabilized by the same physico-chemical determinants as those operating in folded proteins

  17. Protein folding simulations: from coarse-grained model to all-atom model.

    Science.gov (United States)

    Zhang, Jian; Li, Wenfei; Wang, Jun; Qin, Meng; Wu, Lei; Yan, Zhiqiang; Xu, Weixin; Zuo, Guanghong; Wang, Wei

    2009-06-01

    Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL

  18. Coherent topological phenomena in protein folding

    DEFF Research Database (Denmark)

    Bohr, Henrik; Brunak, Søren; Bohr, Jakob

    1997-01-01

    A theory is presented for coherent topological phenomena in protein dynamics with implications for protein folding and stability. We discuss the relationship to the writhing number used in knot diagrams of DNA. The winding state defines a long-range order along the backbone of a protein with long......-range excitations, `wring' modes, that play an important role in protein denaturation and stability. Energy can be pumped into these excitations, either thermally or by an external force....

  19. Melody discrimination and protein fold classification

    Directory of Open Access Journals (Sweden)

    Robert P. Bywater

    2016-10-01

    Full Text Available One of the greatest challenges in theoretical biophysics and bioinformatics is the identification of protein folds from sequence data. This can be regarded as a pattern recognition problem. In this paper we report the use of a melody generation software where the inputs are derived from calculations of evolutionary information, secondary structure, flexibility, hydropathy and solvent accessibility from multiple sequence alignment data. The melodies so generated are derived from the sequence, and by inference, of the fold, in ways that give each fold a sound representation that may facilitate analysis, recognition, or comparison with other sequences.

  20. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  1. From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.

    Science.gov (United States)

    Ignatova, Zoya; Krishnan, Beena; Bombardier, Jeffrey P; Marcelino, Anna Marie C; Hong, Jiang; Gierasch, Lila M

    2007-01-01

    A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

  2. Recent developments in the theory of protein folding: searching for the global energy minimum.

    Science.gov (United States)

    Scheraga, H A

    1996-04-16

    Statistical mechanical theories and computer simulation are being used to gain an understanding of the fundamental features of protein folding. A major obstacle in the computation of protein structures is the multiple-minima problem arising from the existence of many local minima in the multidimensional energy landscape of the protein. This problem has been surmounted for small open-chain and cyclic peptides, and for regular-repeating sequences of models of fibrous proteins. Progress is being made in resolving this problem for globular proteins.

  3. Quantification of Drive-Response Relationships Between Residues During Protein Folding.

    Science.gov (United States)

    Qi, Yifei; Im, Wonpil

    2013-08-13

    Mutual correlation and cooperativity are commonly used to describe residue-residue interactions in protein folding/function. However, these metrics do not provide any information on the causality relationships between residues. Such drive-response relationships are poorly studied in protein folding/function and difficult to measure experimentally due to technical limitations. In this study, using the information theory transfer entropy (TE) that provides a direct measurement of causality between two times series, we have quantified the drive-response relationships between residues in the folding/unfolding processes of four small proteins generated by molecular dynamics simulations. Instead of using a time-averaged single TE value, the time-dependent TE is measured with the Q-scores based on residue-residue contacts and with the statistical significance analysis along the folding/unfolding processes. The TE analysis is able to identify the driving and responding residues that are different from the highly correlated residues revealed by the mutual information analysis. In general, the driving residues have more regular secondary structures, are more buried, and show greater effects on the protein stability as well as folding and unfolding rates. In addition, the dominant driving and responding residues from the TE analysis on the whole trajectory agree with those on a single folding event, demonstrating that the drive-response relationships are preserved in the non-equilibrium process. Our study provides detailed insights into the protein folding process and has potential applications in protein engineering and interpretation of time-dependent residue-based experimental observables for protein function.

  4. Studies of protein structure in solution and protein folding using synchrotron small-angle x-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Lingling [Stanford Univ., CA (United States)

    1996-04-01

    Synchrotron small angle x-ray scattering (SAXS) has been applied to the structural study of several biological systems, including the nitrogenase complex, the heat shock cognate protein (hsc70), and lysozyme folding. The structural information revealed from the SAXS experiments is complementary to information obtained by other physical and biochemical methods, and adds to our knowledge and understanding of these systems.

  5. Protein fold recognition using geometric kernel data fusion.

    Science.gov (United States)

    Zakeri, Pooya; Jeuris, Ben; Vandebril, Raf; Moreau, Yves

    2014-07-01

    Various approaches based on features extracted from protein sequences and often machine learning methods have been used in the prediction of protein folds. Finding an efficient technique for integrating these different protein features has received increasing attention. In particular, kernel methods are an interesting class of techniques for integrating heterogeneous data. Various methods have been proposed to fuse multiple kernels. Most techniques for multiple kernel learning focus on learning a convex linear combination of base kernels. In addition to the limitation of linear combinations, working with such approaches could cause a loss of potentially useful information. We design several techniques to combine kernel matrices by taking more involved, geometry inspired means of these matrices instead of convex linear combinations. We consider various sequence-based protein features including information extracted directly from position-specific scoring matrices and local sequence alignment. We evaluate our methods for classification on the SCOP PDB-40D benchmark dataset for protein fold recognition. The best overall accuracy on the protein fold recognition test set obtained by our methods is ∼ 86.7%. This is an improvement over the results of the best existing approach. Moreover, our computational model has been developed by incorporating the functional domain composition of proteins through a hybridization model. It is observed that by using our proposed hybridization model, the protein fold recognition accuracy is further improved to 89.30%. Furthermore, we investigate the performance of our approach on the protein remote homology detection problem by fusing multiple string kernels. The MATLAB code used for our proposed geometric kernel fusion frameworks are publicly available at http://people.cs.kuleuven.be/∼raf.vandebril/homepage/software/geomean.php?menu=5/. © The Author 2014. Published by Oxford University Press.

  6. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested that prot......The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...

  7. Protein folding: Over half a century lasting quest. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

    Science.gov (United States)

    Krokhotin, Andrey; Dokholyan, Nikolay V.

    2017-07-01

    Most proteins fold into unique three-dimensional (3D) structures that determine their biological functions, such as catalytic activity or macromolecular binding. Misfolded proteins can pose a threat through aberrant interactions with other proteins leading to a number of diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [1,2]. What does determine 3D structure of proteins? The first clue to this question came more than fifty years ago when Anfinsen demonstrated that unfolded proteins can spontaneously fold to their native 3D structures [3,4]. Anfinsen's experiments lead to the conclusion that proteins fold to unique native structure corresponding to the stable and kinetically accessible free energy minimum, and protein native structure is solely determined by its amino acid sequence. The question of how exactly proteins find their free energy minimum proved to be a difficult problem. One of the puzzles, initially pointed out by Levinthal, was an inconsistency between observed protein folding times and theoretical estimates. A self-avoiding polymer model of a globular protein of 100-residues length on a cubic lattice can sample at least 1047 states. Based on the assumption that conformational sampling occurs at the highest vibrational mode of proteins (∼picoseconds), predicted folding time by searching among all the possible conformations leads to ∼1027 years (much larger than the age of the universe) [5]. In contrast, observed protein folding time range from microseconds to minutes. Due to tremendous theoretical progress in protein folding field that has been achieved in past decades, the source of this inconsistency is currently understood that is thoroughly described in the review by Finkelstein et al. [6].

  8. Soft Computing Techniques for the Protein Folding Problem on High Performance Computing Architectures.

    Science.gov (United States)

    Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M

    2016-01-01

    The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.

  9. Protein folding: Defining a standard set of experimental conditions and a preliminary kinetic data set of two-state proteins

    DEFF Research Database (Denmark)

    Maxwell, Karen L.; Wildes, D.; Zarrine-Afsar, A.

    2005-01-01

    Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding ...... efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process....... constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a consensus set of experimental conditions (25°C at pH 7.0, 50 m...... rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized...

  10. Atomic force microscopy and force spectroscopy on the assessment of protein folding and functionality.

    Science.gov (United States)

    Carvalho, Filomena A; Martins, Ivo C; Santos, Nuno C

    2013-03-01

    Atomic force microscopy (AFM) applied to biological systems can, besides generating high-quality and well-resolved images, be employed to study protein folding via AFM-based force spectroscopy. This approach allowed remarkable advances in the measurement of inter- and intramolecular interaction forces with piconewton resolution. The detection of specific interaction forces between molecules based on the AFM sensitivity and the manipulation of individual molecules greatly advanced the understanding of intra-protein and protein-ligand interactions. Apart from the academic interest in the resolution of basic scientific questions, this technique has also key importance on the clarification of several biological questions of immediate biomedical relevance. Force spectroscopy is an especially appropriate technique for "mechanical proteins" that can provide crucial information on single protein molecules and/or domains. Importantly, it also has the potential of combining in a single experiment spatial and kinetic measurements. Here, the main principles of this methodology are described, after which the ability to measure interactions at the single-molecule level is discussed, in the context of relevant protein-folding examples. We intend to demonstrate the potential of AFM-based force spectroscopy in the study of protein folding, especially since this technique is able to circumvent some of the difficulties typically encountered in classical thermal/chemical denaturation studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Periodic and stochastic thermal modulation of protein folding kinetics.

    Science.gov (United States)

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  12. A partially folded intermediate species of the β-sheet protein apo-pseudoazurin ism trapped during proline-limited folding

    NARCIS (Netherlands)

    Reader, J.S.; van Nuland, N.A.J.; Thompson, G.S.; Ferguson, S.J.; Dobson, C.M.; Radford, S.E.

    2001-01-01

    The folding of apo-pseudoazurin, a 123-residue, predominantly -sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using farand near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to

  13. The nature of folded states of globular proteins.

    Science.gov (United States)

    Honeycutt, J D; Thirumalai, D

    1992-06-01

    We suggest, using dynamical simulations of a simple heteropolymer modelling the alpha-carbon sequence in a protein, that generically the folded states of globular proteins correspond to statistically well-defined metastable states. This hypothesis, called the metastability hypothesis, states that there are several free energy minima separated by barriers of various heights such that the folded conformations of a polypeptide chain in each of the minima have similar structural characteristics but have different energies from one another. The calculated structural characteristics, such as bond angle and dihedral angle distribution functions, are assumed to arise from only those configurations belonging to a given minimum. The validity of this hypothesis is illustrated by simulations of a continuum model of a heteropolymer whose low temperature state is a well-defined beta-barrel structure. The simulations were done using a molecular dynamics algorithm (referred to as the "noisy" molecular dynamics method) containing both friction and noise terms. It is shown that for this model there are several distinct metastable minima in which the structural features are similar. Several new methods of analyzing fluctuations in structures belonging to two distinct minima are introduced. The most notable one is a dynamic measure of compactness that can in principle provide the time required for maximal compactness to be achieved. The analysis shows that for a given metastable state in which the protein has a well-defined folded structure the transition to a state of higher compactness occurs very slowly, lending credence to the notion that the system encounters a late barrier in the process of folding to the most compact structure. The examination of the fluctuations in the structures near the unfolding----folding transition temperature indicates that the transition state for the unfolding to folding process occurs closer to the folded state.

  14. Probabilistic analysis for identifying the driving force of protein folding

    Science.gov (United States)

    Tokunaga, Yoshihiko; Yamamori, Yu; Matubayasi, Nobuyuki

    2018-03-01

    Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.

  15. A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2013-06-01

    A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

  16. Conformational dynamics of a protein in the folded and the unfolded state

    Energy Technology Data Exchange (ETDEWEB)

    Fitter, Joerg

    2003-08-01

    In a quasielastic neutron scattering experiment, the picosecond dynamics of {alpha}-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D{sub 2}O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 A; unfolded state, 1.8 A). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.

  17. Thermodynamic properties of an extremely rapid protein folding reaction.

    Science.gov (United States)

    Schindler, T; Schmid, F X

    1996-12-24

    The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to

  18. A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

    Science.gov (United States)

    Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E; Lieberman, Andrew P; Osawa, Yoichi

    2014-11-01

    In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. © 2014 by the Society for Experimental Biology and Medicine.

  19. Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

    Science.gov (United States)

    Solis, Armando D

    2015-12-01

    To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. © 2015 Wiley Periodicals, Inc.

  20. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  1. Transferable coarse-grained potential for de novo protein folding and design.

    Directory of Open Access Journals (Sweden)

    Ivan Coluzza

    Full Text Available Protein folding and design are major biophysical problems, the solution of which would lead to important applications especially in medicine. Here we provide evidence of how a novel parametrization of the Caterpillar model may be used for both quantitative protein design and folding. With computer simulations it is shown that, for a large set of real protein structures, the model produces designed sequences with similar physical properties to the corresponding natural occurring sequences. The designed sequences require further experimental testing. For an independent set of proteins, previously used as benchmark, the correct folded structure of both the designed and the natural sequences is also demonstrated. The equilibrium folding properties are characterized by free energy calculations. The resulting free energy profiles not only are consistent among natural and designed proteins, but also show a remarkable precision when the folded structures are compared to the experimentally determined ones. Ultimately, the updated Caterpillar model is unique in the combination of its fundamental three features: its simplicity, its ability to produce natural foldable designed sequences, and its structure prediction precision. It is also remarkable that low frustration sequences can be obtained with such a simple and universal design procedure, and that the folding of natural proteins shows funnelled free energy landscapes without the need of any potentials based on the native structure.

  2. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

  3. Some physical approaches to protein folding

    Science.gov (United States)

    Bascle, J.; Garel, T.; Orland, H.

    1993-02-01

    To understand how a protein folds is a problem which has important biological implications. In this article, we would like to present a physics-oriented point of view, which is twofold. First of all, we introduce simple statistical mechanics models which display, in the thermodynamic limit, folding and related transitions. These models can be divided into (i) crude spin glass-like models (with their Mattis analogs), where one may look for possible correlations between the chain self-interactions and the folded structure, (ii) glass-like models, where one emphasizes the geometrical competition between one- or two-dimensional local order (mimicking α helix or β sheet structures), and the requirement of global compactness. Both models are too simple to predict the spatial organization of a realistic protein, but are useful for the physicist and should have some feedback in other glassy systems (glasses, collapsed polymers .... ). These remarks lead us to the second physical approach, namely a new Monte-Carlo method, where one grows the protein atom-by-atom (or residue-by-residue), using a standard form (CHARMM .... ) for the total energy. A detailed comparison with other Monte-Carlo schemes, or Molecular Dynamics calculations, is then possible; we will sketch such a comparison for poly-alanines. Our twofold approach illustrates some of the difficulties one encounters in the protein folding problem, in particular those associated with the existence of a large number of metastable states. Le repliement des protéines est un problème qui a de nombreuses implications biologiques. Dans cet article, nous présentons, de deux façons différentes, un point de vue de physicien. Nous introduisons tout d'abord des modèles simples de mécanique statistique qui exhibent, à la limite thermodynamique, des transitions de repliement. Ces modèles peuvent être divisés en (i) verres de spin (éventuellement à la Mattis), où l'on peut chercher des corrélations entre les

  4. An update of the DEF database of protein fold class predictions

    DEFF Research Database (Denmark)

    Reczko, Martin; Karras, Dimitris; Bohr, Henrik

    1997-01-01

    An update is given on the Database of Expected Fold classes (DEF) that contains a collection of fold-class predictions made from protein sequences and a mail server that provides new predictions for new sequences. To any given sequence one of 49 fold-classes is chosen to classify the structure re...... related to the sequence with high accuracy. The updated predictions system is developed using data from the new version of the 3D-ALI database of aligned protein structures and thus is giving more reliable and more detailed predictions than the previous DEF system.......An update is given on the Database of Expected Fold classes (DEF) that contains a collection of fold-class predictions made from protein sequences and a mail server that provides new predictions for new sequences. To any given sequence one of 49 fold-classes is chosen to classify the structure...

  5. Thermodynamics of protein folding: a random matrix formulation.

    Science.gov (United States)

    Shukla, Pragya

    2010-10-20

    The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies. © 2010 IOP Publishing Ltd

  6. Mechanical Modeling and Computer Simulation of Protein Folding

    Science.gov (United States)

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  7. Modulation of the maladaptive stress response to manage diseases of protein folding.

    Directory of Open Access Journals (Sweden)

    Daniela Martino Roth

    2014-11-01

    Full Text Available Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD, Niemann-Pick type C1 disease (NPC1, Alzheimer's disease (AD, and cystic fibrosis (CF. Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR, through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.

  8. Analysis of the free-energy surface of proteins from reversible folding simulations.

    Directory of Open Access Journals (Sweden)

    Lucy R Allen

    2009-07-01

    Full Text Available Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical lambda-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins.

  9. Multiple molecule effects on the cooperativity of protein folding transitions in simulations

    Science.gov (United States)

    Lewis, Jacob I.; Moss, Devin J.; Knotts, Thomas A.

    2012-06-01

    Though molecular simulation of proteins has made notable contributions to the study of protein folding and kinetics, disagreement between simulation and experiment still exists. One of the criticisms levied against simulation is its failure to reproduce cooperative protein folding transitions. This weakness has been attributed to many factors such as a lack of polarizability and adequate capturing of solvent effects. This work, however, investigates how increasing the number of proteins simulated simultaneously can affect the cooperativity of folding transitions — a topic that has received little attention previously. Two proteins are studied in this work: phage T4 lysozyme (Protein Data Bank (PDB) ID: 7LZM) and phage 434 repressor (PDB ID: 1R69). The results show that increasing the number of proteins molecules simulated simultaneously leads to an increase in the macroscopic cooperativity for transitions that are inherently cooperative on the molecular level but has little effect on the cooperativity of other transitions. Taken as a whole, the results identify one area of consideration to improving simulations of protein folding.

  10. A Particle Swarm Optimization-Based Approach with Local Search for Predicting Protein Folding.

    Science.gov (United States)

    Yang, Cheng-Hong; Lin, Yu-Shiun; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2017-10-01

    The hydrophobic-polar (HP) model is commonly used for predicting protein folding structures and hydrophobic interactions. This study developed a particle swarm optimization (PSO)-based algorithm combined with local search algorithms; specifically, the high exploration PSO (HEPSO) algorithm (which can execute global search processes) was combined with three local search algorithms (hill-climbing algorithm, greedy algorithm, and Tabu table), yielding the proposed HE-L-PSO algorithm. By using 20 known protein structures, we evaluated the performance of the HE-L-PSO algorithm in predicting protein folding in the HP model. The proposed HE-L-PSO algorithm exhibited favorable performance in predicting both short and long amino acid sequences with high reproducibility and stability, compared with seven reported algorithms. The HE-L-PSO algorithm yielded optimal solutions for all predicted protein folding structures. All HE-L-PSO-predicted protein folding structures possessed a hydrophobic core that is similar to normal protein folding.

  11. Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

    Science.gov (United States)

    Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin

    2015-03-01

    This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Peptide folding in the presence of interacting protein crowders

    Energy Technology Data Exchange (ETDEWEB)

    Bille, Anna, E-mail: anna.bille@thep.lu.se; Irbäck, Anders, E-mail: anders@thep.lu.se [Computational Biology and Biological Physics, Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, SE-223 62 Lund (Sweden); Mohanty, Sandipan, E-mail: s.mohanty@fz-juelich.de [Jülich Supercomputing Centre, Institute for Advanced Simulation, Forschungszentrum Jülich, D-52425 Jülich (Germany)

    2016-05-07

    Using Monte Carlo methods, we explore and compare the effects of two protein crowders, BPTI and GB1, on the folding thermodynamics of two peptides, the compact helical trp-cage and the β-hairpin-forming GB1m3. The thermally highly stable crowder proteins are modeled using a fixed backbone and rotatable side-chains, whereas the peptides are free to fold and unfold. In the simulations, the crowder proteins tend to distort the trp-cage fold, while having a stabilizing effect on GB1m3. The extent of the effects on a given peptide depends on the crowder type. Due to a sticky patch on its surface, BPTI causes larger changes than GB1 in the melting properties of the peptides. The observed effects on the peptides stem largely from attractive and specific interactions with the crowder surfaces, and differ from those seen in reference simulations with purely steric crowder particles.

  13. The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Moye Wang

    2016-04-01

    Full Text Available As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.

  14. Effects of knot type in the folding of topologically complex lattice proteins

    Science.gov (United States)

    Soler, Miguel A.; Nunes, Ana; Faísca, Patrícia F. N.

    2014-07-01

    The folding properties of a protein whose native structure contains a 52 knot are investigated by means of extensive Monte Carlo simulations of a simple lattice model and compared with those of a 31 knot. A 52 knot embedded in the native structure enhances the kinetic stability of the carrier lattice protein in a way that is clearly more pronounced than in the case of the 31 knot. However, this happens at the expense of a severe loss in folding efficiency, an observation that is consistent with the relative abundance of 31 and 52 knots in the Protein Data Bank. The folding mechanism of the 52 knot shares with that of the 31 knot the occurrence of a threading movement of the chain terminus that lays closer to the knotted core. However, co-concomitant knotting and folding in the 52 knot occurs with negligible probability, in sharp contrast to what is observed for the 31 knot. The study of several single point mutations highlights the importance in the folding of knotted proteins of the so-called structural mutations (i.e., energetic perturbations of native interactions between residues that are critical for knotting but not for folding). On the other hand, the present study predicts that mutations that perturb the folding transition state may significantly enhance the kinetic stability of knotted proteins provided they involve residues located within the knotted core.

  15. Reliable protein folding on non-funneled energy landscapes: the free energy reaction path

    OpenAIRE

    Lois, Gregg; Blawzdziewicz, Jerzy; O'Hern, Corey S.

    2008-01-01

    A theoretical framework is developed to study the dynamics of protein folding. The key insight is that the search for the native protein conformation is influenced by the rate r at which external parameters, such as temperature, chemical denaturant or pH, are adjusted to induce folding. A theory based on this insight predicts that (1) proteins with non-funneled energy landscapes can fold reliably to their native state, (2) reliable folding can occur as an equilibrium or out-of-equilibrium pro...

  16. Visualization of protein folding funnels in lattice models.

    Directory of Open Access Journals (Sweden)

    Antonio B Oliveira

    Full Text Available Protein folding occurs in a very high dimensional phase space with an exponentially large number of states, and according to the energy landscape theory it exhibits a topology resembling a funnel. In this statistical approach, the folding mechanism is unveiled by describing the local minima in an effective one-dimensional representation. Other approaches based on potential energy landscapes address the hierarchical structure of local energy minima through disconnectivity graphs. In this paper, we introduce a metric to describe the distance between any two conformations, which also allows us to go beyond the one-dimensional representation and visualize the folding funnel in 2D and 3D. In this way it is possible to assess the folding process in detail, e.g., by identifying the connectivity between conformations and establishing the paths to reach the native state, in addition to regions where trapping may occur. Unlike the disconnectivity maps method, which is based on the kinetic connections between states, our methodology is based on structural similarities inferred from the new metric. The method was developed in a 27-mer protein lattice model, folded into a 3×3×3 cube. Five sequences were studied and distinct funnels were generated in an analysis restricted to conformations from the transition-state to the native configuration. Consistent with the expected results from the energy landscape theory, folding routes can be visualized to probe different regions of the phase space, as well as determine the difficulty in folding of the distinct sequences. Changes in the landscape due to mutations were visualized, with the comparison between wild and mutated local minima in a single map, which serves to identify different trapping regions. The extension of this approach to more realistic models and its use in combination with other approaches are discussed.

  17. The construction of an amino acid network for understanding protein structure and function.

    Science.gov (United States)

    Yan, Wenying; Zhou, Jianhong; Sun, Maomin; Chen, Jiajia; Hu, Guang; Shen, Bairong

    2014-06-01

    Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein-protein interactions, and for understanding communication within and between proteins.

  18. Towards a systematic classification of protein folds

    DEFF Research Database (Denmark)

    Lindgård, Per-Anker; Bohr, Henrik

    1997-01-01

    structures are given a unique name, which simultaneously represent a linear string of physical coupling constants describing hinge spin interactions. We have defined a metric and a precise distance measure between the fold classes. An automated procedure is constructed in which any protein structure...

  19. Participation of Low Molecular Weight Electron Carriers in Oxidative Protein Folding

    Directory of Open Access Journals (Sweden)

    József Mandl

    2009-03-01

    Full Text Available Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding.

  20. Surfing the free energy landscape of flavodoxin folding

    NARCIS (Netherlands)

    Bollen, Y.J.M.

    2004-01-01

    The research described in this thesis has been carried out to obtain a better understanding of the fundamental rules describing protein folding. Protein folding is the process in which a linear chain of amino acids contracts to a compact state in which it is active. Flavodoxin from Azotobacter

  1. 'Let the phage do the work': Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

    International Nuclear Information System (INIS)

    Teschke, Carolyn M.; Parent, Kristin N.

    2010-01-01

    The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine. DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

  2. New insights into structural determinants of prion protein folding and stability.

    Science.gov (United States)

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  3. Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation.

    Science.gov (United States)

    Madain, Alia; Abu Dalhoum, Abdel Latif; Sleit, Azzam

    2018-06-01

    The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic-hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H-H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H's at even positions and the other side ignores H's at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H-H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.

  4. An Intramolecular Chaperone Inserted in Bacteriophage P22 Coat Protein Mediates Its Chaperonin-independent Folding*

    Science.gov (United States)

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2013-01-01

    The bacteriophage P22 coat protein has the common HK97-like fold but with a genetically inserted domain (I-domain). The role of the I-domain, positioned at the outermost surface of the capsid, is unknown. We hypothesize that the I-domain may act as an intramolecular chaperone because the coat protein folds independently, and many folding mutants are localized to the I-domain. The function of the I-domain was investigated by generating the coat protein core without its I-domain and the isolated I-domain. The core coat protein shows a pronounced folding defect. The isolated I-domain folds autonomously and has a high thermodynamic stability and fast folding kinetics in the presence of a peptidyl prolyl isomerase. Thus, the I-domain provides thermodynamic stability to the full-length coat protein so that it can fold reasonably efficiently while still allowing the HK97-like core to retain the flexibility required for conformational switching during procapsid assembly and maturation. PMID:24126914

  5. Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2008-07-01

    Full Text Available Abstract Background Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins. Results By assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states. Conclusion Our technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold is available at http://faculty.cs.tamu.edu/shsze/ssfold.

  6. Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

    Science.gov (United States)

    Balakrishnan, Swati; Sarma, Siddhartha P

    2017-08-22

    Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b 5 . Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b 5 . The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a C m higher than that of apo-cytochrome b 5 and are more stable to chaotropic agents than apo-cytochrome b 5 . The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

  7. Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis

    NARCIS (Netherlands)

    Krishnappa, Laxmi; Monteferrante, Carmine G; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten

    The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold

  8. Denatured state is critical in determining the properties of model proteins designed on different folds

    DEFF Research Database (Denmark)

    Amatori, Andrea; Ferkinghoff-Borg, Jesper; Tiana, Guido

    2008-01-01

    The thermodynamics of proteins designed on three common folds (SH3, chymotrypsin inhibitor 2 [CI2], and protein G) is studied with a simplified C alpha, model and compared with the thermodynamics of proteins designed on random-generated folds. The model allows to design sequences to fold within a...

  9. Protein Folding Free Energy Landscape along the Committor - the Optimal Folding Coordinate.

    Science.gov (United States)

    Krivov, Sergei V

    2018-06-06

    Recent advances in simulation and experiment have led to dramatic increases in the quantity and complexity of produced data, which makes the development of automated analysis tools very important. A powerful approach to analyze dynamics contained in such data sets is to describe/approximate it by diffusion on a free energy landscape - free energy as a function of reaction coordinates (RC). For the description to be quantitatively accurate, RCs should be chosen in an optimal way. Recent theoretical results show that such an optimal RC exists; however, determining it for practical systems is a very difficult unsolved problem. Here we describe a solution to this problem. We describe an adaptive nonparametric approach to accurately determine the optimal RC (the committor) for an equilibrium trajectory of a realistic system. In contrast to alternative approaches, which require a functional form with many parameters to approximate an RC and thus extensive expertise with the system, the suggested approach is nonparametric and can approximate any RC with high accuracy without system specific information. To avoid overfitting for a realistically sampled system, the approach performs RC optimization in an adaptive manner by focusing optimization on less optimized spatiotemporal regions of the RC. The power of the approach is illustrated on a long equilibrium atomistic folding simulation of HP35 protein. We have determined the optimal folding RC - the committor, which was confirmed by passing a stringent committor validation test. It allowed us to determine a first quantitatively accurate protein folding free energy landscape. We have confirmed the recent theoretical results that diffusion on such a free energy profile can be used to compute exactly the equilibrium flux, the mean first passage times, and the mean transition path times between any two points on the profile. We have shown that the mean squared displacement along the optimal RC grows linear with time as for

  10. Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

    Science.gov (United States)

    Mallik, Saurav; Das, Smita; Kundu, Sudip

    2016-01-01

    Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.

  11. Classification of protein fold classes by knot theory and prediction of folds by neural networks: A combined theoretical and experimental approach

    DEFF Research Database (Denmark)

    Ramnarayan, K.; Bohr, Henrik; Jalkanen, Karl J.

    2008-01-01

    We present different means of classifying protein structure. One is made rigorous by mathematical knot invariants that coincide reasonably well with ordinary graphical fold classification and another classification is by packing analysis. Furthermore when constructing our mathematical fold...... classifications, we utilize standard neural network methods for predicting protein fold classes from amino acid sequences. We also make an analysis of the redundancy of the structural classifications in relation to function and ligand binding. Finally we advocate the use of combining the measurement of the VA...

  12. Simulation of fluorescence resonance energy transfer experiments: effect of the dyes on protein folding

    International Nuclear Information System (INIS)

    Allen, Lucy R; Paci, Emanuele

    2010-01-01

    Fluorescence resonance energy transfer is a powerful technique which is often used to probe the properties of proteins and complex macromolecules. The technique relies on relatively large fluorescent dyes which are engineered into the molecule of interest. In the case of small proteins, these dyes may affect the stability of the protein, and modify the folding kinetics and the folding mechanisms which are being probed. Here we use atomistic simulation to investigate the effect that commonly used fluorescent dyes have on the folding of a four-helix bundle protein. We show that, depending on where the dyes are attached, their effect on the kinetic and thermodynamic properties of the protein may be significant. We find that, while the overall folding mechanism is not affected by the dyes, they can destabilize, or even stabilize, intermediate states.

  13. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Protein folding simulations by generalized-ensemble algorithms.

    Science.gov (United States)

    Yoda, Takao; Sugita, Yuji; Okamoto, Yuko

    2014-01-01

    In the protein folding problem, conventional simulations in physical statistical mechanical ensembles, such as the canonical ensemble with fixed temperature, face a great difficulty. This is because there exist a huge number of local-minimum-energy states in the system and the conventional simulations tend to get trapped in these states, giving wrong results. Generalized-ensemble algorithms are based on artificial unphysical ensembles and overcome the above difficulty by performing random walks in potential energy, volume, and other physical quantities or their corresponding conjugate parameters such as temperature, pressure, etc. The advantage of generalized-ensemble simulations lies in the fact that they not only avoid getting trapped in states of energy local minima but also allows the calculations of physical quantities as functions of temperature or other parameters from a single simulation run. In this article we review the generalized-ensemble algorithms. Four examples, multicanonical algorithm, replica-exchange method, replica-exchange multicanonical algorithm, and multicanonical replica-exchange method, are described in detail. Examples of their applications to the protein folding problem are presented.

  15. Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

    International Nuclear Information System (INIS)

    Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

    2008-01-01

    T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

  16. Discovery of Proteomic Code with mRNA Assisted Protein Folding

    Directory of Open Access Journals (Sweden)

    Jan C. Biro

    2008-12-01

    Full Text Available The 3x redundancy of the Genetic Code is usually explained as a necessity to increase the mutation-resistance of the genetic information. However recent bioinformatical observations indicate that the redundant Genetic Code contains more biological information than previously known and which is additional to the 64/20 definition of amino acids. It might define the physico-chemical and structural properties of amino acids, the codon boundaries, the amino acid co-locations (interactions in the coded proteins and the free folding energy of mRNAs. This additional information, which seems to be necessary to determine the 3D structure of coding nucleic acids as well as the coded proteins, is known as the Proteomic Code and mRNA Assisted Protein Folding.

  17. Self-organization and mismatch tolerance in protein folding: General theory and an application

    Science.gov (United States)

    Fernández, Ariel; Berry, R. Stephen

    2000-03-01

    The folding of a protein is a process both expeditious and robust. The analysis of this process presented here uses a coarse, discretized representation of the evolving form of the backbone chain, based on its torsional states. This coarse description consists of discretizing the torsional coordinates modulo the Ramachandran basins in the local softmode dynamics. Whenever the representation exhibits "contact patterns" that correspond to topological compatibilities with particular structural forms, secondary and then tertiary, the elements constituting the pattern are effectively entrained by a reduction of their rates of exploration of their discretized configuration space. The properties "expeditious and robust" imply that the folding protein must have some tolerance to both torsional "frustrated" and side-chain contact mismatches which may occur during the folding process. The energy-entropy consequences of the staircase or funnel topography of the potential surface should allow the folding protein to correct these mismatches, eventually. This tolerance lends itself to an iterative pattern-recognition-and-feedback description of the folding process that reflects mismatched local torsional states and hydrophobic/polar contacts. The predictive potential of our algorithm is tested by application to the folding of bovine pancreatic trypsin inhibitor (BPTI), a protein whose ability to form its active structure is contingent upon its frustration tolerance.

  18. Prediction of the optimal set of contacts to fold the smallest knotted protein

    International Nuclear Information System (INIS)

    Dabrowski-Tumanski, P; Jarmolinska, A I; Sulkowska, J I

    2015-01-01

    Knotted protein chains represent a new motif in protein folds. They have been linked to various diseases, and recent extensive analysis of the Protein Data Bank shows that they constitute 1.5% of all deposited protein structures. Despite thorough theoretical and experimental investigations, the role of knots in proteins still remains elusive. Nonetheless, it is believed that knots play an important role in mechanical and thermal stability of proteins. Here, we perform a comprehensive analysis of native, shadow-specific and non-native interactions which describe free energy landscape of the smallest knotted protein (PDB id 2efv). We show that the addition of shadow-specific contacts in the loop region greatly enhances folding kinetics, while the addition of shadow-specific contacts along the C-terminal region (H3 or H4) results in a new folding route with slower kinetics. By means of direct coupling analysis (DCA) we predict non-native contacts which also can accelerate kinetics. Next, we show that the length of the C-terminal knot tail is responsible for the shape of the free energy barrier, while the influence of the elongation of the N-terminus is not significant. Finally, we develop a concept of a minimal contact map sufficient for 2efv protein to fold and analyze properties of this protein using this map. (paper)

  19. Prediction of the optimal set of contacts to fold the smallest knotted protein

    Science.gov (United States)

    Dabrowski-Tumanski, P.; Jarmolinska, A. I.; Sulkowska, J. I.

    2015-09-01

    Knotted protein chains represent a new motif in protein folds. They have been linked to various diseases, and recent extensive analysis of the Protein Data Bank shows that they constitute 1.5% of all deposited protein structures. Despite thorough theoretical and experimental investigations, the role of knots in proteins still remains elusive. Nonetheless, it is believed that knots play an important role in mechanical and thermal stability of proteins. Here, we perform a comprehensive analysis of native, shadow-specific and non-native interactions which describe free energy landscape of the smallest knotted protein (PDB id 2efv). We show that the addition of shadow-specific contacts in the loop region greatly enhances folding kinetics, while the addition of shadow-specific contacts along the C-terminal region (H3 or H4) results in a new folding route with slower kinetics. By means of direct coupling analysis (DCA) we predict non-native contacts which also can accelerate kinetics. Next, we show that the length of the C-terminal knot tail is responsible for the shape of the free energy barrier, while the influence of the elongation of the N-terminus is not significant. Finally, we develop a concept of a minimal contact map sufficient for 2efv protein to fold and analyze properties of this protein using this map.

  20. Co-evolutionary constraints of globular proteins correlate with their folding rates.

    Science.gov (United States)

    Mallik, Saurav; Kundu, Sudip

    2015-08-04

    Folding rates (lnkf) of globular proteins correlate with their biophysical properties, but relationship between lnkf and patterns of sequence evolution remains elusive. We introduce 'relative co-evolution order' (rCEO) as length-normalized average primary chain separation of co-evolving pairs (CEPs), which negatively correlates with lnkf. In addition to pairs in native 3D contact, indirectly connected and structurally remote CEPs probably also play critical roles in protein folding. Correlation between rCEO and lnkf is stronger in multi-state proteins than two-state proteins, contrasting the case of contact order (co), where stronger correlation is found in two-state proteins. Finally, rCEO, co and lnkf are fitted into a 3D linear correlation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Equilibrium amide hydrogen exchange and protein folding kinetics

    International Nuclear Information System (INIS)

    Bai Yawen

    1999-01-01

    The classical Linderstrom-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (I↔U↔N). On the other hand, in an on-pathway three-state system (U↔I↔N), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments

  2. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Directory of Open Access Journals (Sweden)

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  3. Bioinformatics analysis identify novel OB fold protein coding genes in C. elegans.

    Directory of Open Access Journals (Sweden)

    Daryanaz Dargahi

    Full Text Available BACKGROUND: The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5-25%. Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n=46 compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n=344 or fruit fly D. melanogaster (n=84. Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies. METHODOLOGY/PRINCIPAL FINDINGS: This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans. CONCLUSIONS/SIGNIFICANCE: This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.

  4. Sampling-based exploration of folded state of a protein under kinematic and geometric constraints

    KAUST Repository

    Yao, Peggy; Zhang, Liangjun; Latombe, Jean-Claude

    2011-01-01

    Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable

  5. Protein folding on the ribosome studied using NMR spectroscopy

    Science.gov (United States)

    Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John

    2013-01-01

    NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462

  6. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  7. Folding Membrane Proteins by Deep Transfer Learning

    KAUST Repository

    Wang, Sheng

    2017-08-29

    Computational elucidation of membrane protein (MP) structures is challenging partially due to lack of sufficient solved structures for homology modeling. Here, we describe a high-throughput deep transfer learning method that first predicts MP contacts by learning from non-MPs and then predicts 3D structure models using the predicted contacts as distance restraints. Tested on 510 non-redundant MPs, our method has contact prediction accuracy at least 0.18 better than existing methods, predicts correct folds for 218 MPs, and generates 3D models with root-mean-square deviation (RMSD) less than 4 and 5 Å for 57 and 108 MPs, respectively. A rigorous blind test in the continuous automated model evaluation project shows that our method predicted high-resolution 3D models for two recent test MPs of 210 residues with RMSD ∼2 Å. We estimated that our method could predict correct folds for 1,345–1,871 reviewed human multi-pass MPs including a few hundred new folds, which shall facilitate the discovery of drugs targeting at MPs.

  8. Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis

    Directory of Open Access Journals (Sweden)

    Qian Zhao

    2018-01-01

    Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead

  9. Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rasia, Rodolfo M. [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France); Lescop, Ewen [CNRS, Institut de Chimie des Substances Naturelles (France); Palatnik, Javier F. [Universidad Nacional de Rosario, Instituto de Biologia Molecular y Celular de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas (Argentina); Boisbouvier, Jerome, E-mail: jerome.boisbouvier@ibs.fr; Brutscher, Bernhard, E-mail: Bernhard.brutscher@ibs.fr [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France)

    2011-11-15

    It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.

  10. Supramolecular Architectures and Mimics of Complex Natural Folds Derived from Rationally Designed alpha-Helical Protein Structures

    Science.gov (United States)

    Tavenor, Nathan Albert

    Protein-based supramolecular polymers (SMPs) are a class of biomaterials which draw inspiration from and expand upon the many examples of complex protein quaternary structures observed in nature: collagen, microtubules, viral capsids, etc. Designing synthetic supramolecular protein scaffolds both increases our understanding of natural superstructures and allows for the creation of novel materials. Similar to small-molecule SMPs, protein-based SMPs form due to self-assembly driven by intermolecular interactions between monomers, and monomer structure determines the properties of the overall material. Using protein-based monomers takes advantage of the self-assembly and highly specific molecular recognition properties encodable in polypeptide sequences to rationally design SMP architectures. The central hypothesis underlying our work is that alpha-helical coiled coils, a well-studied protein quaternary folding motif, are well-suited to SMP design through the addition of synthetic linkers at solvent-exposed sites. Through small changes in the structures of the cross-links and/or peptide sequence, we have been able to control both the nanoscale organization and the macroscopic properties of the SMPs. Changes to the linker and hydrophobic core of the peptide can be used to control polymer rigidity, stability, and dimensionality. The gaps in knowledge that this thesis sought to fill on this project were 1) the relationship between the molecular structure of the cross-linked polypeptides and the macroscopic properties of the SMPs and 2) a means of creating materials exhibiting multi-dimensional net or framework topologies. Separate from the above efforts on supramolecular architectures was work on improving backbone modification strategies for an alpha-helix in the context of a complex protein tertiary fold. Earlier work in our lab had successfully incorporated unnatural building blocks into every major secondary structure (beta-sheet, alpha-helix, loops and beta

  11. PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.

    Science.gov (United States)

    Lowe, Alan R; Perez-Riba, Albert; Itzhaki, Laura S; Main, Ewan R G

    2018-02-06

    For many years, curve-fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large data sets. One field that is reliant on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open-source, and extensible Python framework for graphing, analysis, and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: 1) multiphase kinetic folding fitted to linked equations, 2) global fitting of multiple data sets, and 3) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how PyFolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and among research teams. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Generic framework for mining cellular automata models on protein-folding simulations.

    Science.gov (United States)

    Diaz, N; Tischer, I

    2016-05-13

    Cellular automata model identification is an important way of building simplified simulation models. In this study, we describe a generic architectural framework to ease the development process of new metaheuristic-based algorithms for cellular automata model identification in protein-folding trajectories. Our framework was developed by a methodology based on design patterns that allow an improved experience for new algorithms development. The usefulness of the proposed framework is demonstrated by the implementation of four algorithms, able to obtain extremely precise cellular automata models of the protein-folding process with a protein contact map representation. Dynamic rules obtained by the proposed approach are discussed, and future use for the new tool is outlined.

  13. Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule.

    Science.gov (United States)

    Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan

    2014-10-14

    The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

  14. Theory and simulation of explicit solvent effects on protein folding in vitro and in vivo

    Science.gov (United States)

    England, Jeremy L.

    The aim of this work is to develop theoretical tools for understanding what happens to water that is confined in amphipathic cavities, and for testing the consequences of this understanding for protein folding in vitro and in vivo. We begin in the first chapter with a brief review of the theoretical and simulation literature on the hydrophobic effect and the aqueous solvation of charged species that also puts forward a simple theoretical framework within which various solvation phenomena reported in past studies may be unified. Subsequently, in the second chapter we also review past computational and theoretical work on the specific question of how chaperonin complexes assist the folding of their substrates. With the context set, we turn in Chapter 3 to the case of an open system with water trapped between hydrophobic plates that experiences a uniform electric field normal to and between the plates. Classic bulk theory of electrostriction in polarizable fluids tells us that the electric field should cause an increase in local water density as it rises, yet some simulations have suggested the opposite. We present a mean-field Potts model we have developed to explain this discrepancy, and show how such a simple, coarse-grained lattice description can capture the fundamental consequences of the fact that external electric fields can frustrate the hydrogen bond network in confined water. Chapter 4 continues to pursue the issue of solvent evacuation between hydrophobic plates, but focuses on the impact of chemical denaturants on hydrophobic effects using molecular dynamics simulations of hydrophobic dewetting. We find that while urea and guanidinium have similar qualitative effects at the bulk level, they seem to differ in the microscopic mechanism by which they denature proteins, although both inhibit the onset of dewetting. Lastly, Chapters 5 and 6 examine the potential importance of solvent-mediated forces to protein folding in vivo. Chapter 5 develops a Landau

  15. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    Science.gov (United States)

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  16. Systematic analysis of short internal indels and their impact on protein folding

    Directory of Open Access Journals (Sweden)

    Guo Jun-tao

    2010-08-01

    Full Text Available Abstract Background Protein sequence insertions/deletions (indels can be introduced during evolution or through alternative splicing (AS. Alternative splicing is an important biological phenomenon and is considered as the major means of expanding structural and functional diversity in eukaryotes. Knowledge of the structural changes due to indels is critical to our understanding of the evolution of protein structure and function. In addition, it can help us probe the evolution of alternative splicing and the diversity of functional isoforms. However, little is known about the effects of indels, in particular the ones involving core secondary structures, on the folding of protein structures. The long term goal of our study is to accurately predict the protein AS isoform structures. As a first step towards this goal, we performed a systematic analysis on the structural changes caused by short internal indels through mining highly homologous proteins in Protein Data Bank (PDB. Results We compiled a non-redundant dataset of short internal indels (2-40 amino acids from highly homologous protein pairs and analyzed the sequence and structural features of the indels. We found that about one third of indel residues are in disordered state and majority of the residues are exposed to solvent, suggesting that these indels are generally located on the surface of proteins. Though naturally occurring indels are fewer than engineered ones in the dataset, there are no statistically significant differences in terms of amino acid frequencies and secondary structure types between the "Natural" indels and "All" indels in the dataset. Structural comparisons show that all the protein pairs with short internal indels in the dataset preserve the structural folds and about 85% of protein pairs have global RMSDs (root mean square deviations of 2Å or less, suggesting that protein structures tend to be conserved and can tolerate short insertions and deletions. A few pairs

  17. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

    Science.gov (United States)

    Sato, Yoshimi; Kojima, Rieko; Okumura, Masaki; Hagiwara, Masatoshi; Masui, Shoji; Maegawa, Ken-ichi; Saiki, Masatoshi; Horibe, Tomohisa; Suzuki, Mamoru; Inaba, Kenji

    2013-01-01

    The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

  18. Sampling-based exploration of folded state of a protein under kinematic and geometric constraints

    KAUST Repository

    Yao, Peggy

    2011-10-04

    Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable conformations to bind to. Therefore, one of the best options to study protein-ligand binding is to sample conformations broadly distributed over the protein-folded state. This article presents a new sampler, called kino-geometric sampler (KGS). This sampler encodes dominant energy terms implicitly by simple kinematic and geometric constraints. Two key technical contributions of KGS are (1) a robotics-inspired Jacobian-based method to simultaneously deform a large number of interdependent kinematic cycles without any significant break-up of the closure constraints, and (2) a diffusive strategy to generate conformation distributions that diffuse quickly throughout the protein folded state. Experiments on four very different test proteins demonstrate that KGS can efficiently compute distributions containing conformations close to target (e.g., functional) conformations. These targets are not given to KGS, hence are not used to bias the sampling process. In particular, for a lysine-binding protein, KGS was able to sample conformations in both the intermediate and functional states without the ligand, while previous work using molecular dynamics simulation had required the ligand to be taken into account in the potential function. Overall, KGS demonstrates that kino-geometric constraints characterize the folded subset of a protein conformation space and that this subset is small enough to be approximated by a relatively small distribution of conformations. © 2011 Wiley Periodicals, Inc.

  19. Right- and left-handed three-helix proteins. I. Experimental and simulation analysis of differences in folding and structure.

    Science.gov (United States)

    Glyakina, Anna V; Pereyaslavets, Leonid B; Galzitskaya, Oxana V

    2013-09-01

    Despite the large number of publications on three-helix protein folding, there is no study devoted to the influence of handedness on the rate of three-helix protein folding. From the experimental studies, we make a conclusion that the left-handed three-helix proteins fold faster than the right-handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three-helix proteins (four right-handed and four left-handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary-structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left-handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right-handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three-helix proteins (238 right- and 94 left-handed). From our analysis, we found that the left-handed three-helix proteins have some less-dense packing that should result in faster folding for some proteins as compared to the case of right-handed proteins. Copyright © 2013 Wiley Periodicals, Inc.

  20. Targeting the OB-Folds of Replication Protein A with Small Molecules

    Directory of Open Access Journals (Sweden)

    Victor J. Anciano Granadillo

    2010-01-01

    Full Text Available Replication protein A (RPA is the main eukaryotic single-strand (ss DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

  1. Two states or not two states: Single-molecule folding studies of protein L

    Science.gov (United States)

    Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

    2018-03-01

    Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

  2. Protein folding optimization based on 3D off-lattice model via an improved artificial bee colony algorithm.

    Science.gov (United States)

    Li, Bai; Lin, Mu; Liu, Qiao; Li, Ya; Zhou, Changjun

    2015-10-01

    Protein folding is a fundamental topic in molecular biology. Conventional experimental techniques for protein structure identification or protein folding recognition require strict laboratory requirements and heavy operating burdens, which have largely limited their applications. Alternatively, computer-aided techniques have been developed to optimize protein structures or to predict the protein folding process. In this paper, we utilize a 3D off-lattice model to describe the original protein folding scheme as a simplified energy-optimal numerical problem, where all types of amino acid residues are binarized into hydrophobic and hydrophilic ones. We apply a balance-evolution artificial bee colony (BE-ABC) algorithm as the minimization solver, which is featured by the adaptive adjustment of search intensity to cater for the varying needs during the entire optimization process. In this work, we establish a benchmark case set with 13 real protein sequences from the Protein Data Bank database and evaluate the convergence performance of BE-ABC algorithm through strict comparisons with several state-of-the-art ABC variants in short-term numerical experiments. Besides that, our obtained best-so-far protein structures are compared to the ones in comprehensive previous literature. This study also provides preliminary insights into how artificial intelligence techniques can be applied to reveal the dynamics of protein folding. Graphical Abstract Protein folding optimization using 3D off-lattice model and advanced optimization techniques.

  3. Ab initio folding of mixed-fold FSD-EY protein using formula-based polarizable hydrogen bond (PHB) charge model

    Science.gov (United States)

    Zhang, Dawei; Lazim, Raudah; Mun Yip, Yew

    2017-09-01

    We conducted an all-atom ab initio folding of FSD-EY, a protein with a ββα configuration using non-polarizable (AMBER) and polarizable force fields (PHB designed by Gao et al.) in implicit solvent. The effect of reducing the polarization effect integrated into the force field by the PHB model, termed the PHB0.7 was also examined in the folding of FSD-EY. This model incorporates into the force field 70% of the original polarization effect to minimize the likelihood of over-stabilizing the backbone hydrogen bonds. Precise folding of the β-sheet of FSD-EY was further achieved by relaxing the REMD structure obtained in explicit water.

  4. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Dabney-Smith, Carole [Miami Univ., Oxford, OH (United States)

    2016-07-18

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembled proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cp

  5. Structure of the thioredoxin-fold domain of human phosducin-like protein 2

    International Nuclear Information System (INIS)

    Lou, Xiaochu; Bao, Rui; Zhou, Cong-Zhao; Chen, Yuxing

    2009-01-01

    The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence

  6. Identification of a key structural element for protein folding within beta-hairpin turns.

    Science.gov (United States)

    Kim, Jaewon; Brych, Stephen R; Lee, Jihun; Logan, Timothy M; Blaber, Michael

    2003-05-09

    Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

  7. Protein folding includes oligomerization – examples from the endoplasmic reticulum and cytosol

    NARCIS (Netherlands)

    Christis, C.; Lubsen, N.H.; Braakman, I.

    2008-01-01

    A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma

  8. Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori

    Science.gov (United States)

    Wang, Liang-Wei; Liu, Yu-Nan; Lyu, Ping-Chiang; Jackson, Sophie E.; Hsu, Shang-Te Danny

    2015-09-01

    Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.

  9. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  10. In vivo labelling of proteins associated with folded chromosomes of yeast

    International Nuclear Information System (INIS)

    Litske Petersen, J.G.; Pinon, R.

    1980-01-01

    Proteins associated with the pre-replicative (g 1 ) and post-replicative (g 2 ) folded chromosomes of Saccharomyces cerevisiae can be labelled in vivo by growing cells in acetate vegetative medium containing [ 35 S]methionine. In both sporulating (MATa/MATα) and non-sporulating (MATa/MATa, MATα/MATα) diploids proteins associated with the resting stage genome (g 0 ) can be labelled with [ 35 S]methionine during nitrogen starvation and in sporulation medium. In addition, in MATa/MATα diploids proteins associated with the meiotic replication form (r) can also be labelled. SDS-polyacrylamide gel electrophoresis and autoradiography of the labelled proteins from the various folded genome forms showed that the g 1 and g 2 patterns are, with the exception of one polypeptide band, essentially identical. Several differences distinguished the r and g 0 patterns from those of the g 1 and g 2 structures. At least four polypeptide bands distinguish the r and g 0 patterns. No significant differences were observed between the g 0 proteins of sporulating and non-sporulating diploids. (author)

  11. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  12. FoldEco: A Model for Proteostasis in E. coli

    Directory of Open Access Journals (Sweden)

    Evan T. Powers

    2012-03-01

    Full Text Available To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide questions and shows how to convert these questions to experimentally measurable quantities, such as changes in protein concentrations with chaperone or protease levels, which can then be used to improve our current understanding of proteostasis and refine the model. A web version of FoldEco is available at http://foldeco.scripps.edu.

  13. Fast mapping of global protein folding states by multivariate NMR:

    DEFF Research Database (Denmark)

    Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel

    2010-01-01

    To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method......-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat....

  14. Can Natural Proteins Designed with ‘Inverted’ Peptide Sequences Adopt Native-Like Protein Folds?

    Science.gov (United States)

    Sridhar, Settu; Guruprasad, Kunchur

    2014-01-01

    We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to ‘swap’ certain short peptide sequences in naturally occurring proteins with their corresponding ‘inverted’ peptides and generate ‘artificial’ proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5–12 and 18 amino acid residues. Our analysis illustrates with examples that such ‘artificial’ proteins may be generated by identifying peptides with ‘similar structural environment’ and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides. PMID:25210740

  15. Adaptive local learning in sampling based motion planning for protein folding.

    Science.gov (United States)

    Ekenna, Chinwe; Thomas, Shawna; Amato, Nancy M

    2016-08-01

    Simulating protein folding motions is an important problem in computational biology. Motion planning algorithms, such as Probabilistic Roadmap Methods, have been successful in modeling the folding landscape. Probabilistic Roadmap Methods and variants contain several phases (i.e., sampling, connection, and path extraction). Most of the time is spent in the connection phase and selecting which variant to employ is a difficult task. Global machine learning has been applied to the connection phase but is inefficient in situations with varying topology, such as those typical of folding landscapes. We develop a local learning algorithm that exploits the past performance of methods within the neighborhood of the current connection attempts as a basis for learning. It is sensitive not only to different types of landscapes but also to differing regions in the landscape itself, removing the need to explicitly partition the landscape. We perform experiments on 23 proteins of varying secondary structure makeup with 52-114 residues. We compare the success rate when using our methods and other methods. We demonstrate a clear need for learning (i.e., only learning methods were able to validate against all available experimental data) and show that local learning is superior to global learning producing, in many cases, significantly higher quality results than the other methods. We present an algorithm that uses local learning to select appropriate connection methods in the context of roadmap construction for protein folding. Our method removes the burden of deciding which method to use, leverages the strengths of the individual input methods, and it is extendable to include other future connection methods.

  16. Effect of the geometry of confining media on the stability and folding rate of α -helix proteins

    Science.gov (United States)

    Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad

    2018-05-01

    Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding

  17. Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein

    Science.gov (United States)

    Day, Ryan; Paschek, Dietmar; Garcia, Angel E.

    2012-01-01

    We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs per replica timescale. The total simulation time employed in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P,T), over the whole region of temperature and pressures sampled in the simulations. The ΔG(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. PMID:20408169

  18. Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

    Science.gov (United States)

    Dal Molin, J. P.; Caliri, A.

    2018-01-01

    Here we focus on the conformational search for the native structure when it is ruled by the hydrophobic effect and steric specificities coming from amino acids. Our main tool of investigation is a 3D lattice model provided by a ten-letter alphabet, the stereochemical model. This minimalist model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. We have three central goals here. The first one is to characterize the folding time (τ) by two distinct sampling methods, so we present two sets of 103 MC simulations for a fast protein-like sequence. The resulting sets of characteristic folding times, τ and τq were obtained by the application of the standard Metropolis algorithm (MA), as well as by an enhanced algorithm (Mq A). The finding for τq shows two things: (i) the chain-solvent hydrophobic interactions {hk } plus a set of inter-residues steric constraints {ci,j } are able to emulate the conformational search for the native structure. For each one of the 103MC performed simulations, the target is always found within a finite time window; (ii) the ratio τq / τ ≅ 1 / 10 suggests that the effect of local thermal fluctuations, encompassed by the Tsallis weight, provides to the chain an innate efficiency to escape from energetic and steric traps. We performed additional MC simulations with variations of our design rule to attest this first result, both algorithms the MA and the Mq A were applied to a restricted set of targets, a physical insight is provided. Our second finding was obtained by a set of 600 independent MC simulations, only performed with the Mq A applied to an extended set of 200 representative targets, our native structures. The results show how structural patterns should modulate τq, which cover four orders of magnitude; this finding is our second goal. The third, and last result, was obtained with a special kind of simulation performed with the purpose to explore a

  19. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    International Nuclear Information System (INIS)

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N.; Schug, Alexander

    2015-01-01

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism

  20. Characterisation of transition state structures for protein folding using 'high', 'medium' and 'low' {Phi}-values.

    Science.gov (United States)

    Geierhaas, Christian D; Salvatella, Xavier; Clarke, Jane; Vendruscolo, Michele

    2008-03-01

    It has been suggested that Phi-values, which allow structural information about transition states (TSs) for protein folding to be obtained, are most reliably interpreted when divided into three classes (high, medium and low). High Phi-values indicate almost completely folded regions in the TS, intermediate Phi-values regions with a detectable amount of structure and low Phi-values indicate mostly unstructured regions. To explore the extent to which this classification can be used to characterise in detail the structure of TSs for protein folding, we used Phi-values divided into these classes as restraints in molecular dynamics simulations. This type of procedure is related to that used in NMR spectroscopy to define the structure of native proteins from the measurement of inter-proton distances derived from nuclear Overhauser effects. We illustrate this approach by determining the TS ensembles of five proteins and by showing that the results are similar to those obtained by using as restraints the actual numerical Phi-values measured experimentally. Our results indicate that the simultaneous consideration of a set of low-resolution Phi-values can provide sufficient information for characterising the architecture of a TS for folding of a protein.

  1. Introducing the Levinthal's Protein Folding Paradox and Its Solution

    Science.gov (United States)

    Martínez, Leandro

    2014-01-01

    The protein folding (Levinthal's) paradox states that it would not be possible in a physically meaningful time to a protein to reach the native (functional) conformation by a random search of the enormously large number of possible structures. This paradox has been solved: it was shown that small biases toward the native conformation result…

  2. Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

    Science.gov (United States)

    Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

    2006-11-28

    We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

  3. Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding

    NARCIS (Netherlands)

    Engel, R.; Westphal, A.H.; Huberts, D.; Nabuurs, S.M.; Lindhoud, S.; Visser, A.J.W.G.; Mierlo, van C.P.M.

    2008-01-01

    To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is

  4. A replica exchange Monte Carlo algorithm for protein folding in the HP model

    Directory of Open Access Journals (Sweden)

    Shmygelska Alena

    2007-09-01

    Full Text Available Abstract Background The ab initio protein folding problem consists of predicting protein tertiary structure from a given amino acid sequence by minimizing an energy function; it is one of the most important and challenging problems in biochemistry, molecular biology and biophysics. The ab initio protein folding problem is computationally challenging and has been shown to be NP MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaat0uy0HwzTfgDPnwy1egaryqtHrhAL1wy0L2yHvdaiqaacqWFneVtcqqGqbauaaa@3961@-hard even when conformations are restricted to a lattice. In this work, we implement and evaluate the replica exchange Monte Carlo (REMC method, which has already been applied very successfully to more complex protein models and other optimization problems with complex energy landscapes, in combination with the highly effective pull move neighbourhood in two widely studied Hydrophobic Polar (HP lattice models. Results We demonstrate that REMC is highly effective for solving instances of the square (2D and cubic (3D HP protein folding problem. When using the pull move neighbourhood, REMC outperforms current state-of-the-art algorithms for most benchmark instances. Additionally, we show that this new algorithm provides a larger ensemble of ground-state structures than the existing state-of-the-art methods. Furthermore, it scales well with sequence length, and it finds significantly better conformations on long biological sequences and sequences with a provably unique ground-state structure, which is believed to be a characteristic of real proteins. We also present evidence that our REMC algorithm can fold sequences which exhibit significant interaction between termini in the hydrophobic core relatively easily. Conclusion We demonstrate that REMC utilizing the pull move

  5. The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

    Directory of Open Access Journals (Sweden)

    Bani Kumar Pathak

    Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

  6. What determines the structures of native folds of proteins?

    International Nuclear Information System (INIS)

    Trovato, Antonio; Hoang, Trinh X; Banavar, Jayanth R; Maritan, Amos; Seno, Flavio

    2005-01-01

    We review a simple physical model (Hoang et al 2004 Proc. Natl Acad. Sci. USA 101 7960, Banavar et al 2004 Phys. Rev. E at press) which captures the essential physico-chemical ingredients that determine protein structure, such as the inherent anisotropy of a chain molecule, the geometrical and energetic constraints placed by hydrogen bonds, sterics, and hydrophobicity. Within this framework, marginally compact conformations resembling the native state folds of proteins emerge as competing minima in the free energy landscape. Here we demonstrate that a hydrophobic-polar (HP) sequence composed of regularly repeated patterns has as its ground state a β-helical structure remarkably similar to a known architecture in the Protein Data Bank

  7. Improving decoy databases for protein folding algorithms

    KAUST Repository

    Lindsey, Aaron

    2014-01-01

    Copyright © 2014 ACM. Predicting protein structures and simulating protein folding are two of the most important problems in computational biology today. Simulation methods rely on a scoring function to distinguish the native structure (the most energetically stable) from non-native structures. Decoy databases are collections of non-native structures used to test and verify these functions. We present a method to evaluate and improve the quality of decoy databases by adding novel structures and removing redundant structures. We test our approach on 17 different decoy databases of varying size and type and show significant improvement across a variety of metrics. We also test our improved databases on a popular modern scoring function and show that they contain a greater number of native-like structures than the original databases, thereby producing a more rigorous database for testing scoring functions.

  8. Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2014-03-01

    We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

  9. Understanding curcumin-induced modulation of protein aggregation.

    Science.gov (United States)

    Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

    2017-07-01

    Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Development and application of a free energy force field for all atom protein folding

    International Nuclear Information System (INIS)

    Verma, A.

    2007-11-01

    Proteins are the workhorses of all cellular life. They constitute the building blocks and the machinery of all cells and typically function in specific three-dimensional conformations into which each protein folds. Currently over one million protein sequences are known, compared to about 40,000 structures deposited in the Protein Data Bank (the world-wide database of protein structures). Reliable theoretical methods for protein structure prediction could help to reduce the gap between sequence and structural databases and elucidate the biological information in structurally unresolved sequences. In this thesis we explore an approach for protein structure prediction and folding that is based on the Anfinsen's hypothesis that most proteins in their native state are in thermodynamic equilibrium with their environment. We have developed a free energy forcefield (PFF02) that locates the native conformation of many proteins from all structural classes at the global minimum of the free-energy model. We have validated the forcefield against a large decoy set (Rosetta). The average root mean square deviation (RMSD) for the lowest energy structure for the 32 proteins of the decoy set was only 2.14 Aa from the experimental conformation. We have successfully implemented and used stochastic optimization methods, such as the basin hopping technique and evolutionary algorithms for all atom protein structure prediction. The evolutionary algorithm performs exceptionally well on large supercomputational architectures, such as BlueGene and MareNostrum. Using the PFF02 forcefield, we were able to fold 13 proteins (12-56 amino acids), which include helix, sheet and mixed secondary structure. On average the predicted structure of these proteins deviated from their experimental conformation by only 2.89 Aa RMSD. (orig.)

  11. Multiple scales and phases in discrete chains with application to folded proteins

    Science.gov (United States)

    Sinelnikova, A.; Niemi, A. J.; Nilsson, Johan; Ulybyshev, M.

    2018-05-01

    Chiral heteropolymers such as large globular proteins can simultaneously support multiple length scales. The interplay between the different scales brings about conformational diversity, determines the phase properties of the polymer chain, and governs the structure of the energy landscape. Most importantly, multiple scales produce complex dynamics that enable proteins to sustain live matter. However, at the moment there is incomplete understanding of how to identify and distinguish the various scales that determine the structure and dynamics of a complex protein. Here we address this impending problem. We develop a methodology with the potential to systematically identify different length scales, in the general case of a linear polymer chain. For this we introduce and analyze the properties of an order parameter that can both reveal the presence of different length scales and can also probe the phase structure. We first develop our concepts in the case of chiral homopolymers. We introduce a variant of Kadanoff's block-spin transformation to coarse grain piecewise linear chains, such as the C α backbone of a protein. We derive analytically, and then verify numerically, a number of properties that the order parameter can display, in the case of a chiral polymer chain. In particular, we propose that in the case of a chiral heteropolymer the order parameter can reveal traits of several different phases, contingent on the length scale at which it is scrutinized. We confirm that this is the case with crystallographic protein structures in the Protein Data Bank. Thus our results suggest relations between the scales, the phases, and the complexity of folding pathways.

  12. Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

    Science.gov (United States)

    Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

    2017-08-08

    To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

  13. Long range correlations and folding angle with applications to α-helical proteins

    Science.gov (United States)

    Krokhotin, Andrey; Nicolis, Stam; Niemi, Antti J.

    2014-03-01

    The conformational complexity of chain-like macromolecules such as proteins and other linear polymers is much larger than that of point-like atoms and molecules. Unlike particles, chains can bend, twist, and even become knotted. Thus chains might also display a much richer phase structure. Unfortunately, it is not very easy to characterize the phase of a long chain. Essentially, the only known attribute is the radius of gyration. The way how it changes when the degree of polymerization becomes different, and how it evolves when the ambient temperature and solvent properties change, is commonly used to disclose the phase. But in any finite length chain there are corrections to scaling that complicate the detailed analysis of the phase structure. Here we introduce a quantity that we call the folding angle to identify and scrutinize the phase structure, as a complement to the radius of gyration. We argue for a mean-field level relationship between the folding angle and the scaling exponent in the radius of gyration. We then estimate the value of the folding angle in the case of crystallographic α-helical protein structures in the Protein Data Bank. We also show how the experimental value of the folding angle can be obtained computationally, using a semiclassical Born-Oppenheimer description of α-helical chiral chains.

  14. Using the Computer Game "FoldIt" to Entice Students to Explore External Representations of Protein Structure in a Biochemistry Course for Nonmajors

    Science.gov (United States)

    Farley, Peter C.

    2013-01-01

    This article describes a novel approach to teaching novice Biochemistry students visual literacy skills and understanding of some aspects of protein structure using the internet resource FoldIt and a worksheet based on selected Introductory Puzzles from this computer game. In responding to a questionnaire, students indicated that they (94%)…

  15. Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT

    Directory of Open Access Journals (Sweden)

    Gregory R. Kowald

    2016-01-01

    Full Text Available Earthworms express, as most animals, metallothioneins (MTs—small, cysteine-rich proteins that bind d10 metal ions (Zn(II, Cd(II, or Cu(I in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II and Zn(II. Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II, expressions in the presence of Zn(II yielded mixtures. The average affinities of wMT-2 determined for either Cd(II or Zn(II are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

  16. The Dynameomics Entropy Dictionary: A Large-Scale Assessment of Conformational Entropy across Protein Fold Space.

    Science.gov (United States)

    Towse, Clare-Louise; Akke, Mikael; Daggett, Valerie

    2017-04-27

    Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing data set of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identifies deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, α-helices have lower entropy on average than do β-sheets, and both are lower than coil regions.

  17. Steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins.

    Science.gov (United States)

    Huang, Wenxi; Liu, Wanting; Jin, Jingjie; Xiao, Qilan; Lu, Ruibin; Chen, Wei; Xiong, Sheng; Zhang, Gong

    2018-03-25

    Translational pausing coordinates protein synthesis and co-translational folding. It is a common factor that facilitates the correct folding of large, multi-domain proteins. For small proteins, pausing sites rarely occurs in the gene body, and the 3'-end pausing sites are only essential for the folding of a fraction of proteins. The determinant of the necessity of the pausings remains obscure. In this study, we demonstrated that the steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins. Validated by experiments with 5 model proteins, we found that the rigid protein structures do not, while the flexible structures do need 3'-end pausings to fold correctly. Therefore, rational optimization of translational pausing can improve soluble expression of small proteins with flexible structures, but not the rigid ones. The rigidity of the structure can be quantitatively estimated in silico using molecular dynamic simulation. Nevertheless, we also found that the translational pausing optimization increases the fitness of the expression host, and thus benefits the recombinant protein production, independent from the soluble expression. These results shed light on the structural basis of the translational pausing and provided a practical tool for industrial protein fermentation. Copyright © 2017. Published by Elsevier Inc.

  18. On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1.

    Science.gov (United States)

    Wu, Chun; Shea, Joan-Emma

    2010-11-18

    FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures.

  19. Phase transition in polypeptides: a step towards the understanding of protein folding

    DEFF Research Database (Denmark)

    Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

    2006-01-01

    We present a formalism which turns out to be very successful in the description of the polypeptide folding. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely on fundamental physical principles. It describes...... essential thermodynamical properties of the system such as heat capacity, the phase transition temperature and others from the analysis of the polypeptide potential energy surface calculated within ab initio density functional theory and parameterized by two dihedral angles. This problem is viewed...

  20. Recognition of secretory proteins in Escherichia coli requires signals in addition to the signal sequence and slow folding

    Directory of Open Access Journals (Sweden)

    Flower Ann M

    2002-11-01

    Full Text Available Abstract Background The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. Results In the current work, we tested this hypothesis using a mutant form of λ repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant λ repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase. Conclusions These results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.

  1. Discrete Frenet frame, inflection point solitons, and curve visualization with applications to folded proteins

    Science.gov (United States)

    Hu, Shuangwei; Lundgren, Martin; Niemi, Antti J.

    2011-06-01

    We develop a transfer matrix formalism to visualize the framing of discrete piecewise linear curves in three-dimensional space. Our approach is based on the concept of an intrinsically discrete curve. This enables us to more effectively describe curves that in the limit where the length of line segments vanishes approach fractal structures in lieu of continuous curves. We verify that in the case of differentiable curves the continuum limit of our discrete equation reproduces the generalized Frenet equation. In particular, we draw attention to the conceptual similarity between inflection points where the curvature vanishes and topologically stable solitons. As an application we consider folded proteins, their Hausdorff dimension is known to be fractal. We explain how to employ the orientation of Cβ carbons of amino acids along a protein backbone to introduce a preferred framing along the backbone. By analyzing the experimentally resolved fold geometries in the Protein Data Bank we observe that this Cβ framing relates intimately to the discrete Frenet framing. We also explain how inflection points (a.k.a. soliton centers) can be located in the loops and clarify their distinctive rôle in determining the loop structure of folded proteins.

  2. An essential nonredundant role for mycobacterial DnaK in native protein folding.

    Directory of Open Access Journals (Sweden)

    Allison Fay

    2014-07-01

    Full Text Available Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.

  3. The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain.

    Directory of Open Access Journals (Sweden)

    Søren W. Pedersen

    Full Text Available Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

  4. A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

    DEFF Research Database (Denmark)

    Lyngsø, C.; Kjaerulff, S.; Muller, S.

    2010-01-01

    in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality...

  5. Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

    Science.gov (United States)

    Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

    2016-01-01

    While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

  6. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

    Science.gov (United States)

    Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin

    2012-01-01

    Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740

  7. TMFoldWeb: a web server for predicting transmembrane protein fold class.

    Science.gov (United States)

    Kozma, Dániel; Tusnády, Gábor E

    2015-09-17

    Here we present TMFoldWeb, the web server implementation of TMFoldRec, a transmembrane protein fold recognition algorithm. TMFoldRec uses statistical potentials and utilizes topology filtering and a gapless threading algorithm. It ranks template structures and selects the most likely candidates and estimates the reliability of the obtained lowest energy model. The statistical potential was developed in a maximum likelihood framework on a representative set of the PDBTM database. According to the benchmark test the performance of TMFoldRec is about 77 % in correctly predicting fold class for a given transmembrane protein sequence. An intuitive web interface has been developed for the recently published TMFoldRec algorithm. The query sequence goes through a pipeline of topology prediction and a systematic sequence to structure alignment (threading). Resulting templates are ordered by energy and reliability values and are colored according to their significance level. Besides the graphical interface, a programmatic access is available as well, via a direct interface for developers or for submitting genome-wide data sets. The TMFoldWeb web server is unique and currently the only web server that is able to predict the fold class of transmembrane proteins while assigning reliability scores for the prediction. This method is prepared for genome-wide analysis with its easy-to-use interface, informative result page and programmatic access. Considering the info-communication evolution in the last few years, the developed web server, as well as the molecule viewer, is responsive and fully compatible with the prevalent tablets and mobile devices.

  8. Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.

    Science.gov (United States)

    Kukic, Predrag; Kannan, Arvind; Dijkstra, Maurits J J; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

    2015-10-01

    It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.

  9. FRAN and RBF-PSO as two components of a hyper framework to recognize protein folds.

    Science.gov (United States)

    Abbasi, Elham; Ghatee, Mehdi; Shiri, M E

    2013-09-01

    In this paper, an intelligent hyper framework is proposed to recognize protein folds from its amino acid sequence which is a fundamental problem in bioinformatics. This framework includes some statistical and intelligent algorithms for proteins classification. The main components of the proposed framework are the Fuzzy Resource-Allocating Network (FRAN) and the Radial Bases Function based on Particle Swarm Optimization (RBF-PSO). FRAN applies a dynamic method to tune up the RBF network parameters. Due to the patterns complexity captured in protein dataset, FRAN classifies the proteins under fuzzy conditions. Also, RBF-PSO applies PSO to tune up the RBF classifier. Experimental results demonstrate that FRAN improves prediction accuracy up to 51% and achieves acceptable multi-class results for protein fold prediction. Although RBF-PSO provides reasonable results for protein fold recognition up to 48%, it is weaker than FRAN in some cases. However the proposed hyper framework provides an opportunity to use a great range of intelligent methods and can learn from previous experiences. Thus it can avoid the weakness of some intelligent methods in terms of memory, computational time and static structure. Furthermore, the performance of this system can be enhanced throughout the system life-cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1.

    Directory of Open Access Journals (Sweden)

    Chun Wu

    Full Text Available FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96 coupled with the implicit solvent model (IGB = 5, we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K as well as the denatured structures.

  11. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  12. The energy landscapes of repeat-containing proteins: topology, cooperativity, and the folding funnels of one-dimensional architectures.

    Directory of Open Access Journals (Sweden)

    Diego U Ferreiro

    2008-05-01

    Full Text Available Repeat-proteins are made up of near repetitions of 20- to 40-amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi-one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete "domain" (the stability and cooperativity of the repeating array can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (DeltaG(water and the cooperativity of denaturation (m-value, which do not ordinarily apply for globular proteins. We show how the parameters for the "coarse-grained" description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are "poised" at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions.

  13. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

    Directory of Open Access Journals (Sweden)

    Tyo Keith EJ

    2012-03-01

    Full Text Available Abstract Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor or a larger protein (α-amylase. Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a degradation of protein/recycling amino acids, (b overall transcription/translation repression, and (c oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

  14. Thermostability in endoglucanases is fold-specific

    Science.gov (United States)

    2011-01-01

    Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

  15. Thermostability in endoglucanases is fold-specific

    Directory of Open Access Journals (Sweden)

    Wolt Jeffrey D

    2011-02-01

    Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

  16. Insights into the fold organization of TIM barrel from interaction energy based structure networks.

    Science.gov (United States)

    Vijayabaskar, M S; Vishveshwara, Saraswathi

    2012-01-01

    There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

  17. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    Science.gov (United States)

    Atkinson, Holly J; Babbitt, Patricia C

    2009-10-01

    The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  18. New protein structures provide an updated understanding of phenylketonuria.

    Science.gov (United States)

    Jaffe, Eileen K

    2017-08-01

    Phenylketonuria (PKU) and less severe hyperphenylalaninemia (HPA) constitute the most common inborn error of amino acid metabolism, and is most often caused by defects in phenylalanine hydroxylase (PAH) function resulting in accumulation of Phe to neurotoxic levels. Despite the success of dietary intervention in preventing permanent neurological damage, individuals living with PKU clamor for additional non-dietary therapies. The bulk of disease-associated mutations are PAH missense variants, which occur throughout the entire 452 amino acid human PAH protein. While some disease-associated mutations affect protein structure (e.g. truncations) and others encode catalytically dead variants, most have been viewed as defective in protein folding/stability. Here we refine this view to address how PKU-associated missense variants can perturb the equilibrium among alternate native PAH structures (resting-state PAH and activated PAH), thus shifting the tipping point of this equilibrium to a neurotoxic Phe concentration. This refined view of PKU introduces opportunities for the design or discovery of therapeutic pharmacological chaperones that can help restore the tipping point to healthy Phe levels and how such a therapeutic might work with or without the inhibitory pharmacological chaperone BH 4 . Dysregulation of an equilibrium of architecturally distinct native PAH structures departs from the concept of "misfolding", provides an updated understanding of PKU, and presents an enhanced foundation for understanding genotype/phenotype relationships. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The Complexity of Folding Self-Folding Origami

    Science.gov (United States)

    Stern, Menachem; Pinson, Matthew B.; Murugan, Arvind

    2017-10-01

    Why is it difficult to refold a previously folded sheet of paper? We show that even crease patterns with only one designed folding motion inevitably contain an exponential number of "distractor" folding branches accessible from a bifurcation at the flat state. Consequently, refolding a sheet requires finding the ground state in a glassy energy landscape with an exponential number of other attractors of higher energy, much like in models of protein folding (Levinthal's paradox) and other NP-hard satisfiability (SAT) problems. As in these problems, we find that refolding a sheet requires actuation at multiple carefully chosen creases. We show that seeding successful folding in this way can be understood in terms of subpatterns that fold when cut out ("folding islands"). Besides providing guidelines for the placement of active hinges in origami applications, our results point to fundamental limits on the programmability of energy landscapes in sheets.

  20. CATHEDRAL: a fast and effective algorithm to predict folds and domain boundaries from multidomain protein structures.

    Directory of Open Access Journals (Sweden)

    Oliver C Redfern

    2007-11-01

    Full Text Available We present CATHEDRAL, an iterative protocol for determining the location of previously observed protein folds in novel multidomain protein structures. CATHEDRAL builds on the features of a fast secondary-structure-based method (using graph theory to locate known folds within a multidomain context and a residue-based, double-dynamic programming algorithm, which is used to align members of the target fold groups against the query protein structure to identify the closest relative and assign domain boundaries. To increase the fidelity of the assignments, a support vector machine is used to provide an optimal scoring scheme. Once a domain is verified, it is excised, and the search protocol is repeated in an iterative fashion until all recognisable domains have been identified. We have performed an initial benchmark of CATHEDRAL against other publicly available structure comparison methods using a consensus dataset of domains derived from the CATH and SCOP domain classifications. CATHEDRAL shows superior performance in fold recognition and alignment accuracy when compared with many equivalent methods. If a novel multidomain structure contains a known fold, CATHEDRAL will locate it in 90% of cases, with <1% false positives. For nearly 80% of assigned domains in a manually validated test set, the boundaries were correctly delineated within a tolerance of ten residues. For the remaining cases, previously classified domains were very remotely related to the query chain so that embellishments to the core of the fold caused significant differences in domain sizes and manual refinement of the boundaries was necessary. To put this performance in context, a well-established sequence method based on hidden Markov models was only able to detect 65% of domains, with 33% of the subsequent boundaries assigned within ten residues. Since, on average, 50% of newly determined protein structures contain more than one domain unit, and typically 90% or more of these

  1. Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

    International Nuclear Information System (INIS)

    Giesen, Alexander W.; Homans, Steve W.; Brown, Jonathan Miles

    2003-01-01

    We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N i -H N i , N i -C' i-1 , H N i - C' i-1 ) in two tensor frames and only backbone H N -H N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 A with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms

  2. The Complexity of Folding Self-Folding Origami

    Directory of Open Access Journals (Sweden)

    Menachem Stern

    2017-12-01

    Full Text Available Why is it difficult to refold a previously folded sheet of paper? We show that even crease patterns with only one designed folding motion inevitably contain an exponential number of “distractor” folding branches accessible from a bifurcation at the flat state. Consequently, refolding a sheet requires finding the ground state in a glassy energy landscape with an exponential number of other attractors of higher energy, much like in models of protein folding (Levinthal’s paradox and other NP-hard satisfiability (SAT problems. As in these problems, we find that refolding a sheet requires actuation at multiple carefully chosen creases. We show that seeding successful folding in this way can be understood in terms of subpatterns that fold when cut out (“folding islands”. Besides providing guidelines for the placement of active hinges in origami applications, our results point to fundamental limits on the programmability of energy landscapes in sheets.

  3. A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds.

    Directory of Open Access Journals (Sweden)

    Konstantin B Zeldovich

    2007-07-01

    Full Text Available In this work we develop a microscopic physical model of early evolution where phenotype--organism life expectancy--is directly related to genotype--the stability of its proteins in their native conformations-which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the "Big Bang" scenario whereby exponential population growth ensues as soon as favorable sequence-structure combinations (precursors of stable proteins are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species--subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution.

  4. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    Directory of Open Access Journals (Sweden)

    Holly J Atkinson

    2009-10-01

    Full Text Available The group of proteins that contain a thioredoxin (Trx fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  5. Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Aksyuk, Anastasia A.; Kurochkina, Lidia P.; Fokine, Andrei; Forouhar, Farhad; Mesyanzhinov, Vadim V.; Tong, Liang; Rossmann, Michael G. (SOIBC); (Purdue); (Columbia)

    2012-02-21

    Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 {angstrom} diameter icosahedral head and a 2000 {angstrom}-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 {angstrom} resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 {angstrom} resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.

  6. Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

    Science.gov (United States)

    Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

    2012-01-01

    Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

  7. MFIB: a repository of protein complexes with mutual folding induced by binding.

    Science.gov (United States)

    Fichó, Erzsébet; Reményi, István; Simon, István; Mészáros, Bálint

    2017-11-15

    It is commonplace that intrinsically disordered proteins (IDPs) are involved in crucial interactions in the living cell. However, the study of protein complexes formed exclusively by IDPs is hindered by the lack of data and such analyses remain sporadic. Systematic studies benefited other types of protein-protein interactions paving a way from basic science to therapeutics; yet these efforts require reliable datasets that are currently lacking for synergistically folding complexes of IDPs. Here we present the Mutual Folding Induced by Binding (MFIB) database, the first systematic collection of complexes formed exclusively by IDPs. MFIB contains an order of magnitude more data than any dataset used in corresponding studies and offers a wide coverage of known IDP complexes in terms of flexibility, oligomeric composition and protein function from all domains of life. The included complexes are grouped using a hierarchical classification and are complemented with structural and functional annotations. MFIB is backed by a firm development team and infrastructure, and together with possible future community collaboration it will provide the cornerstone for structural and functional studies of IDP complexes. MFIB is freely accessible at http://mfib.enzim.ttk.mta.hu/. The MFIB application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. simon.istvan@ttk.mta.hu, meszaros.balint@ttk.mta.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

  8. The Role of Short-Chain Conjugated Poly-(R-3-Hydroxybutyrate (cPHB in Protein Folding

    Directory of Open Access Journals (Sweden)

    Rosetta N. Reusch

    2013-05-01

    Full Text Available Poly-(R-3-hydroxybutyrate (PHB, a linear polymer of R-3-hydroxybutyrate (R-3HB, is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues, which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues which is conjugated to proteins (cPHB, primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA of Escherichia coli.

  9. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    Science.gov (United States)

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  10. Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type

    Directory of Open Access Journals (Sweden)

    Fujiwara Kazuo

    2012-08-01

    Full Text Available Abstract Background A large number of studies have been carried out to obtain amino acid propensities for α-helices and β-sheets. The obtained propensities for α-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the β-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects β-sheet propensity. Results We calculated amino acid propensities for α-helices and β-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that α-helix propensities do not differ significantly by fold, but β-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for α-helix, but such is not the case for the β-sheet propensities. We also found some fold dependence on amino acid frequency in β-strands. Folds with a high Ser, Thr and Asn content at exposed sites in β-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = −0.90 and to have flat β-sheets. At buried sites in β-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = −0.93. "All-β" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas "α/β" proteins tend to have a higher content of Val, Ile and Leu. Conclusions The α-helix propensities are similar for all folds and for exposed and buried residues. However, β-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in β-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding

  11. Production, purification and oxidative folding of the mouse recombinant prion protein

    Czech Academy of Sciences Publication Activity Database

    Pavlíček, A.; Bednárová, Lucie; Holada, K.

    2007-01-01

    Roč. 52, č. 4 (2007), s. 391-397 ISSN 0015-5632 R&D Projects: GA ČR GD310/05/H533 Grant - others:GA ČR(CZ) GA310/04/0419 Institutional research plan: CEZ:AV0Z40550506 Keywords : recombinant prion protein * production * purification * folding Subject RIV: CE - Biochemistry Impact factor: 0.989, year: 2007 http://www.biomed.cas.cz/mbu/folia/

  12. RNA folding: structure prediction, folding kinetics and ion electrostatics.

    Science.gov (United States)

    Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

    2015-01-01

    Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.

  13. A structural basis for cellular uptake of GST-fold proteins.

    Directory of Open Access Journals (Sweden)

    Melanie J Morris

    Full Text Available It has recently emerged that glutathione transferase enzymes (GSTs and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.

  14. Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

    International Nuclear Information System (INIS)

    Roder, H.; Wuethrich, K.

    1986-01-01

    A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1 H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1 H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes

  15. Vocal fold ion transport and mucin expression following acrolein exposure.

    Science.gov (United States)

    Levendoski, Elizabeth Erickson; Sivasankar, M Preeti

    2014-05-01

    The vocal fold epithelium is exposed to inhaled particulates including pollutants during breathing in everyday environments. Yet, our understanding of the effects of pollutants on vocal fold epithelial function is extremely limited. The objective of this study was to investigate the effect of the pollutant acrolein on two vocal fold epithelial mechanisms: ion transport and mucin (MUC) synthesis. These mechanisms were chosen as each plays a critical role in vocal defense and in maintaining surface hydration which is necessary for optimal voice production. Healthy, native porcine vocal folds (N = 85) were excised and exposed to an acrolein or sham challenge. A 60-min acrolein, but not sham challenge significantly reduced ion transport and inhibited cyclic adenosine monophosphate-dependent, increases in ion transport. Decreases in ion transport were associated with reduced sodium absorption. Within the same timeline, no significant acrolein-induced changes in MUC gene or protein expression were observed. These results improve our understanding of the effects of acrolein on key vocal fold epithelial functions and inform the development of future investigations that seek to elucidate the impact of a wide range of pollutant exposures on vocal fold health.

  16. Thermodynamics of protein folding using a modified Wako-Saitô-Muñoz-Eaton model.

    Science.gov (United States)

    Tsai, Min-Yeh; Yuan, Jian-Min; Teranishi, Yoshiaki; Lin, Sheng Hsien

    2012-09-01

    Herein, we propose a modified version of the Wako-Saitô-Muñoz-Eaton (WSME) model. The proposed model introduces an empirical temperature parameter for the hypothetical structural units (i.e., foldons) in proteins to include site-dependent thermodynamic behavior. The thermodynamics for both our proposed model and the original WSME model were investigated. For a system with beta-hairpin topology, a mathematical treatment (contact-pair treatment) to facilitate the calculation of its partition function was developed. The results show that the proposed model provides better insight into the site-dependent thermodynamic behavior of the system, compared with the original WSME model. From this site-dependent point of view, the relationship between probe-dependent experimental results and model's thermodynamic predictions can be explained. The model allows for suggesting a general principle to identify foldon behavior. We also find that the backbone hydrogen bonds may play a role of structural constraints in modulating the cooperative system. Thus, our study may contribute to the understanding of the fundamental principles for the thermodynamics of protein folding.

  17. Method of generating ploynucleotides encoding enhanced folding variants

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.

    2017-05-02

    The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

  18. MICROFLUIDIC MIXERS FOR THE INVESTIGATION OF PROTEIN FOLDING USING SYNCHROTRON RADIATION CIRCULAR DICHROISM SPECTROSCOPY

    International Nuclear Information System (INIS)

    Kane, A; Hertzog, D; Baumgartel, P; Lengefeld, J; Horsley, D; Schuler, B; Bakajin, O

    2006-01-01

    The purpose of this study is to design, fabricate and optimize microfluidic mixers to investigate the kinetics of protein secondary structure formation with Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The mixers are designed to rapidly initiate protein folding reaction through the dilution of denaturant. The devices are fabricated out of fused silica, so that they are transparent in the UV. We present characterization of mixing in the fabricated devices, as well as the initial SRCD data on proteins inside the mixers

  19. Coarsely resolved topography along protein folding pathways

    Science.gov (United States)

    Fernández, Ariel; Kostov, Konstantin S.; Berry, R. Stephen

    2000-03-01

    The kinetic data from the coarse representation of polypeptide torsional dynamics described in the preceding paper [Fernandez and Berry, J. Chem. Phys. 112, 5212 (2000), preceding paper] is inverted by using detailed balance to obtain a topographic description of the potential-energy surface (PES) along the dominant folding pathway of the bovine pancreatic trypsin inhibitor (BPTI). The topography is represented as a sequence of minima and effective saddle points. The dominant folding pathway displays an overall monotonic decrease in energy with a large number of staircaselike steps, a clear signature of a good structure-seeker. The diversity and availability of alternative folding pathways is analyzed in terms of the Shannon entropy σ(t) associated with the time-dependent probability distribution over the kinetic ensemble of contact patterns. Several stages in the folding process are evident. Initially misfolded states form and dismantle revealing no definite pattern in the topography and exhibiting high Shannon entropy. Passage down a sequence of staircase steps then leads to the formation of a nativelike intermediate, for which σ(t) is much lower and fairly constant. Finally, the structure of the intermediate is refined to produce the native state of BPTI. We also examine how different levels of tolerance to mismatches of side chain contacts influence the folding kinetics, the topography of the dominant folding pathway, and the Shannon entropy. This analysis yields upper and lower bounds of the frustration tolerance required for the expeditious and robust folding of BPTI.

  20. The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Osmark, Peter; Neergaard, Thomas B.

    1999-01-01

    The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24...... probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding...... step and that a sequential framework model can describe the protein folding reaction....

  1. The role of atomic level steric effects and attractive forces in protein folding.

    Science.gov (United States)

    Lammert, Heiko; Wolynes, Peter G; Onuchic, José N

    2012-02-01

    Protein folding into tertiary structures is controlled by an interplay of attractive contact interactions and steric effects. We investigate the balance between these contributions using structure-based models using an all-atom representation of the structure combined with a coarse-grained contact potential. Tertiary contact interactions between atoms are collected into a single broad attractive well between the C(β) atoms between each residue pair in a native contact. Through the width of these contact potentials we control their tolerance for deviations from the ideal structure and the spatial range of attractive interactions. In the compact native state dominant packing constraints limit the effects of a coarse-grained contact potential. During folding, however, the broad attractive potentials allow an early collapse that starts before the native local structure is completely adopted. As a consequence the folding transition is broadened and the free energy barrier is decreased. Eventually two-state folding behavior is lost completely for systems with very broad attractive potentials. The stabilization of native-like residue interactions in non-perfect geometries early in the folding process frequently leads to structural traps. Global mirror images are a notable example. These traps are penalized by the details of the repulsive interactions only after further collapse. Successful folding to the native state requires simultaneous guidance from both attractive and repulsive interactions. Copyright © 2011 Wiley Periodicals, Inc.

  2. BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints

    Science.gov (United States)

    Fischer, Axel W.; Alexander, Nathan S.; Woetzel, Nils; Karakaş, Mert; Weiner, Brian E.; Meiler, Jens

    2016-01-01

    For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The protein-size-normalized root-mean-square-deviation (RMSD100) value of the most accurate model is better than 8 Å for twenty-seven, better than 6 Å for twenty-two, and better than 4 Å for fifteen out of twenty-nine proteins, demonstrating the algorithm’s ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data. PMID:25820805

  3. A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

    Science.gov (United States)

    2015-01-01

    Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

  4. In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

    Directory of Open Access Journals (Sweden)

    Dref C De Moura

    Full Text Available The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L. plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

  5. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  6. The ModFOLD4 server for the quality assessment of 3D protein models

    OpenAIRE

    McGuffin, Liam J.; Buenavista, Maria T.; Roche, Daniel B.

    2013-01-01

    Once you have generated a 3D model of a protein,\\ud how do you know whether it bears any resemblance\\ud to the actual structure? To determine the usefulness\\ud of 3D models of proteins, they must be assessed in\\ud terms of their quality by methods that predict their\\ud similarity to the native structure. The ModFOLD4\\ud server is the latest version of our leading independent\\ud server for the estimation of both the global and\\ud local (per-residue) quality of 3D protein models. The\\ud server ...

  7. Conserved nucleation sites reinforce the significance of Phi value analysis in protein-folding studies.

    Science.gov (United States)

    Gianni, Stefano; Jemth, Per

    2014-07-01

    The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.

  8. Polymer collapse, protein folding, and the percolation threshold.

    Science.gov (United States)

    Meirovitch, Hagai

    2002-01-15

    We study the transition of polymers in the dilute regime from a swollen shape at high temperatures to their low-temperature structures. The polymers are modeled by a single self-avoiding walk (SAW) on a lattice for which l of the monomers (the H monomers) are self-attracting, i.e., if two nonbonded H monomers become nearest neighbors on the lattice they gain energy of interaction (epsilon = -/epsilon/); the second type of monomers, denoted P, are neutral. This HP model was suggested by Lau and Dill (Macromolecules 1989, 22, 3986-3997) to study protein folding, where H and P are the hydrophobic and polar amino acid residues, respectively. The model is simulated on the square and simple cubic (SC) lattices using the scanning method. We show that the ground state and the sharpness of the transition depend on the lattice, the fraction g of the H monomers, as well as on their arrangement along the chain. In particular, if the H monomers are distributed at random and g is larger than the site percolation threshold of the lattice, a collapsed transition is very likely to occur. This conclusion, drawn for the lattice models, is also applicable to proteins where an effective lattice with coordination number between that of the SC lattice and the body centered cubic lattice is defined. Thus, the average fraction of hydrophobic amino acid residues in globular proteins is found to be close to the percolation threshold of the effective lattice.

  9. Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

    2012-03-01

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Multi-scaled explorations of binding-induced folding of intrinsically disordered protein inhibitor IA3 to its target enzyme.

    Directory of Open Access Journals (Sweden)

    Jin Wang

    2011-04-01

    Full Text Available Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change - protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.

  11. Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

    Directory of Open Access Journals (Sweden)

    Lee Dae-Hee

    2009-03-01

    Full Text Available Abstract Background Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A and cyclohexanone monooxygenase (CHMO. Results We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively. Conclusion The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.

  12. ModFOLD6: an accurate web server for the global and local quality estimation of 3D protein models.

    Science.gov (United States)

    Maghrabi, Ali H A; McGuffin, Liam J

    2017-07-03

    Methods that reliably estimate the likely similarity between the predicted and native structures of proteins have become essential for driving the acceptance and adoption of three-dimensional protein models by life scientists. ModFOLD6 is the latest version of our leading resource for Estimates of Model Accuracy (EMA), which uses a pioneering hybrid quasi-single model approach. The ModFOLD6 server integrates scores from three pure-single model methods and three quasi-single model methods using a neural network to estimate local quality scores. Additionally, the server provides three options for producing global score estimates, depending on the requirements of the user: (i) ModFOLD6_rank, which is optimized for ranking/selection, (ii) ModFOLD6_cor, which is optimized for correlations of predicted and observed scores and (iii) ModFOLD6 global for balanced performance. The ModFOLD6 methods rank among the top few for EMA, according to independent blind testing by the CASP12 assessors. The ModFOLD6 server is also continuously automatically evaluated as part of the CAMEO project, where significant performance gains have been observed compared to our previous server and other publicly available servers. The ModFOLD6 server is freely available at: http://www.reading.ac.uk/bioinf/ModFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

    Science.gov (United States)

    Miller, Craig R; Lee, Kuo Hao; Wichman, Holly A; Ytreberg, F Marty

    2014-01-01

    Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

  14. SAAFEC: Predicting the Effect of Single Point Mutations on Protein Folding Free Energy Using a Knowledge-Modified MM/PBSA Approach.

    Science.gov (United States)

    Getov, Ivan; Petukh, Marharyta; Alexov, Emil

    2016-04-07

    Folding free energy is an important biophysical characteristic of proteins that reflects the overall stability of the 3D structure of macromolecules. Changes in the amino acid sequence, naturally occurring or made in vitro, may affect the stability of the corresponding protein and thus could be associated with disease. Several approaches that predict the changes of the folding free energy caused by mutations have been proposed, but there is no method that is clearly superior to the others. The optimal goal is not only to accurately predict the folding free energy changes, but also to characterize the structural changes induced by mutations and the physical nature of the predicted folding free energy changes. Here we report a new method to predict the Single Amino Acid Folding free Energy Changes (SAAFEC) based on a knowledge-modified Molecular Mechanics Poisson-Boltzmann (MM/PBSA) approach. The method is comprised of two main components: a MM/PBSA component and a set of knowledge based terms delivered from a statistical study of the biophysical characteristics of proteins. The predictor utilizes a multiple linear regression model with weighted coefficients of various terms optimized against a set of experimental data. The aforementioned approach yields a correlation coefficient of 0.65 when benchmarked against 983 cases from 42 proteins in the ProTherm database. the webserver can be accessed via http://compbio.clemson.edu/SAAFEC/.

  15. Using protein design algorithms to understand the molecular basis of disease caused by protein-DNA interactions: the Pax6 example

    DEFF Research Database (Denmark)

    Alibes, A.; Nadra, A.; De Masi, Federico

    2010-01-01

    diseases such as aniridia. The validity of FoldX to deal with protein-DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos......Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein's function is not trivial. Protein design tools are commonly used to explain mutations that affect protein...... stability, or protein-protein interaction, but not for mutations that could affect protein-DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several...

  16. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

    Science.gov (United States)

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction.

    Science.gov (United States)

    Roche, Daniel B; Buenavista, Maria T; Tetchner, Stuart J; McGuffin, Liam J

    2011-07-01

    The IntFOLD server is a novel independent server that integrates several cutting edge methods for the prediction of structure and function from sequence. Our guiding principles behind the server development were as follows: (i) to provide a simple unified resource that makes our prediction software accessible to all and (ii) to produce integrated output for predictions that can be easily interpreted. The output for predictions is presented as a simple table that summarizes all results graphically via plots and annotated 3D models. The raw machine readable data files for each set of predictions are also provided for developers, which comply with the Critical Assessment of Methods for Protein Structure Prediction (CASP) data standards. The server comprises an integrated suite of five novel methods: nFOLD4, for tertiary structure prediction; ModFOLD 3.0, for model quality assessment; DISOclust 2.0, for disorder prediction; DomFOLD 2.0 for domain prediction; and FunFOLD 1.0, for ligand binding site prediction. Predictions from the IntFOLD server were found to be competitive in several categories in the recent CASP9 experiment. The IntFOLD server is available at the following web site: http://www.reading.ac.uk/bioinf/IntFOLD/.

  18. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  19. Improving protein fold recognition and structural class prediction accuracies using physicochemical properties of amino acids.

    Science.gov (United States)

    Raicar, Gaurav; Saini, Harsh; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

    2016-08-07

    Predicting the three-dimensional (3-D) structure of a protein is an important task in the field of bioinformatics and biological sciences. However, directly predicting the 3-D structure from the primary structure is hard to achieve. Therefore, predicting the fold or structural class of a protein sequence is generally used as an intermediate step in determining the protein's 3-D structure. For protein fold recognition (PFR) and structural class prediction (SCP), two steps are required - feature extraction step and classification step. Feature extraction techniques generally utilize syntactical-based information, evolutionary-based information and physicochemical-based information to extract features. In this study, we explore the importance of utilizing the physicochemical properties of amino acids for improving PFR and SCP accuracies. For this, we propose a Forward Consecutive Search (FCS) scheme which aims to strategically select physicochemical attributes that will supplement the existing feature extraction techniques for PFR and SCP. An exhaustive search is conducted on all the existing 544 physicochemical attributes using the proposed FCS scheme and a subset of physicochemical attributes is identified. Features extracted from these selected attributes are then combined with existing syntactical-based and evolutionary-based features, to show an improvement in the recognition and prediction performance on benchmark datasets. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Bactericidal/Permeability-increasing protein fold-containing family member A1 in airway host protection and respiratory disease.

    Science.gov (United States)

    Britto, Clemente J; Cohn, Lauren

    2015-05-01

    Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.

  1. Protein P7 of the cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.

    Directory of Open Access Journals (Sweden)

    Garrett Katz

    Full Text Available The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase, and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.

  2. Natural triple beta-stranded fibrous folds.

    Science.gov (United States)

    Mitraki, Anna; Papanikolopoulou, Katerina; Van Raaij, Mark J

    2006-01-01

    A distinctive family of beta-structured folds has recently been described for fibrous proteins from viruses. Virus fibers are usually involved in specific host-cell recognition. They are asymmetric homotrimeric proteins consisting of an N-terminal virus-binding tail, a central shaft or stalk domain, and a C-terminal globular receptor-binding domain. Often they are entirely or nearly entirely composed of beta-structure. Apart from their biological relevance and possible gene therapy applications, their shape, stability, and rigidity suggest they may be useful as blueprints for biomechanical design. Folding and unfolding studies suggest their globular C-terminal domain may fold first, followed by a "zipping-up" of the shaft domains. The C-terminal domains appear to be important for registration because peptides corresponding to shaft domains alone aggregate into nonnative fibers and/or amyloid structures. C-terminal domains can be exchanged between different fibers and the resulting chimeric proteins are useful as a way to solve structures of unknown parts of the shaft domains. The following natural triple beta-stranded fibrous folds have been discovered by X-ray crystallography: the triple beta-spiral, triple beta-helix, and T4 short tail fiber fold. All have a central longitudinal hydrophobic core and extensive intermonomer polar and nonpolar interactions. Now that a reasonable body of structural and folding knowledge has been assembled about these fibrous proteins, the next challenge and opportunity is to start using this information in medical and industrial applications such as gene therapy and nanotechnology.

  3. Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

    Science.gov (United States)

    Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

    2012-12-15

    Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured

  4. Web-based computational chemistry education with CHARMMing II: Coarse-grained protein folding.

    Directory of Open Access Journals (Sweden)

    Frank C Pickard

    2014-07-01

    Full Text Available A lesson utilizing a coarse-grained (CG Gō-like model has been implemented into the CHARMM INterface and Graphics (CHARMMing web portal (www.charmming.org to the Chemistry at HARvard Macromolecular Mechanics (CHARMM molecular simulation package. While widely used to model various biophysical processes, such as protein folding and aggregation, CG models can also serve as an educational tool because they can provide qualitative descriptions of complex biophysical phenomena for a relatively cheap computational cost. As a proof of concept, this lesson demonstrates the construction of a CG model of a small globular protein, its simulation via Langevin dynamics, and the analysis of the resulting data. This lesson makes connections between modern molecular simulation techniques and topics commonly presented in an advanced undergraduate lecture on physical chemistry. It culminates in a straightforward analysis of a short dynamics trajectory of a small fast folding globular protein; we briefly describe the thermodynamic properties that can be calculated from this analysis. The assumptions inherent in the model and the data analysis are laid out in a clear, concise manner, and the techniques used are consistent with those employed by specialists in the field of CG modeling. One of the major tasks in building the Gō-like model is determining the relative strength of the nonbonded interactions between coarse-grained sites. New functionality has been added to CHARMMing to facilitate this process. The implementation of these features into CHARMMing helps automate many of the tedious aspects of constructing a CG Gō model. The CG model builder and its accompanying lesson should be a valuable tool to chemistry students, teachers, and modelers in the field.

  5. Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

    Science.gov (United States)

    Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

    2013-03-01

    Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

  6. Competition between folding and glycosylation in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Bruun, A W; Kielland-Brandt, Morten

    1996-01-01

    Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

  7. Can a pairwise contact potential stabilize native protein folds against decoys obtained by threading?

    Science.gov (United States)

    Vendruscolo, M; Najmanovich, R; Domany, E

    2000-02-01

    We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading.

  8. Combinatorial pattern discovery approach for the folding trajectory analysis of a beta-hairpin.

    Directory of Open Access Journals (Sweden)

    Laxmi Parida

    2005-06-01

    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity c in RO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  9. Combinatorial Pattern Discovery Approach for the Folding Trajectory Analysis of a beta-Hairpin.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity cinRO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  10. Supersymmetric quantum mechanics method for the Fokker-Planck equation with applications to protein folding dynamics

    Science.gov (United States)

    Polotto, Franciele; Drigo Filho, Elso; Chahine, Jorge; Oliveira, Ronaldo Junio de

    2018-03-01

    This work developed analytical methods to explore the kinetics of the time-dependent probability distributions over thermodynamic free energy profiles of protein folding and compared the results with simulation. The Fokker-Planck equation is mapped onto a Schrödinger-type equation due to the well-known solutions of the latter. Through a semi-analytical description, the supersymmetric quantum mechanics formalism is invoked and the time-dependent probability distributions are obtained with numerical calculations by using the variational method. A coarse-grained structure-based model of the two-state protein Tm CSP was simulated at a Cα level of resolution and the thermodynamics and kinetics were fully characterized. Analytical solutions from non-equilibrium conditions were obtained with the simulated double-well free energy potential and kinetic folding times were calculated. It was found that analytical folding time as a function of temperature agrees, quantitatively, with simulations and experiments from the literature of Tm CSP having the well-known 'U' shape of the Chevron Plots. The simple analytical model developed in this study has a potential to be used by theoreticians and experimentalists willing to explore, quantitatively, rates and the kinetic behavior of their system by informing the thermally activated barrier. The theory developed describes a stochastic process and, therefore, can be applied to a variety of biological as well as condensed-phase two-state systems.

  11. A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Pineda-Lucena, Antonio; Liao, Jack; Cort, John R.; Yee, Adelinda; Kennedy, Michael A.; Edwards, Aled M.

    2003-05-01

    As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by open reading frame YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded b-sheet with strand order 2143 and two a-helices, with an overall topology of bbabba. Strand b1 runs parallel to b4, and b2:b1 and b4:b3 pairs are arranged in an antiparallel fashion. While this fold belongs to the split bab family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds

  12. WW Domain Folding Complexity Revealed by Infrared Spectroscopy

    OpenAIRE

    Davis, Caitlin M.; Dyer, R. Brian

    2014-01-01

    Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescen...

  13. Emerging role of N- and C-terminal interactions in stabilizing (β/α8 fold with special emphasis on Family 10 xylanases

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    2012-09-01

    Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

  14. EMERGING ROLE OF N- AND C-TERMINAL INTERACTIONS IN STABILIZING (β;/α8 FOLD WITH SPECIAL EMPHASIS ON FAMILY 10 XYLANASES

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    2012-09-01

    Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

  15. Design of an Efficient Turbulent Micro-Mixer for Protein Folding Experiments

    Science.gov (United States)

    Inguva, Venkatesh; Perot, Blair

    2015-11-01

    Protein folding studies require the development of micro-mixers that require less sample, mix at faster rates, and still provide a high signal to noise ratio. Chaotic to marginally turbulent micro-mixers are promising candidates for this application. In this study, various turbulence and unsteadiness generation concepts are explored that avoid cavitation. The mixing enhancements include flow turning regions, flow splitters, and vortex shedding. The relative effectiveness of these different approaches for rapid micro-mixing is discussed. Simulations found that flow turning regions provided the best mixing profile. Experimental validation of the optimal design is verified through laser confocal microscopy experiments. This work is support by the National Science Foundation.

  16. Chaotic Multiquenching Annealing Applied to the Protein Folding Problem

    Directory of Open Access Journals (Sweden)

    Juan Frausto-Solis

    2014-01-01

    Full Text Available The Chaotic Multiquenching Annealing algorithm (CMQA is proposed. CMQA is a new algorithm, which is applied to protein folding problem (PFP. This algorithm is divided into three phases: (i multiquenching phase (MQP, (ii annealing phase (AP, and (iii dynamical equilibrium phase (DEP. MQP enforces several stages of quick quenching processes that include chaotic functions. The chaotic functions can increase the exploration potential of solutions space of PFP. AP phase implements a simulated annealing algorithm (SA with an exponential cooling function. MQP and AP are delimited by different ranges of temperatures; MQP is applied for a range of temperatures which goes from extremely high values to very high values; AP searches for solutions in a range of temperatures from high values to extremely low values. DEP phase finds the equilibrium in a dynamic way by applying least squares method. CMQA is tested with several instances of PFP.

  17. How old is your fold?

    NARCIS (Netherlands)

    Winstanley, Henry F.; Abeln, Sanne; Deane, Charlotte M.

    Motivation: At present there exists no age estimate for the different protein structures found in nature. It has become clear from occurrence studies that different folds arose at different points in evolutionary time. An estimation of the age of different folds would be a starting point for many

  18. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most...... diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed...

  19. Probing slowly exchanging protein systems via {sup 13}C{sup {alpha}}-CEST: monitoring folding of the Im7 protein

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, Alexandar L.; Bouvignies, Guillaume; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

    2013-03-15

    A {sup 13}C{sup {alpha}} chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-{sup 13}C]-glucose is the sole carbon source is exploited, producing proteins with {sup 13}C at the C{sup {alpha}} position, while the majority of residues remain unlabeled at CO or C{sup {beta}}. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state {sup 13}C{sup {alpha}} chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

  20. Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: Comparison between hard-sphere solvent and water

    International Nuclear Information System (INIS)

    Oshima, Hiraku; Kinoshita, Masahiro

    2015-01-01

    In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient

  1. A collaborative visual analytics suite for protein folding research.

    Science.gov (United States)

    Harvey, William; Park, In-Hee; Rübel, Oliver; Pascucci, Valerio; Bremer, Peer-Timo; Li, Chenglong; Wang, Yusu

    2014-09-01

    Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind important biochemical processes such as protein folding and molecular interaction. With the rapidly increasing power of modern computers, large-scale MD simulation experiments can be performed regularly, generating huge amounts of MD data. An important question is how to analyze and interpret such massive and complex data. One of the (many) challenges involved in analyzing MD simulation data computationally is the high-dimensionality of such data. Given a massive collection of molecular conformations, researchers typically need to rely on their expertise and prior domain knowledge in order to retrieve certain conformations of interest. It is not easy to make and test hypotheses as the data set as a whole is somewhat "invisible" due to its high dimensionality. In other words, it is hard to directly access and examine individual conformations from a sea of molecular structures, and to further explore the entire data set. There is also no easy and convenient way to obtain a global view of the data or its various modalities of biochemical information. To this end, we present an interactive, collaborative visual analytics tool for exploring massive, high-dimensional molecular dynamics simulation data sets. The most important utility of our tool is to provide a platform where researchers can easily and effectively navigate through the otherwise "invisible" simulation data sets, exploring and examining molecular conformations both as a whole and at individual levels. The visualization is based on the concept of a topological landscape, which is a 2D terrain metaphor preserving certain topological and geometric properties of the high dimensional protein energy landscape. In addition to facilitating easy exploration of conformations, this 2D terrain metaphor also provides a platform where researchers can visualize and analyze various properties (such as contact density) overlayed on the

  2. Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

    Energy Technology Data Exchange (ETDEWEB)

    DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel; Beese, Lorena S.; Barany, George; Distefano, Mark D. (Duke); (UMM)

    2010-11-15

    Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

  3. Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

    Science.gov (United States)

    Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A

    2011-04-28

    Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded

  4. Folding and membrane insertion of the pore-forming peptide gramicidin occur as a concerted process.

    Science.gov (United States)

    Hicks, Matthew R; Damianoglou, Angeliki; Rodger, Alison; Dafforn, Timothy R

    2008-11-07

    Many antibiotic peptides function by binding and inserting into membranes. Understanding this process provides an insight into the fundamentals of both membrane protein folding and antibiotic peptide function. For the first time, in this work, flow-aligned linear dichroism (LD) is used to study the folding of the antibiotic peptide gramicidin. LD provides insight into the combined processes of peptide folding and insertion and has the advantage over other similar techniques of being insensitive to off-membrane aggregation events. By combining LD data with conventional measurements of protein fluorescence and circular dichroism, the mechanism of gramicidin insertion is elucidated. The mechanism consists of five separately assignable steps that include formation of a water-insoluble gramicidin aggregate, dissociation from the aggregate, partitioning of peptide to the membrane surface, oligomerisation on the surface and concerted insertion and folding of the peptide to the double-helical form of gramicidin. Measurement of the rates of each step shows that although changes in the fluorescence signal cease 10 s after the initiation of the process, the insertion of the peptide into the membrane is actually not complete for a further 60 min. This last membrane insertion phase is only apparent by measurement of LD and circular dichroism signal changes. In summary, this study demonstrates the importance of multi-technique approaches, including LD, in studies of membrane protein folding.

  5. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  6. New variants of known folds: do they bring new biology?

    International Nuclear Information System (INIS)

    Koonin, Eugene V.

    2010-01-01

    New distinct versions of known protein folds provide a powerful means of protein-function prediction that complements sequence and genomic context analysis. New distinct versions of known protein folds provide a powerful means of protein-function prediction that complements sequence and genomic context analysis. These structures do not supplant direct biochemical experiments, but are indispensable for the complete characterization of proteins

  7. My 65 years in protein chemistry.

    Science.gov (United States)

    Scheraga, Harold A

    2015-05-01

    This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein-protein interactions and to nucleic acids and to protein-nucleic acid interactions. The ultimate results led to an understanding

  8. Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo

    Directory of Open Access Journals (Sweden)

    Qingzhong Kong

    2013-07-01

    Full Text Available Prion diseases, or transmissible spongiform encephalopathies (TSEs, are associated with the conformational conversion of the cellular prion protein, PrPC, into a protease-resistant form, PrPSc. Here, we show that mutation-induced thermodynamic stabilization of the folded, α-helical domain of PrPC has a dramatic inhibitory effect on the conformational conversion of prion protein in vitro, as well as on the propagation of TSE disease in vivo. Transgenic mice expressing a human prion protein variant with increased thermodynamic stability were found to be much more resistant to infection with the TSE agent than those expressing wild-type human prion protein, in both the primary passage and three subsequent subpassages. These findings not only provide a line of evidence in support of the protein-only model of TSEs but also yield insight into the molecular nature of the PrPC→PrPSc conformational transition, and they suggest an approach to the treatment of prion diseases.

  9. Complete fold annotation of the human proteome using a novel structural feature space.

    Science.gov (United States)

    Middleton, Sarah A; Illuminati, Joseph; Kim, Junhyong

    2017-04-13

    Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.

  10. Structural anatomy of telomere OB proteins.

    Science.gov (United States)

    Horvath, Martin P

    2011-10-01

    Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.

  11. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C’ scalar couplings (3hbJNC’)

    NARCIS (Netherlands)

    Bonvin, A.M.J.J.; Houben, K.; Guenneugues, M.N.L.; Kaptein, R.; Boelens, R.

    2001-01-01

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein

  12. Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

    Science.gov (United States)

    Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

  13. Conformational disorder in folded and intrinsically disordered proteins from nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Salmon, Loic

    2010-01-01

    Biological macromolecules are, by essence, dynamical systems. While the importance of this flexibility is nowadays well established, the accurate characterization of the conformational disorder of these systems remains an important challenge. Nuclear magnetic resonance spectroscopy is a unique tool to probe these motions at atomic level, through the analysis of spin relaxation or residual dipolar couplings. The latter allows all motions occurring at timescales faster than the millisecond to be investigated, including physiologically important timescales. The information presents in those couplings is interpreted here using mainly analytical approaches in order to quantify the amounts of dynamics present in folded protein, to determine the direction of those motions and to obtain structural information within this conformational disorder. These analytical approaches are complemented by numerical methods, that allowed the observation of phenomena from a different point of view or the investigation of other systems such as intrinsically disordered proteins. All of these studies demonstrate an important complementarity between structural order and conformational disorder. (author)

  14. Protein folding and non-conventional drug design: a primer for nuclear structure physicists

    International Nuclear Information System (INIS)

    Broglia, R.A.; Tiana, G.; Provasi, D.

    2004-01-01

    Some of the paradigms emerging from the study of the phenomena of phase transitions in finite many-body systems, like e.g. the atomic nucleus can be used at profit to solve the protein folding problem within the framework of simple (although not oversimplified) models. From this solution a paradigm emerges for the design of non-conventional drugs, which inhibit enzymatic action without inducing resistance (mutations). The application of these concepts to the design of an inhibitor to the HIV-protease central in the life cycle of the HIV virus is discussed

  15. WW domain folding complexity revealed by infrared spectroscopy.

    Science.gov (United States)

    Davis, Caitlin M; Dyer, R Brian

    2014-09-02

    Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.

  16. Folding behavior of four silks of giant honey bee reflects the evolutionary conservation of aculeate silk proteins.

    Science.gov (United States)

    Maitip, Jakkrawut; Trueman, Holly E; Kaehler, Benjamin D; Huttley, Gavin A; Chantawannakul, Panuwan; Sutherland, Tara D

    2015-04-01

    Multiple gene duplication events in the precursor of the Aculeata (bees, ants, hornets) gave rise to four silk genes. Whilst these homologs encode proteins with similar amino acid composition and coiled coil structure, the retention of all four homologs implies they each are important. In this study we identified, produced and characterized the four silk proteins from Apis dorsata, the giant Asian honeybee. The proteins were readily purified, allowing us to investigate the folding behavior of solutions of individual proteins in comparison to mixtures of all four proteins at concentrations where they assemble into their native coiled coil structure. In contrast to solutions of any one protein type, solutions of a mixture of the four proteins formed coiled coils that were stable against dilution and detergent denaturation. The results are consistent with the formation of a heteromeric coiled coil protein complex. The mechanism of silk protein coiled coil formation and evolution is discussed in light of these results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    International Nuclear Information System (INIS)

    Helander, Sara; Montecchio, Meri; Lemak, Alexander; Farès, Christophe; Almlöf, Jonas; Li, Yanjun; Yee, Adelinda; Arrowsmith, Cheryl H.; Dhe-Paganon, Sirano; Sunnerhagen, Maria

    2014-01-01

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25 1–73 , a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains

  18. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    Energy Technology Data Exchange (ETDEWEB)

    Helander, Sara; Montecchio, Meri [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Lemak, Alexander [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Farès, Christophe [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Almlöf, Jonas [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Li, Yanjun [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Yee, Adelinda [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Arrowsmith, Cheryl H. [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Dhe-Paganon, Sirano [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Sunnerhagen, Maria, E-mail: maria.sunnerhagen@liu.se [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)

    2014-04-25

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

  19. Investigation of the three-dimensional lattice HP protein folding model using a genetic algorithm

    Directory of Open Access Journals (Sweden)

    Fábio L. Custódio

    2004-01-01

    Full Text Available An approach to the hydrophobic-polar (HP protein folding model was developed using a genetic algorithm (GA to find the optimal structures on a 3D cubic lattice. A modification was introduced to the scoring system of the original model to improve the model's capacity to generate more natural-like structures. The modification was based on the assumption that it may be preferable for a hydrophobic monomer to have a polar neighbor than to be in direct contact with the polar solvent. The compactness and the segregation criteria were used to compare structures created by the original HP model and by the modified one. An islands' algorithm, a new selection scheme and multiple-points crossover were used to improve the performance of the algorithm. Ten sequences, seven with length 27 and three with length 64 were analyzed. Our results suggest that the modified model has a greater tendency to form globular structures. This might be preferable, since the original HP model does not take into account the positioning of long polar segments. The algorithm was implemented in the form of a program with a graphical user interface that might have a didactical potential in the study of GA and on the understanding of hydrophobic core formation.

  20. Improved understanding of protein complex offers insight into DNA

    Science.gov (United States)

    Summer Science Writing Internship Improved understanding of protein complex offers insight into DNA clearer understanding of the origin recognition complex (ORC) - a protein complex that directs DNA replication - through its crystal structure offers new insight into fundamental mechanisms of DNA replication

  1. The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease.

    Science.gov (United States)

    Karmi, Ola; Marjault, Henri-Baptiste; Pesce, Luca; Carloni, Paolo; Onuchic, Jose' N; Jennings, Patricia A; Mittler, Ron; Nechushtai, Rachel

    2018-02-12

    NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT's backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins' [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs' [2Fe-2S] cluster and the implication of the latter to the NEET proteins' cellular and systemic function in health and disease.

  2. Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles

    Directory of Open Access Journals (Sweden)

    Peter Schmidt

    2018-01-01

    Full Text Available Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

  3. Topologies to geometries in protein folding: Hierarchical and nonhierarchical scenarios

    Science.gov (United States)

    Fernández, Ariel; Colubri, Andrés; Berry, R. Stephen

    2001-04-01

    This work presents a method to portray protein folding dynamics at a coarse resolution, based on a pattern-recognition-and-feedback description of the evolution of torsional motions of the backbone chain in the hydrophobic collapse of the protein. The approach permits theory and computation to treat the search of conformation space from picoseconds to the millisecond time scale or longer, the time scales of adiabatic evolution of soft-mode dynamics. The procedure tracks the backbone torsional coordinates modulo the basins of attraction to which they belong in the Ramachandran maps. The state and history of the backbone are represented in a map of local torsional states and hydrophobicity/hydrophilicity matching of the residues comprising the chain, the local topology matrix (LTM). From this map, we infer allowable structural features by recognizing patterns in the LTM as topologically compatible with particular structural forms within a level of frustration tolerance. Each such 3D realization of an LTM leads to a contact map, from which one can infer one or more structures. Introduction of energetic and entropic terms allow elimination of all but the most favored of these structures at each new juncture. The method's predictive power is first established by comparing "final," stable LTMs for natural sequences of intermediate length (N⩽120) with PDB data. The method is extended further to β-lactoglobulin (β-LG, N=162), the quintessential nonhierarchical folder.

  4. Folding and Function of Proteorhodopsins in Photoenergy Transducing Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Spudich, John L. [University of Texas Medical School, Houston, TX (United States). Health Science Center, Dept. of Biochemistry and Molecular Biology

    2012-08-10

    The overall research objectives are to develop proteorhodopsin (PR) proteins as a model system for α-helical membrane protein insertion and folding, and to advance understanding of the diversity and mechanisms of PRs, a large family of photoenergy transducers (~4000 identified) abundant in the world’s oceans. Specific aims are: (1) To develop a high-efficiency genetic selection procedure for light-driven proton-pumping in E. coli cells. Such a procedure would provide a positive selection method for proper folding and function of PRs in the E. coli membrane. (2) Characterize flash-induced absorption changes and photocurrents in PR variants in organisms from various environments, and their expression level and function when expressed in E. coli. Subaims are to: (a) elucidate the relationship of the transport mechanism to mechanisms of other microbial rhodopsins, some of which like PRs function as ion transporters and some of which use light energy to activate signaling pathways (sensory rhodopsins); and (b) identify important residues and chemical events in light-driven proton transport by PRs. In addition to their importance to the energy of the biosphere PRs have attracted interest for their potential for use in making photoenergy-transducing membranes for bioengineering applications.

  5. Comparing the Folding and Misfolding Energy Landscapes of Phosphoglycerate Kinase

    OpenAIRE

    Agocs, Gergely; Szabo, Bence T.; Koehler, Gottfried; Osvath, Szabolcs

    2012-01-01

    Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the prop...

  6. An expanded view of the protein folding landscape of PDZ domains

    DEFF Research Database (Denmark)

    Hultqvist, Greta; Pedersen, Søren W; Chi, Celestine N.

    2012-01-01

    state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding...

  7. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

    Directory of Open Access Journals (Sweden)

    Florence Guillière

    Full Text Available While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  8. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    is a well-recognized classification system of proteins, which is based on manual in- ... can easily correspond to the information in the 2D matrix. ..... [7] U K Muppirala and Zhijun Li, Protein Engineering, Design & Selection 19, 265 (2006).

  9. Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

    Energy Technology Data Exchange (ETDEWEB)

    K Kucera; L Harrison; M Cappello; Y Modis

    2011-12-31

    Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

  10. Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo

    Directory of Open Access Journals (Sweden)

    Baik L. Seong

    2011-03-01

    Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

  11. Protein folding on a chip

    CERN Multimedia

    2004-01-01

    "Scientists at the U.S. Department of Energy's Brookhaven National Laboratory are proposing to use a super- computer originally developed to simulate elementary particles in high- energy physics to help determine the structures and functions of proteins, including, for example, the 30,000 or so proteins encoded by the human genome" (1 page)

  12. Guiding the folding pathway of DNA origami.

    Science.gov (United States)

    Dunn, Katherine E; Dannenberg, Frits; Ouldridge, Thomas E; Kwiatkowska, Marta; Turberfield, Andrew J; Bath, Jonathan

    2015-09-03

    DNA origami is a robust assembly technique that folds a single-stranded DNA template into a target structure by annealing it with hundreds of short 'staple' strands. Its guiding design principle is that the target structure is the single most stable configuration. The folding transition is cooperative and, as in the case of proteins, is governed by information encoded in the polymer sequence. A typical origami folds primarily into the desired shape, but misfolded structures can kinetically trap the system and reduce the yield. Although adjusting assembly conditions or following empirical design rules can improve yield, well-folded origami often need to be separated from misfolded structures. The problem could in principle be avoided if assembly pathway and kinetics were fully understood and then rationally optimized. To this end, here we present a DNA origami system with the unusual property of being able to form a small set of distinguishable and well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape, thus allowing us to probe the assembly process. The obtained high yield of well-folded origami structures confirms the existence of efficient folding pathways, while the shape distribution provides information about individual trajectories through the folding landscape. We find that, similarly to protein folding, the assembly of DNA origami is highly cooperative; that reversible bond formation is important in recovering from transient misfoldings; and that the early formation of long-range connections can very effectively enforce particular folds. We use these insights to inform the design of the system so as to steer assembly towards desired structures. Expanding the rational design process to include the assembly pathway should thus enable more reproducible synthesis, particularly when targeting more complex structures. We anticipate that this expansion will be essential if DNA origami is to continue its

  13. A de novo designed monomeric, compact three helix bundle protein on a carbohydrate template

    DEFF Research Database (Denmark)

    Malik, Leila; Nygård, Jesper; Christensen, Niels Johan

    2015-01-01

    De novo design and chemical synthesis of proteins and of other artificial structures, which mimic them, is a central strategy for understanding protein folding and for accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial...... the template could facilitate protein folding. Here we report the design and synthesis of 3-helix bundle carboproteins on deoxy-hexopyranosides. The carboproteins were analyzed by CD, AUC, SAXS, and NMR, which revealed the formation of the first compact, and folded monomeric carboprotein distinctly different...

  14. High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

    Science.gov (United States)

    Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

    2018-04-27

    Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Adapted to roar: functional morphology of tiger and lion vocal folds.

    Directory of Open Access Journals (Sweden)

    Sarah A Klemuk

    Full Text Available Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice.

  16. Muscular anatomy of the human ventricular folds.

    Science.gov (United States)

    Moon, Jerald; Alipour, Fariborz

    2013-09-01

    Our purpose in this study was to better understand the muscular anatomy of the ventricular folds in order to help improve biomechanical modeling of phonation and to better understand the role of these muscles during phonatory and nonphonatory tasks. Four human larynges were decalcified, sectioned coronally from posterior to anterior by a CryoJane tape transfer system, and stained with Masson's trichrome. The total and relative areas of muscles observed in each section were calculated and used for characterizing the muscle distribution within the ventricular folds. The ventricular folds contained anteriorly coursing thyroarytenoid and ventricularis muscle fibers that were in the lower half of the ventricular fold posteriorly, and some ventricularis muscle was evident in the upper and lateral portions of the fold more anteriorly. Very little muscle tissue was observed in the medial half of the fold, and the anterior half of the ventricular fold was largely devoid of any muscle tissue. All 4 larynges contained muscle bundles that coursed superiorly and medially through the upper half of the fold, toward the lateral margin of the epiglottis. Although variability of expression was evident, a well-defined thyroarytenoid muscle was readily apparent lateral to the arytenoid cartilage in all specimens.

  17. The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state

    NARCIS (Netherlands)

    Wierenga, P.A.; Egmond, M.R.; Voragen, A.G.J.; Jongh, H.H.J.de

    2006-01-01

    Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends

  18. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  19. Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation

    Science.gov (United States)

    Fluitt, Aaron Michael

    Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.

  20. Fiscal 1999 achievement report on research and development project on intellectual infrastructure creation and utilization technologies. Development of efficient protein expression system (Development of efficient protein expression system utilizing protein folding mechanism of hyperthermophilic bacteria); 1999 nendo kokoritsu tanpakushitsu hatsugen system no kaihatsu seika hokokusho. Chokonetsukin no tanpakushitsu oritatami kiko wo riyoshita kokoritsu tanpakushitsu hatsugen system no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Efforts were exerted to achieve efficient expression of proteins of hyperthermophilic bacteria, hyperthermophilic archaeabacteria in particular, using a heterogene expression system in which Escherichia coli was the host. In an effort to search for genes related to protein folding and to elucidate the mechanism of folding, chaperonin and prefoldin subunit genes, out of various factors participating in protein folding in hyperthermophilic archaeabacteria, were cloned, and expressed in Escherichia coli. As a system for analyzing protein folding reaction, an experimental system was established on a substrate comprising isopropyl malate dehydrogenase, citrate synthase, glucose dehydrogenase, and a green fluorescent protein. Studies were further conducted to elucidate the mechanism of expression of enzyme genes in Escherichia coli for the establishment of a mass production method for useful enzymes. Also carried out was the research and development of an element technology evaluation system involving protein expression. (NEDO)

  1. NoFold: RNA structure clustering without folding or alignment.

    Science.gov (United States)

    Middleton, Sarah A; Kim, Junhyong

    2014-11-01

    Structures that recur across multiple different transcripts, called structure motifs, often perform a similar function-for example, recruiting a specific RNA-binding protein that then regulates translation, splicing, or subcellular localization. Identifying common motifs between coregulated transcripts may therefore yield significant insight into their binding partners and mechanism of regulation. However, as most methods for clustering structures are based on folding individual sequences or doing many pairwise alignments, this results in a tradeoff between speed and accuracy that can be problematic for large-scale data sets. Here we describe a novel method for comparing and characterizing RNA secondary structures that does not require folding or pairwise alignment of the input sequences. Our method uses the idea of constructing a distance function between two objects by their respective distances to a collection of empirical examples or models, which in our case consists of 1973 Rfam family covariance models. Using this as a basis for measuring structural similarity, we developed a clustering pipeline called NoFold to automatically identify and annotate structure motifs within large sequence data sets. We demonstrate that NoFold can simultaneously identify multiple structure motifs with an average sensitivity of 0.80 and precision of 0.98 and generally exceeds the performance of existing methods. We also perform a cross-validation analysis of the entire set of Rfam families, achieving an average sensitivity of 0.57. We apply NoFold to identify motifs enriched in dendritically localized transcripts and report 213 enriched motifs, including both known and novel structures. © 2014 Middleton and Kim; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  2. Protein degradation and protection against misfolded or damaged proteins

    Science.gov (United States)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  3. How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

    Science.gov (United States)

    Bonetti, Daniela; Troilo, Francesca; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano

    2018-04-24

    The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils......, is a general hallmark. They also include the α1-antitrypsin deficiency, where disease-causing mutations in the serine protease inhibitor, α1-antitrypsin (α1AT), leads to accumulation of the aberrant protein in the liver of these patients. The native metastable structure of α1AT constitutes a molecular trap...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding...

  5. Light-activated control of protein channel assembly mediated by membrane mechanics

    Science.gov (United States)

    Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

    2016-12-01

    Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

  6. A Self-Assisting Protein Folding Model for Teaching Structural Molecular Biology.

    Science.gov (United States)

    Davenport, Jodi; Pique, Michael; Getzoff, Elizabeth; Huntoon, Jon; Gardner, Adam; Olson, Arthur

    2017-04-04

    Structural molecular biology is now becoming part of high school science curriculum thus posing a challenge for teachers who need to convey three-dimensional (3D) structures with conventional text and pictures. In many cases even interactive computer graphics does not go far enough to address these challenges. We have developed a flexible model of the polypeptide backbone using 3D printing technology. With this model we have produced a polypeptide assembly kit to create an idealized model of the Triosephosphate isomerase mutase enzyme (TIM), which forms a structure known as TIM barrel. This kit has been used in a laboratory practical where students perform a step-by-step investigation into the nature of protein folding, starting with the handedness of amino acids to the formation of secondary and tertiary structure. Based on the classroom evidence we collected, we conclude that these models are valuable and inexpensive resource for teaching structural molecular biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Folding pathways explored with artificial potential functions

    International Nuclear Information System (INIS)

    Ulutaş, B; Bozma, I; Haliloglu, T

    2009-01-01

    This paper considers the generation of trajectories to a given protein conformation and presents a novel approach based on artificial potential functions—originally proposed for multi-robot navigation. The artificial potential function corresponds to a simplified energy model, but with the novelty that—motivated by work on robotic navigation—a nonlinear compositional scheme of constructing the energy model is adapted instead of an additive formulation. The artificial potential naturally gives rise to a dynamic system for the protein structure that ensures collision-free motion to an equilibrium point. In cases where the equilibrium point is the native conformation, the motion trajectory corresponds to the folding pathway. This framework is used to investigate folding in a variety of protein structures, and the results are compared with those of other approaches including experimental studies

  8. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC')

    Energy Technology Data Exchange (ETDEWEB)

    Bonvin, Alexandre M.J.J.; Houben, Klaartje; Guenneugues, Marc; Kaptein, Robert; Boelens, Rolf [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)

    2001-11-15

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small {alpha}/{beta} protein chymotrypsin inhibitor 2. Dihedral angle restraints for the {phi} and {psi} angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond {sup 3hb}J{sub NC'} coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

  9. Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding.

    Science.gov (United States)

    Jacob, Maik H; D'Souza, Roy N; Schwarzlose, Thomas; Wang, Xiaojuan; Huang, Fang; Haas, Elisha; Nau, Werner M

    2018-04-26

    Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.

  10. Bifurcation of self-folded polygonal bilayers

    Science.gov (United States)

    Abdullah, Arif M.; Braun, Paul V.; Hsia, K. Jimmy

    2017-09-01

    Motivated by the self-assembly of natural systems, researchers have investigated the stimulus-responsive curving of thin-shell structures, which is also known as self-folding. Self-folding strategies not only offer possibilities to realize complicated shapes but also promise actuation at small length scales. Biaxial mismatch strain driven self-folding bilayers demonstrate bifurcation of equilibrium shapes (from quasi-axisymmetric doubly curved to approximately singly curved) during their stimulus-responsive morphing behavior. Being a structurally instable, bifurcation could be used to tune the self-folding behavior, and hence, a detailed understanding of this phenomenon is appealing from both fundamental and practical perspectives. In this work, we investigated the bifurcation behavior of self-folding bilayer polygons. For the mechanistic understanding, we developed finite element models of planar bilayers (consisting of a stimulus-responsive and a passive layer of material) that transform into 3D curved configurations. Our experiments with cross-linked Polydimethylsiloxane samples that change shapes in organic solvents confirmed our model predictions. Finally, we explored a design scheme to generate gripper-like architectures by avoiding the bifurcation of stimulus-responsive bilayers. Our research contributes to the broad field of self-assembly as the findings could motivate functional devices across multiple disciplines such as robotics, artificial muscles, therapeutic cargos, and reconfigurable biomedical devices.

  11. Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.

    Science.gov (United States)

    Jethva, Prashant N; Udgaonkar, Jayant B

    2017-09-07

    Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

  12. Ca-Dependent Folding of Human Calumenin

    Science.gov (United States)

    Mazzorana, Marco; Hussain, Rohanah; Sorensen, Thomas

    2016-01-01

    Human calumenin (hCALU) is a six EF-hand protein belonging to the CREC family. As other members of the family, it is localized in the secretory pathway and regulates the activity of SERCA2a and of the ryanodine receptor in the endoplasmic reticulum (ER). We have studied the effects of Ca2+ binding to the protein and found it to attain a more compact structure upon ion binding. Circular Dichroism (CD) measurements suggest a major rearrangement of the protein secondary structure, which reversibly switches from disordered at low Ca2+ concentrations to predominantly alpha-helical when Ca2+ is added. SAXS experiments confirm the transition from an unfolded to a compact structure, which matches the structural prediction of a trilobal fold. Overall our experiments suggest that calumenin is a Ca2+ sensor, which folds into a compact structure, capable of interacting with its molecular partners, when Ca2+ concentration within the ER reaches the millimolar range. PMID:26991433

  13. Using analyses of amino Acid coevolution to understand protein structure and function.

    Science.gov (United States)

    Ashenberg, Orr; Laub, Michael T

    2013-01-01

    Determining which residues of a protein contribute to a specific function is a difficult problem. Analyses of amino acid covariation within a protein family can serve as a useful guide by identifying residues that are functionally coupled. Covariation analyses have been successfully used on several different protein families to identify residues that work together to promote folding, enable protein-protein interactions, or contribute to an enzymatic activity. Covariation is a statistical signal that can be measured in a multiple sequence alignment of homologous proteins. As sequence databases have expanded dramatically, covariation analyses have become easier and more powerful. In this chapter, we describe how functional covariation arises during the evolution of proteins and how this signal can be distinguished from various background signals. We discuss the basic methodology for performing amino acid covariation analysis, using bacterial two-component signal transduction proteins as an example. We provide practical suggestions for each step of the process including assembly of protein sequences, construction of a multiple sequence alignment, measurement of covariation, and analysis of results. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Understanding Protein Synthesis: An Interactive Card Game Discussion

    Science.gov (United States)

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a…

  15. Mitochondrial associated ubiquitin fold modifier-1 mediated protein conjugation in Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Sreenivas Gannavaram

    2011-01-01

    Full Text Available In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1 and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2 specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

  16. Force generation by titin folding.

    Science.gov (United States)

    Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós

    2017-07-01

    Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.

  17. Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

    Science.gov (United States)

    Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R; Poulsen, Heidi I; Jensen, Peter H; Knudsen, Jens; Kragelund, Birthe B; Poulsen, Flemming M

    2005-04-01

    Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues. (c) 2005 Wiley-Liss, Inc.

  18. Energy Landscapes: From Protein Folding to Molecular Assembly

    Science.gov (United States)

    Databases National Security Education Center (NSEC) Center for Nonlinear Studies Engineering Institute assembly is very common in biology and in nanotechnology. Simple examples of self-assembly are the folding efflux pump machinery, ATP synthase, the ribosome, and many others. In nanotechnology, self-assembly has

  19. PROTEINS IN VACUO . A MORE EFFICIENT MEANS OF ...

    African Journals Online (AJOL)

    With the aim of understanding solvent effects in protein folding, unfolding, stability and dynamic behavior, studies of protein ions in vacuo have become popular in recent years. One experimental descriptor which gives a general overview of ionic structure is the orientationally-averaged collision cross section , which is ...

  20. NAPS: Network Analysis of Protein Structures

    Science.gov (United States)

    Chakrabarty, Broto; Parekh, Nita

    2016-01-01

    Traditionally, protein structures have been analysed by the secondary structure architecture and fold arrangement. An alternative approach that has shown promise is modelling proteins as a network of non-covalent interactions between amino acid residues. The network representation of proteins provide a systems approach to topological analysis of complex three-dimensional structures irrespective of secondary structure and fold type and provide insights into structure-function relationship. We have developed a web server for network based analysis of protein structures, NAPS, that facilitates quantitative and qualitative (visual) analysis of residue–residue interactions in: single chains, protein complex, modelled protein structures and trajectories (e.g. from molecular dynamics simulations). The user can specify atom type for network construction, distance range (in Å) and minimal amino acid separation along the sequence. NAPS provides users selection of node(s) and its neighbourhood based on centrality measures, physicochemical properties of amino acids or cluster of well-connected residues (k-cliques) for further analysis. Visual analysis of interacting domains and protein chains, and shortest path lengths between pair of residues are additional features that aid in functional analysis. NAPS support various analyses and visualization views for identifying functional residues, provide insight into mechanisms of protein folding, domain-domain and protein–protein interactions for understanding communication within and between proteins. URL:http://bioinf.iiit.ac.in/NAPS/. PMID:27151201

  1. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

    International Nuclear Information System (INIS)

    Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta; Benach, Jordi; Riera, Antoni; Pous, Joan; Macias, Maria J.

    2015-01-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily

  2. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

    Energy Technology Data Exchange (ETDEWEB)

    Beich-Frandsen, Mads; Aragón, Eric [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Llimargas, Marta [Institut de Biologia Molecular de Barcelona, IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Benach, Jordi [ALBA Synchrotron, BP 1413, km 3.3, Cerdanyola del Vallès (Spain); Riera, Antoni [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Universitat de Barcelona, Martí i Franqués 1-11, 08028 Barcelona (Spain); Pous, Joan [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Platform of Crystallography IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Macias, Maria J., E-mail: maria.macias@irbbarcelona.org [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08010 Barcelona (Spain)

    2015-04-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

  3. Extreme Mechanics: Self-Folding Origami

    Science.gov (United States)

    Santangelo, Christian D.

    2017-03-01

    Origami has emerged as a tool for designing three-dimensional structures from flat films. Because they can be fabricated by lithographic or roll-to-roll processing techniques, they have great potential for the manufacture of complicated geometries and devices. This article discusses the mechanics of origami and kirigami with a view toward understanding how to design self-folding origami structures. Whether an origami structure can be made to fold autonomously depends strongly on the geometry and kinematics of the origami fold pattern. This article collects some of the results on origami rigidity into a single framework, and discusses how these aspects affect the foldability of origami. Despite recent progress, most problems in origami and origami design remain completely open.

  4. Protein Design Using Unnatural Amino Acids

    Science.gov (United States)

    Bilgiçer, Basar; Kumar, Krishna

    2003-11-01

    With the increasing availability of whole organism genome sequences, understanding protein structure and function is of capital importance. Recent developments in the methodology of incorporation of unnatural amino acids into proteins allow the exploration of proteins at a very detailed level. Furthermore, de novo design of novel protein structures and function is feasible with unprecedented sophistication. Using examples from the literature, this article describes the available methods for unnatural amino acid incorporation and highlights some recent applications including the design of hyperstable protein folds.

  5. Nonlinear vs. linear biasing in Trp-cage folding simulations

    Energy Technology Data Exchange (ETDEWEB)

    Spiwok, Vojtěch, E-mail: spiwokv@vscht.cz; Oborský, Pavel; Králová, Blanka [Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, Prague 6 166 28 (Czech Republic); Pazúriková, Jana [Institute of Computer Science, Masaryk University, Botanická 554/68a, 602 00 Brno (Czech Republic); Křenek, Aleš [Institute of Computer Science, Masaryk University, Botanická 554/68a, 602 00 Brno (Czech Republic); Center CERIT-SC, Masaryk Univerzity, Šumavská 416/15, 602 00 Brno (Czech Republic)

    2015-03-21

    Biased simulations have great potential for the study of slow processes, including protein folding. Atomic motions in molecules are nonlinear, which suggests that simulations with enhanced sampling of collective motions traced by nonlinear dimensionality reduction methods may perform better than linear ones. In this study, we compare an unbiased folding simulation of the Trp-cage miniprotein with metadynamics simulations using both linear (principle component analysis) and nonlinear (Isomap) low dimensional embeddings as collective variables. Folding of the mini-protein was successfully simulated in 200 ns simulation with linear biasing and non-linear motion biasing. The folded state was correctly predicted as the free energy minimum in both simulations. We found that the advantage of linear motion biasing is that it can sample a larger conformational space, whereas the advantage of nonlinear motion biasing lies in slightly better resolution of the resulting free energy surface. In terms of sampling efficiency, both methods are comparable.

  6. Synchrotron radiation circular dichroism spectroscopy study of recombinant T β4 folding

    Science.gov (United States)

    Huang, Yung-Chin; Chu, Hsueh-Liang; Chen, Peng-Jen; Chang, Chia-Ching

    Thymosin beta 4 (T β4) is a 43-amino acid small peptide, has been demonstrated that it can promote cardiac repair, wound repair, tissue protection, and involve in the proliferation of blood cell precursor stem cells of bone marrow. Moreover, T β4 has been identified as a multifunction intrinsically disordered protein, which is lacking the stable tertiary structure. Owing to the small size and disordered character, the T β4 protein degrades rapidly and the storage condition is critical. Therefore, it is not easy to reveal its folding mechanism of native T β4. However, recombinant T β4 protein (rT β4), which fused with a 5-kDa peptide in its amino-terminal, is stable and possesses identical function of T β4. Therefore, rT β4 can be used to study its folding mechanism. By using over-critical folding process, stable folding intermediates of rT β4 can be obtained. Structure analysis of folding intermediates by synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies indicate that rT β4 is a random coli major protein and its hydrophobic region becomes compact gradually. Moreover, the rT β4 folding is a two state transition. Thermal denaturation analysis indicates that rT β4 lacks stable tertiary structure. These results indicated that rT β4, similar to T β4, is an intrinsically disordered protein. Research is supported by MOST, Taiwan. MOST 103-2112-M-009-011-MY3. Corresponding author: Chia-Ching Chang; ccchang01@faculty.nctu.edu.tw.

  7. Mechanical Models of Fault-Related Folding

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, A. M.

    2003-01-09

    The subject of the proposed research is fault-related folding and ground deformation. The results are relevant to oil-producing structures throughout the world, to understanding of damage that has been observed along and near earthquake ruptures, and to earthquake-producing structures in California and other tectonically-active areas. The objectives of the proposed research were to provide both a unified, mechanical infrastructure for studies of fault-related foldings and to present the results in computer programs that have graphical users interfaces (GUIs) so that structural geologists and geophysicists can model a wide variety of fault-related folds (FaRFs).

  8. Golf-course and funnel energy landscapes: Protein folding concepts in martensites

    Science.gov (United States)

    Shankaraiah, N.

    2017-06-01

    We use protein folding energy landscape concepts such as golf course and funnel to study re-equilibration in athermal martensites under systematic temperature quench Monte Carlo simulations. On quenching below a transition temperature, the seeded high-symmetry parent-phase austenite that converts to the low-symmetry product-phase martensite, through autocatalytic twinning or elastic photocopying, has both rapid conversions and incubation delays in the temperature-time-transformation phase diagram. We find the rapid (incubation delays) conversions at low (high) temperatures arises from the presence of large (small) size of golf-course edge that has the funnel inside for negative energy states. In the incubating state, the strain structure factor enters into the Brillouin-zone golf course through searches for finite transitional pathways which close off at the transition temperature with Vogel-Fulcher divergences that are insensitive to Hamiltonian energy scales and log-normal distributions, as signatures of dominant entropy barriers. The crossing of the entropy barrier is identified through energy occupancy distributions, Monte Carlo acceptance fractions, heat emission, and internal work.

  9. The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.

    Science.gov (United States)

    Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

    2006-04-01

    The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

  10. Structural proteomics of minimal organisms: conservation ofprotein fold usage and evolutionary implications

    Energy Technology Data Exchange (ETDEWEB)

    Chandonia, John-Marc; Kim, Sung-Hou

    2006-03-15

    Background: Determining the complete repertoire of proteinstructures for all soluble, globular proteins in a single organism hasbeen one of the major goals of several structural genomics projects inrecent years. Results: We report that this goal has nearly been reachedfor several "minimal organisms"--parasites or symbionts with reducedgenomes--for which over 95 percent of the soluble, globular proteins maynow be assigned folds, overall 3-D backbone structures. We analyze thestructures of these proteins as they relate to cellular functions, andcompare conservation off old usage between functional categories. We alsocompare patterns in the conservation off olds among minimal organisms andthose observed between minimal organisms and other bacteria. Conclusion:We find that proteins performing essential cellular functions closelyrelated to transcription and translation exhibit a higher degree ofconservation in fold usage than proteins in other functional categories.Folds related to transcription and translation functional categories werealso over represented in minimal organisms compared to otherbacteria.

  11. Unique Features of Halophilic Proteins.

    Science.gov (United States)

    Arakawa, Tsutomu; Yamaguchi, Rui; Tokunaga, Hiroko; Tokunaga, Masao

    2017-01-01

    Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.

  12. Insights into the role of elastin in vocal fold health and disease

    Science.gov (United States)

    Moore, Jaime

    2011-01-01

    Elastic fibers are large, complex and surprisingly poorly understood extracellular matrix (ECM) macromolecules. The elastin fiber, generated from a single human gene - elastin (ELN), is a self assembling integral protein that endows critical mechanic proprieties to elastic tissues and organs such as the skin, lungs, and arteries. The biology of elastic fibers is complex because they have multiple components, a tightly regulated developmental deposition, a multi-step hierarchical assembly and unique biomechanical functions. Elastin is present in vocal folds, where it plays a pivotal role in the quality of phonation. This review article provides an overview of the genesis of elastin and its wide- ranging structure and function. Specific distribution within the vocal fold lamina propria across the lifespan in normal and pathological states and its contribution to vocal fold biomechanics will be examined. Elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. The properties of various elastin– based materials will be discussed and their current and future applications evaluated. A new level of understanding of the biomechanical properties of vocal fold elastin composites and their molecular basis should lead to new strategies for elastic fiber repair and regeneration in aging and disease. PMID:21708449

  13. Self-Folding Textiles through Manipulation of Knit Stitch Architecture

    Directory of Open Access Journals (Sweden)

    Chelsea E. Knittel

    2015-12-01

    Full Text Available This research presents a preliminary study on finding predictable methods of controlling the self-folding behaviors of weft knit textiles for use in the development of smart textiles and garment devices, such as those with shape memory, auxetic behavior or transformation abilities. In this work, Shima Seiki SDS-One Apex computer-aided knitting technology, Shima Seiki industrial knitting machines, and the study of paper origami tessellation patterns were used as tools to understand and predict the self-folding abilities of weft knit textiles. A wide range of self-folding weft knit structures was produced, and relationships between the angles and ratios of the knit and purl stitch types were determined. Mechanical testing was used as a means to characterize differences produced by stitch patterns, and to further understand the relationships between angles and folding abilities. By defining a formulaic method for predicting the nature of the folds that occur due to stitch architecture patterns, we can better design self-folding fabrics for smart textile applications.

  14. Protein folding pathology in domestic animals.

    Science.gov (United States)

    Gruys, Erik

    2004-10-01

    Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7-10 nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAI, AApoAII, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Abeta and in sheep APrPsc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the 'amyloid enhancing factor' (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. Abeta-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis

  15. On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu.

    Science.gov (United States)

    Vielberg, Marie-Theres; Bauer, Verena C; Groll, Michael

    2018-03-02

    The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O γ , Asp17O δ1 and Lys32N ε , it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH 2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Early Events, Kinetic Intermediates and the Mechanism of Protein Folding in Cytochrome c

    Directory of Open Access Journals (Sweden)

    David S. Kliger

    2009-04-01

    Full Text Available Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t £ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-ms conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 ms timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

  17. Numerical construction of the p(fold) (committor) reaction coordinate for a Markov process.

    Science.gov (United States)

    Krivov, Sergei V

    2011-10-06

    To simplify the description of a complex multidimensional dynamical process, one often projects it onto a single reaction coordinate. In protein folding studies, the folding probability p(fold) is an optimal reaction coordinate which preserves many important properties of the dynamics. The construction of the coordinate is difficult. Here, an efficient numerical approach to construct the p(fold) reaction coordinate for a Markov process (satisfying the detailed balance) is described. The coordinate is obtained by optimizing parameters of a chosen functional form to make a generalized cut-based free energy profile the highest. The approach is illustrated by constructing the p(fold) reaction coordinate for the equilibrium folding simulation of FIP35 protein reported by Shaw et al. (Science 2010, 330, 341-346). © 2011 American Chemical Society

  18. Classification of proteins: available structural space for molecular modeling.

    Science.gov (United States)

    Andreeva, Antonina

    2012-01-01

    The wealth of available protein structural data provides unprecedented opportunity to study and better understand the underlying principles of protein folding and protein structure evolution. A key to achieving this lies in the ability to analyse these data and to organize them in a coherent classification scheme. Over the past years several protein classifications have been developed that aim to group proteins based on their structural relationships. Some of these classification schemes explore the concept of structural neighbourhood (structural continuum), whereas other utilize the notion of protein evolution and thus provide a discrete rather than continuum view of protein structure space. This chapter presents a strategy for classification of proteins with known three-dimensional structure. Steps in the classification process along with basic definitions are introduced. Examples illustrating some fundamental concepts of protein folding and evolution with a special focus on the exceptions to them are presented.

  19. Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

    International Nuclear Information System (INIS)

    Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

    2005-01-01

    Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

  20. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  1. Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: Assignment of aromatic resonances with application to protein folding, structure, and dynamics

    International Nuclear Information System (INIS)

    Weiss, M.A.; Shoelson, S.E.; Nguyen, D.T.; O'Shea, E.; Karplus, M.; Khait, I.; Neuringer, L.J.; Inouye, K.; Frank, B.H.; Beckage, M.

    1989-01-01

    The aromatic 1 H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25 → Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constrains in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures

  2. The Multiple-Minima Problem in Protein Folding

    Science.gov (United States)

    Scheraga, Harold A.

    1991-10-01

    The conformational energy surface of a polypeptide or protein has many local minima, and conventional energy minimization procedures reach only a local minimum (near the starting point of the optimization algorithm) instead of the global minimum (the multiple-minima problem). Several procedures have been developed to surmount this problem, the most promising of which are: (a) build up procedure, (b) optimization of electrostatics, (c) Monte Carlo-plus-energy minimization, (d) electrostatically-driven Monte Carlo, (e) inclusion of distance restraints, (f) adaptive importance-sampling Monte Carlo, (g) relaxation of dimensionality, (h) pattern-recognition, and (i) diffusion equation method. These procedures have been applied to a variety of polypeptide structural problems, and the results of such computations are presented. These include the computation of the structures of open-chain and cyclic peptides, fibrous proteins and globular proteins. Present efforts are being devoted to scaling up these procedures from small polypeptides to proteins, to try to compute the three-dimensional structure of a protein from its amino sequence.

  3. A reduced amino acid alphabet for understanding and designing protein adaptation to mutation.

    Science.gov (United States)

    Etchebest, C; Benros, C; Bornot, A; Camproux, A-C; de Brevern, A G

    2007-11-01

    Protein sequence world is considerably larger than structure world. In consequence, numerous non-related sequences may adopt similar 3D folds and different kinds of amino acids may thus be found in similar 3D structures. By grouping together the 20 amino acids into a smaller number of representative residues with similar features, sequence world simplification may be achieved. This clustering hence defines a reduced amino acid alphabet (reduced AAA). Numerous works have shown that protein 3D structures are composed of a limited number of building blocks, defining a structural alphabet. We previously identified such an alphabet composed of 16 representative structural motifs (5-residues length) called Protein Blocks (PBs). This alphabet permits to translate the structure (3D) in sequence of PBs (1D). Based on these two concepts, reduced AAA and PBs, we analyzed the distributions of the different kinds of amino acids and their equivalences in the structural context. Different reduced sets were considered. Recurrent amino acid associations were found in all the local structures while other were specific of some local structures (PBs) (e.g Cysteine, Histidine, Threonine and Serine for the alpha-helix Ncap). Some similar associations are found in other reduced AAAs, e.g Ile with Val, or hydrophobic aromatic residues Trp with Phe and Tyr. We put into evidence interesting alternative associations. This highlights the dependence on the information considered (sequence or structure). This approach, equivalent to a substitution matrix, could be useful for designing protein sequence with different features (for instance adaptation to environment) while preserving mainly the 3D fold.

  4. Folding and Fracturing of Rocks: the background

    Science.gov (United States)

    Ramsay, John G.

    2017-04-01

    This book was generated by structural geology teaching classes at Imperial College. I was appointed lecturer during 1957 and worked together with Dr Gilbert Wilson teaching basic structural geology at B.Sc level. I became convinced that the subject, being essentially based on geometric field observations, required a firm mathematical basis for its future development. In particular it seemed to me to require a very sound understanding of stress and strain. My field experience suggested that a knowledge of two- and three-demensional strain was critical in understanding natural tectonic processes. I found a rich confirmation for this in early publications of deformed fossils, oolitic limestones and spotted slates made by several geologists around the beginning of the 20th century (Sorby, Philips, Haughton, Harker) often using surprisingly sophisticated mathematical methods. These methods were discussed and elaborated in Folding and Fracturing of Rocks in a practical way. The geometric features of folds were related to folding mechanisms and the fold related small scale structures such as cleavage, schistosity and lineation explained in terms of rock strain. My work in the Scottish Highlands had shown just how repeated fold superposition could produce very complex geometric features, while further work in other localities suggested that such geometric complications are common in many orogenic zones. From the development of structural geological studies over the past decades it seems that the readers of this book have found many of the ideas set out are still of practical application. The mapping of these outcrop-scale structures should be emphasised in all field studies because they can be seen as ''fingerprints'' of regional scale tectonic processes. My own understanding of structural geology has been inspired by field work and I am of the opinion that future progress in understanding will be likewise based on careful observation and measurement of the features of

  5. The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins[W

    Science.gov (United States)

    Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

    2006-01-01

    The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. PMID:16531499

  6. Domains of Pyrococcus furiosus L-asparaginase fold sequentially and assemble through strong intersubunit associative forces.

    Science.gov (United States)

    Garg, Dushyant K; Tomar, Rachana; Dhoke, Reema R; Srivastava, Ankit; Kundu, Bishwajit

    2015-05-01

    Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, L-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.

  7. New force replica exchange method and protein folding pathways probed by force-clamp technique.

    Science.gov (United States)

    Kouza, Maksim; Hu, Chin-Kun; Li, Mai Suan

    2008-01-28

    We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes

  8. Small Scaffolds, Big Potential: Developing Miniature Proteins as Therapeutic Agents.

    Science.gov (United States)

    Holub, Justin M

    2017-09-01

    Preclinical Research Miniature proteins are a class of oligopeptide characterized by their short sequence lengths and ability to adopt well-folded, three-dimensional structures. Because of their biomimetic nature and synthetic tractability, miniature proteins have been used to study a range of biochemical processes including fast protein folding, signal transduction, catalysis and molecular transport. Recently, miniature proteins have been gaining traction as potential therapeutic agents because their small size and ability to fold into defined tertiary structures facilitates their development as protein-based drugs. This research overview discusses emerging developments involving the use of miniature proteins as scaffolds to design novel therapeutics for the treatment and study of human disease. Specifically, this review will explore strategies to: (i) stabilize miniature protein tertiary structure; (ii) optimize biomolecular recognition by grafting functional epitopes onto miniature protein scaffolds; and (iii) enhance cytosolic delivery of miniature proteins through the use of cationic motifs that facilitate endosomal escape. These objectives are discussed not only to address challenges in developing effective miniature protein-based drugs, but also to highlight the tremendous potential miniature proteins hold for combating and understanding human disease. Drug Dev Res 78 : 268-282, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Evaluating the effects of cutoffs and treatment of long-range electrostatics in protein folding simulations.

    Directory of Open Access Journals (Sweden)

    Stefano Piana

    Full Text Available The use of molecular dynamics simulations to provide atomic-level descriptions of biological processes tends to be computationally demanding, and a number of approximations are thus commonly employed to improve computational efficiency. In the past, the effect of these approximations on macromolecular structure and stability has been evaluated mostly through quantitative studies of small-molecule systems or qualitative observations of short-timescale simulations of biological macromolecules. Here we present a quantitative evaluation of two commonly employed approximations, using a test system that has been the subject of a number of previous protein folding studies--the villin headpiece. In particular, we examined the effect of (i the use of a cutoff-based force-shifting technique rather than an Ewald summation for the treatment of electrostatic interactions, and (ii the length of the cutoff used to determine how many pairwise interactions are included in the calculation of both electrostatic and van der Waals forces. Our results show that the free energy of folding is relatively insensitive to the choice of cutoff beyond 9 Å, and to whether an Ewald method is used to account for long-range electrostatic interactions. In contrast, we find that the structural properties of the unfolded state depend more strongly on the two approximations examined here.

  10. Adaptive enhanced sampling with a path-variable for the simulation of protein folding and aggregation

    Science.gov (United States)

    Peter, Emanuel K.

    2017-12-01

    In this article, we present a novel adaptive enhanced sampling molecular dynamics (MD) method for the accelerated simulation of protein folding and aggregation. We introduce a path-variable L based on the un-biased momenta p and displacements dq for the definition of the bias s applied to the system and derive 3 algorithms: general adaptive bias MD, adaptive path-sampling, and a hybrid method which combines the first 2 methodologies. Through the analysis of the correlations between the bias and the un-biased gradient in the system, we find that the hybrid methodology leads to an improved force correlation and acceleration in the sampling of the phase space. We apply our method on SPC/E water, where we find a conservation of the average water structure. We then use our method to sample dialanine and the folding of TrpCage, where we find a good agreement with simulation data reported in the literature. Finally, we apply our methodologies on the initial stages of aggregation of a hexamer of Alzheimer's amyloid β fragment 25-35 (Aβ 25-35) and find that transitions within the hexameric aggregate are dominated by entropic barriers, while we speculate that especially the conformation entropy plays a major role in the formation of the fibril as a rate limiting factor.

  11. Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1.

    Science.gov (United States)

    de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter

    2016-06-08

    The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

  12. Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.

    Directory of Open Access Journals (Sweden)

    Nathan V Welham

    2011-03-01

    Full Text Available The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match. The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

  13. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH–YopH chaperone–effector complex

    International Nuclear Information System (INIS)

    Vujanac, Milos; Stebbins, C. Erec

    2013-01-01

    The structure of a SycH–YopH chaperone–effector complex from Yersinia reveals the bacterial state of a protein that adopts different folds in the host and pathogen environments. Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH–YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed

  14. Connecting Protein Structure to Intermolecular Interactions: A Computer Modeling Laboratory

    Science.gov (United States)

    Abualia, Mohammed; Schroeder, Lianne; Garcia, Megan; Daubenmire, Patrick L.; Wink, Donald J.; Clark, Ginevra A.

    2016-01-01

    An understanding of protein folding relies on a solid foundation of a number of critical chemical concepts, such as molecular structure, intra-/intermolecular interactions, and relating structure to function. Recent reports show that students struggle on all levels to achieve these understandings and use them in meaningful ways. Further, several…

  15. Can understanding the packing of side chains improve the design of protein-protein interactions?

    Science.gov (United States)

    Zhou, Alice; O'Hern, Corey; Regan, Lynne

    2011-03-01

    With the long-term goal to improve the design of protein-protein interactions, we have begun extensive computational studies to understand how side-chains of key residues of binding partners geometrically fit together at protein-peptide interfaces, e.g. the tetratrico-peptide repeat protein and its cognate peptide). We describe simple atomic-scale models of hydrophobic dipeptides, which include hard-core repulsion, bond length and angle constraints, and Van der Waals attraction. By completely enumerating all minimal energy structures in these systems, we are able to reproduce important features of the probability distributions of side chain dihedral angles of hydrophic residues in the protein data bank. These results are the crucial first step in developing computational models that can predict the side chain conformations of residues at protein-peptide interfaces. CSO acknowledges support from NSF grant no. CMMT-1006527.

  16. Communication: Role of explicit water models in the helix folding/unfolding processes

    Science.gov (United States)

    Palazzesi, Ferruccio; Salvalaglio, Matteo; Barducci, Alessandro; Parrinello, Michele

    2016-09-01

    In the last years, it has become evident that computer simulations can assume a relevant role in modelling protein dynamical motions for their ability to provide a full atomistic image of the processes under investigation. The ability of the current protein force-fields in reproducing the correct thermodynamics and kinetics systems behaviour is thus an essential ingredient to improve our understanding of many relevant biological functionalities. In this work, employing the last developments of the metadynamics framework, we compare the ability of state-of-the-art all-atom empirical functions and water models to consistently reproduce the folding and unfolding of a helix turn motif in a model peptide. This theoretical study puts in evidence that the choice of the water models can influence the thermodynamic and the kinetics of the system under investigation, and for this reason cannot be considered trivial.

  17. Folding and insertion thermodynamics of the transmembrane WALP peptide

    NARCIS (Netherlands)

    Bereau, T.; Bennett, W.F.D.; Pfaendtner, J.; Deserno, M.; Karttunen, M.E.J.

    2015-01-01

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)$_n$(L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural

  18. Folding and insertion thermodynamics of the transmembrane WALP peptide

    NARCIS (Netherlands)

    Bereau, T.; Bennett, W.F.D. Drew; Pfaendtner, J.; Deserno, M.; Karttunen, M.

    2015-01-01

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural

  19. Protein Structure Prediction by Protein Threading

    Science.gov (United States)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  20. Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein.

    Science.gov (United States)

    Leliveld, S Rutger; Stitz, Lothar; Korth, Carsten

    2008-06-10

    A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.

  1. Use of designed sequences in protein structure recognition.

    Science.gov (United States)

    Kumar, Gayatri; Mudgal, Richa; Srinivasan, Narayanaswamy; Sandhya, Sankaran

    2018-05-09

    Knowledge of the protein structure is a pre-requisite for improved understanding of molecular function. The gap in the sequence-structure space has increased in the post-genomic era. Grouping related protein sequences into families can aid in narrowing the gap. In the Pfam database, structure description is provided for part or full-length proteins of 7726 families. For the remaining 52% of the families, information on 3-D structure is not yet available. We use the computationally designed sequences that are intermediately related to two protein domain families, which are already known to share the same fold. These strategically designed sequences enable detection of distant relationships and here, we have employed them for the purpose of structure recognition of protein families of yet unknown structure. We first measured the success rate of our approach using a dataset of protein families of known fold and achieved a success rate of 88%. Next, for 1392 families of yet unknown structure, we made structural assignments for part/full length of the proteins. Fold association for 423 domains of unknown function (DUFs) are provided as a step towards functional annotation. The results indicate that knowledge-based filling of gaps in protein sequence space is a lucrative approach for structure recognition. Such sequences assist in traversal through protein sequence space and effectively function as 'linkers', where natural linkers between distant proteins are unavailable. This article was reviewed by Oliviero Carugo, Christine Orengo and Srikrishna Subramanian.

  2. In-cell thermodynamics and a new role for protein surfaces.

    Science.gov (United States)

    Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

    2016-02-16

    There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

  3. Malfolded protein structure and proteostasis in lung diseases.

    Science.gov (United States)

    Balch, William E; Sznajder, Jacob I; Budinger, Scott; Finley, Daniel; Laposky, Aaron D; Cuervo, Ana Maria; Benjamin, Ivor J; Barreiro, Esther; Morimoto, Richard I; Postow, Lisa; Weissman, Allan M; Gail, Dorothy; Banks-Schlegel, Susan; Croxton, Thomas; Gan, Weiniu

    2014-01-01

    Recent discoveries indicate that disorders of protein folding and degradation play a particularly important role in the development of lung diseases and their associated complications. The overarching purpose of the National Heart, Lung, and Blood Institute workshop on "Malformed Protein Structure and Proteostasis in Lung Diseases" was to identify mechanistic and clinical research opportunities indicated by these recent discoveries in proteostasis science that will advance our molecular understanding of lung pathobiology and facilitate the development of new diagnostic and therapeutic strategies for the prevention and treatment of lung disease. The workshop's discussion focused on identifying gaps in scientific knowledge with respect to proteostasis and lung disease, discussing new research advances and opportunities in protein folding science, and highlighting novel technologies with potential therapeutic applications for diagnosis and treatment.

  4. Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

    Science.gov (United States)

    Kinjo, Akira R; Nakamura, Haruki

    2013-01-01

    Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

  5. Mechanism and function of the chaperonin from Methanococcus maripaludis: implications for archaeal protein homeostasis and energy production

    Energy Technology Data Exchange (ETDEWEB)

    frydman, judith

    2018-03-23

    Archaea offer a potentially cost effective and renewable source of energy. The methanogen M. maripaludis, a fast growing archaea that obtains energy by sequestering H2 and reducing CO2 to methane by the methanogenic pathway, is an attractive source for biofuel production. More recently, it has also been suggested that the methanogenesis pathway could be run in reverse, to produce H2 growing the organism in formate. A multi-level understanding of archaeal protein homeostasis, should be instrumental for improving the functionality and design of the enzyme pathways and complexes involved in energy production and storage. One additional importance consequence of a better understanding of archaeal protein homeostasis will be to increase their stress resistance, since their utilization for the efficient large-scale production of methane (and eventually also of H2) requires that the organisms are resistance to a range of growth conditions. This proposal was focused on understanding how archaea achieve protein folding and assembly and maintain protein homeostasis, which are essential for function and viability. We hypothesize that the homo-oligomeric ring shaped chaperonin from M. maripaludis, Mm-Cpn, is central to achaeal protein homeostasis and assists folding of a wide spectrum of metabolic, structural and regulatory archaeal proteins. Through a combination of biochemistry, systems biology, computational and structural biology, we have been testing this hypothesis through two complementary efforts: (i) identify the archaeal substrate repertoire of Mm-Cpn, and (ii) define mechanistic and structural principles of Mm-Cpn mediated protein folding.

  6. Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

    Science.gov (United States)

    Sharma, Shruti; Gupta, Ravi; Deswal, Renu

    2017-05-01

    In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Synthetic oligorotaxanes exert high forces when folding under mechanical load

    Science.gov (United States)

    Sluysmans, Damien; Hubert, Sandrine; Bruns, Carson J.; Zhu, Zhixue; Stoddart, J. Fraser; Duwez, Anne-Sophie

    2018-01-01

    Folding is a ubiquitous process that nature uses to control the conformations of its molecular machines, allowing them to perform chemical and mechanical tasks. Over the years, chemists have synthesized foldamers that adopt well-defined and stable folded architectures, mimicking the control expressed by natural systems1,2. Mechanically interlocked molecules, such as rotaxanes and catenanes, are prototypical molecular machines that enable the controlled movement and positioning of their component parts3-5. Recently, combining the exquisite complexity of these two classes of molecules, donor-acceptor oligorotaxane foldamers have been synthesized, in which interactions between the mechanically interlocked component parts dictate the single-molecule assembly into a folded secondary structure6-8. Here we report on the mechanochemical properties of these molecules. We use atomic force microscopy-based single-molecule force spectroscopy to mechanically unfold oligorotaxanes, made of oligomeric dumbbells incorporating 1,5-dioxynaphthalene units encircled by cyclobis(paraquat-p-phenylene) rings. Real-time capture of fluctuations between unfolded and folded states reveals that the molecules exert forces of up to 50 pN against a mechanical load of up to 150 pN, and displays transition times of less than 10 μs. While the folding is at least as fast as that observed in proteins, it is remarkably more robust, thanks to the mechanically interlocked structure. Our results show that synthetic oligorotaxanes have the potential to exceed the performance of natural folding proteins.

  8. Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

    Science.gov (United States)

    Ebrahimi, Mansour; Aghagolzadeh, Parisa; Shamabadi, Narges; Tahmasebi, Ahmad; Alsharifi, Mohammed; Adelson, David L; Hemmatzadeh, Farhid; Ebrahimie, Esmaeil

    2014-01-01

    The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and

  9. Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

    Directory of Open Access Journals (Sweden)

    Mansour Ebrahimi

    Full Text Available The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896 of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms, percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for

  10. Analysis of residuals from enzyme kinetic and protein folding experiments in the presence of correlated experimental noise.

    Science.gov (United States)

    Kuzmic, Petr; Lorenz, Thorsten; Reinstein, Jochen

    2009-12-01

    Experimental data from continuous enzyme assays or protein folding experiments often contain hundreds, or even thousands, of densely spaced data points. When the sampling interval is extremely short, the experimental data points might not be statistically independent. The resulting neighborhood correlation invalidates important theoretical assumptions of nonlinear regression analysis. As a consequence, certain goodness-of-fit criteria, such as the runs-of-signs test and the autocorrelation function, might indicate a systematic lack of fit even if the experiment does agree very well with the underlying theoretical model. A solution to this problem is to analyze only a subset of the residuals of fit, such that any excessive neighborhood correlation is eliminated. Substrate kinetics of the HIV protease and the unfolding kinetics of UMP/CMP kinase, a globular protein from Dictyostelium discoideum, serve as two illustrative examples. A suitable data-reduction algorithm has been incorporated into software DYNAFIT [P. Kuzmic, Anal. Biochem. 237 (1996) 260-273], freely available to all academic researchers from http://www.biokin.com.

  11. Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

    Science.gov (United States)

    Meuzelaar, Heleen; Tros, Martijn; Huerta-Viga, Adriana; van Dijk, Chris N; Vreede, Jocelyne; Woutersen, Sander

    2014-03-06

    Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu - and Arg + are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.

  12. F-Type Lectins: A Highly Diversified Family of Fucose-Binding Proteins with a Unique Sequence Motif and Structural Fold, Involved in Self/Non-Self-Recognition

    Directory of Open Access Journals (Sweden)

    Gerardo R. Vasta

    2017-11-01

    Full Text Available The F-type lectin (FTL family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD] that displays a novel jellyroll fold (“F-type” fold and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option.

  13. Rate constant and reaction coordinate of Trp-cage folding in explicit water

    NARCIS (Netherlands)

    Juraszek, J.; Bolhuis, P.G.

    2008-01-01

    We report rate constant calculations and a reaction coordinate analysis of the rate-limiting folding and unfolding process of the Trp-cage mini-protein in explicit solvent using transition interface sampling. Previous transition path sampling simulations revealed that in this (un)folding process the

  14. Evidence of non-coincidence of normalized sigmoidal curves of two different structural properties for two-state protein folding/unfolding

    International Nuclear Information System (INIS)

    Rahaman, Hamidur; Khan, Md. Khurshid Alam; Hassan, Md. Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan

    2013-01-01

    Highlights: ► Non-coincidence of normalized sigmoidal curves of two different structural properties is consistence with the two-state protein folding/unfolding. ► DSC measurements of denaturation show a two-state behavior of g-cyt-c at pH 6.0. ► Urea-induced denaturation of g-cyt-c is a variable two- state process at pH 6.0. ► GdmCl-induced denaturation of g-cyt-c is a fixed two- state process at pH 6.0. -- Abstract: In practice, the observation of non-coincidence of normalized sigmoidal transition curves measured by two different structural properties constitutes a proof of existence of thermodynamically stable intermediate(s) on the folding ↔ unfolding pathway of a protein. Here we give first experimental evidence that this non-coincidence is also observed for a two-state protein denaturation. Proof of this evidence comes from our studies of denaturation of goat cytochrome-c (g-cyt-c) at pH 6.0. These studies involve differential scanning calorimetry (DSC) measurements in the absence of urea and measurements of urea-induced denaturation curves monitored by observing changes in absorbance at 405, 530, and 695 nm and circular dichroism (CD) at 222, 405, and 416 nm. DSC measurements showed that denaturation of the protein is a two-state process, for calorimetric and van’t Hoff enthalpy changes are, within experimental errors, identical. Normalization of urea-induced denaturation curves monitored by optical properties leads to noncoincident sigmoidal curves. Heat-induced transition of g-cyt-c in the presence of different urea concentrations was monitored by CD at 222 nm and absorption at 405 nm. It was observed that these two different structural probes gave not only identical values of T m (transition temperature), ΔH m (change in enthalpy at T m ) and ΔC p (constant-pressure heat capacity change), but these thermodynamic parameters in the absence of urea are also in agreement with those obtained from DSC measurements

  15. SDEM modelling of fault-propagation folding

    DEFF Research Database (Denmark)

    Clausen, O.R.; Egholm, D.L.; Poulsen, Jane Bang

    2009-01-01

    and variations in Mohr-Coulomb parameters including internal friction. Using SDEM modelling, we have mapped the propagation of the tip-line of the fault, as well as the evolution of the fold geometry across sedimentary layers of contrasting rheological parameters, as a function of the increased offset......Understanding the dynamics and kinematics of fault-propagation-folding is important for evaluating the associated hydrocarbon play, for accomplishing reliable section balancing (structural reconstruction), and for assessing seismic hazards. Accordingly, the deformation style of fault-propagation...... a precise indication of when faults develop and hence also the sequential evolution of secondary faults. Here we focus on the generation of a fault -propagated fold with a reverse sense of motion at the master fault, and varying only the dip of the master fault and the mechanical behaviour of the deformed...

  16. Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

    Science.gov (United States)

    Finkelstein, A. V.

    1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

  17. Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif.

    Science.gov (United States)

    Papanikolopoulou, Katerina; Teixeira, Susana; Belrhali, Hassan; Forsyth, V Trevor; Mitraki, Anna; van Raaij, Mark J

    2004-09-03

    Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.

  18. [Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

    Science.gov (United States)

    Melnik, T N; Nagibina, G S; Surin, A K; Glukhova, K A; Melnik, B S

    2018-01-01

    Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

  19. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    Science.gov (United States)

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  20. MOLECULAR DOCKING AND DYNAMICS STUDIES ON THE PROTEIN-PROTEIN INTERACTIONS OF ELECTRICALLY ACTIVE PILIN NANOWIRES OF GEOBACTER SULFURREDUCENS.

    Directory of Open Access Journals (Sweden)

    D. Jeya Sundara Sharmila1 *

    2017-06-01

    Full Text Available Molecular interactions are key aspects in biological recognitions applicable in nano/micro systems. Bacterial nanowires are pilus filament based structures that can conduct electrons. The transport of electron is proposed to be facilitated by filamentous fibers made up of polymeric assemblies of proteins called pilin. Geobacter sulfurreducens is capable of delivering electrons through extracellular electron transport (EET by employing conductive nanowires, which are pilin proteins composed of type IV subunit PilA. Protein-protein interactions play an important role in the stabilization of the pilin nanowire assembly complex and it contains transmembrane (TM domain. In current study, protein-protein docking and multiple molecular dynamic (MD simulations were performed to understand the binding mode of pilin nanowires. The MD result explains the conformational behavior and folding of pilin nanowires in water environment in different time scale duration 20, 5, 5, 10 and 20ns (total of 60ns. Direct hydrogen bonds and water mediated hydrogen bonds that play a crucial role during the simulation were investigated. The conformational state, folding, end-toend distance profile and hydrogen bonding behavior had indicated that the Geobacter sulfurreducens pilin nanowires have electrical conductivity properties.

  1. The Crystal Structure of a Maxi/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages

    Energy Technology Data Exchange (ETDEWEB)

    Cornell, Thomas A. [Department; Division; Srivastava, Yogesh [Genome; Jauch, Ralf [Genome Institute of Singapore, Singapore; Genome; Fan, Rongli [Division; Orner, Brendan P. [Department; Division

    2017-07-19

    Cage proteins assemble into nanoscale structures with large central cavities. They play roles, including those as virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage. Two ferritin subfamilies have strongly similar tertiary structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around 4-fold symmetry axes, and mini-ferritins are 12-meric, tetrahedral cages with 3-fold axes defined by C-termini lacking E-domains. To understand the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between those of the maxi- and mini-ferritin. The research described herein sets out to understand (a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and (b) whether the symmetrical preference of the E-domain in the maxi-ferritin (4-fold axis) overrules the C-terminal preference in the mini-ferritin (3-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage that can be nearly superimposed with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the 3-fold symmetry axes.

  2. Multiple routes and milestones in the folding of HIV-1 protease monomer.

    Directory of Open Access Journals (Sweden)

    Massimiliano Bonomi

    Full Text Available Proteins fold on a time scale incompatible with a mechanism of random search in conformational space thus indicating that somehow they are guided to the native state through a funneled energetic landscape. At the same time the heterogeneous kinetics suggests the existence of several different folding routes. Here we propose a scenario for the folding mechanism of the monomer of HIV-1 protease in which multiple pathways and milestone events coexist. A variety of computational approaches supports this picture. These include very long all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of clusters found in multiple high-temperature unfolding simulations and a complete characterization of free-energy surfaces carried out using a structure-based potential at atomistic resolution and a combination of metadynamics and parallel tempering. Our results confirm that the monomer in solution is stable toward unfolding and show that at least two unfolding pathways exist. In our scenario, the formation of a hydrophobic core is a milestone in the folding process which must occur along all the routes that lead this protein towards its native state. Furthermore, the ensemble of folding pathways proposed here substantiates a rational drug design strategy based on inhibiting the folding of HIV-1 protease.

  3. Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function.

    Directory of Open Access Journals (Sweden)

    Xinxin Liu

    Full Text Available Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3 expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001. Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (p<0.05 and a reduction in transepithelial electrical resistance (TEER by 180.0% (p<0.001. While the expression of tight junctional protein did not change in acrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (p<0.05. Taken together, these data provide evidence that acute acrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium.

  4. Effects of Polymer Hydrophobicity on Protein Structure and Aggregation Kinetics in Crowded Milieu.

    Science.gov (United States)

    Breydo, Leonid; Sales, Amanda E; Frege, Telma; Howell, Mark C; Zaslavsky, Boris Y; Uversky, Vladimir N

    2015-05-19

    We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect.

  5. Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

    Science.gov (United States)

    Finka, Andrija; Goloubinoff, Pierre

    2013-09-01

    In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

  6. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of

  7. Oral and vocal fold diadochokinesis in dysphonic women

    OpenAIRE

    Louzada,Talita; Beraldinelle,Roberta; Berretin-Felix,Giédre; Brasolotto,Alcione Ghedini

    2011-01-01

    The evaluation of oral and vocal fold diadochokinesis (DDK) in individuals with voice disorders may contribute to the understanding of factors that affect the balanced vocal production. Scientific studies that make use of this assessment tool support the knowledge advance of this area, reflecting the development of more appropriate therapeutic planning. Objective: To compare the results of oral and vocal fold DDK in dysphonic women and in women without vocal disorders. Material and methods: F...

  8. Disulfide bonds in folding and transport of the mouse hepatitis virus glycoproteins

    NARCIS (Netherlands)

    Horzinek, M.C.; Opstelten, D.-J.E.; Groote, P. de; Vennema, H.; Rottier, P.J.M.

    1993-01-01

    We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum

  9. Flips for 3-folds and 4-folds

    CERN Document Server

    Corti, Alessio

    2007-01-01

    This edited collection of chapters, authored by leading experts, provides a complete and essentially self-contained construction of 3-fold and 4-fold klt flips. A large part of the text is a digest of Shokurov's work in the field and a concise, complete and pedagogical proof of the existence of 3-fold flips is presented. The text includes a ten page glossary and is accessible to students and researchers in algebraic geometry.

  10. RNAslider: a faster engine for consecutive windows folding and its application to the analysis of genomic folding asymmetry.

    Science.gov (United States)

    Horesh, Yair; Wexler, Ydo; Lebenthal, Ilana; Ziv-Ukelson, Michal; Unger, Ron

    2009-03-04

    Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L free energy (MFE) for the folding of each of the L-sized substrings of S. The consecutive windows folding problem can be naively solved in O(NL3) by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2) solution for this problem has been described. Here, we describe and implement an O(NLpsi(L)) engine for the consecutive windows folding problem, where psi(L) is shown to converge to O(1) under the assumption of a standard probabilistic polymer folding model, yielding an O(L) speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5') folding bias, i.e. that the minimal free energy (MFE) of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.

  11. Self-folding polymeric containers for encapsulation and delivery of drugs.

    Science.gov (United States)

    Fernandes, Rohan; Gracias, David H

    2012-11-01

    Self-folding broadly refers to self-assembly processes wherein thin films or interconnected planar templates curve, roll-up or fold into three dimensional (3D) structures such as cylindrical tubes, spirals, corrugated sheets or polyhedra. The process has been demonstrated with metallic, semiconducting and polymeric films and has been used to curve tubes with diameters as small as 2nm and fold polyhedra as small as 100nm, with a surface patterning resolution of 15nm. Self-folding methods are important for drug delivery applications since they provide a means to realize 3D, biocompatible, all-polymeric containers with well-tailored composition, size, shape, wall thickness, porosity, surface patterns and chemistry. Self-folding is also a highly parallel process, and it is possible to encapsulate or self-load therapeutic cargo during assembly. A variety of therapeutic cargos such as small molecules, peptides, proteins, bacteria, fungi and mammalian cells have been encapsulated in self-folded polymeric containers. In this review, we focus on self-folding of all-polymeric containers. We discuss the mechanistic aspects of self-folding of polymeric containers driven by differential stresses or surface tension forces, the applications of self-folding polymers in drug delivery and we outline future challenges. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The Ever-Evolving Concept of the Gene: The Use of RNA/Protein Experimental Techniques to Understand Genome Functions

    Directory of Open Access Journals (Sweden)

    Andrea Cipriano

    2018-03-01

    Full Text Available The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as “junk” DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs, which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years.

  13. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  14. A Soluble, Folded Protein without Charged Amino Acid Residues

    DEFF Research Database (Denmark)

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall

    2016-01-01

    side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find......Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably...

  15. PROSPECTIVE TEACHERS’ COGNITIVE STRUCTURES CONCERNING PROTEIN SYNTHESIS AND THEIR DEGREE OF UNDERSTANDING

    Directory of Open Access Journals (Sweden)

    Cem Gerçek

    2018-02-01

    Full Text Available The purpose of education is to actualise meaningful learning. Therefore, researching the issues on how students process information and how they configure it is important for meaningful learning. The issue of protein synthesis contains a number of abstract topics and concepts. Hence, it is important in biology teaching to be informed of students’ cognitive structures concerning protein synthesis. This research aims to analyse prospective teachers’ cognitive structures about protein synthesis and their degree of understanding the subject. The research group was composed of 17 volunteering prospective teachers who had been chosen through purposeful sampling. The data were collected via semi-structured interviews. Flow maps and content analysis were used in analysing the data. The results demonstrated that prospective teachers had too many misconceptions about protein synthesis and that their knowledge extent and rich connection are inadequate. The prospective teachers’ degree of understanding protein synthesis was divided into three categories. The results obtained in this research suggested that teachers should be careful in teaching the subject of protein synthesis. Students’ prior knowledge and their misconceptions should be determined and content or contexts to facilitate them to learn an abstract subject such as protein synthesis should be presented.

  16. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    International Nuclear Information System (INIS)

    Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

    2006-01-01

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

  17. Differential Precipitation and Solubilization of Proteins.

    Science.gov (United States)

    Ryan, Barry J; Kinsella, Gemma K

    2017-01-01

    Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded proteins. Here, appropriate bioinformatics tools to aid in understanding a protein's propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein.

  18. Protein structure recognition: From eigenvector analysis to structural threading method

    Science.gov (United States)

    Cao, Haibo

    In this work, we try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. We found a strong correlation between amino acid sequence and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, we give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part include discussions of interactions among amino acids residues, lattice HP model, and the designablity principle. In the second part, we try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in our eigenvector study of protein contact matrix. We believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, we discuss a threading method based on the correlation between amino acid sequence and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, we list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

  19. Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Haibo [Iowa State Univ., Ames, IA (United States)

    2003-01-01

    In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

  20. Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

    International Nuclear Information System (INIS)

    Haibo Cao

    2003-01-01

    In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches

  1. Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

    Science.gov (United States)

    Ren, Weitong; Ji, Dongqing; Xu, Xiulian

    2018-01-01

    The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

  2. Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

    Directory of Open Access Journals (Sweden)

    Weitong Ren

    Full Text Available The HIV-1 nucleocapsid 7 (NCp7 plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3 recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

  3. Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH.

    Science.gov (United States)

    McDonald, Sarah K; Fleming, Karen G

    2016-11-08

    Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.

  4. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  5. High-resolution structure of a retroviral protease folded as a monomer

    International Nuclear Information System (INIS)

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-01-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C α deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  6. The influence of thyroarytenoid and cricothyroid muscle activation on vocal fold stiffness and eigenfrequencies

    OpenAIRE

    Yin, Jun; Zhang, Zhaoyan

    2013-01-01

    The influence of the thyroarytenoid (TA) and cricothyroid (CT) muscle activation on vocal fold stiffness and eigenfrequencies was investigated in a muscularly controlled continuum model of the vocal folds. Unlike the general understanding that vocal fold fundamental frequency was determined by vocal fold tension, this study showed that vocal fold eigenfrequencies were primarily determined by vocal fold stiffness. This study further showed that, with reference to the resting state of zero stra...

  7. Protein and solute distribution in drug substance containers during frozen storage and post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development.

    Science.gov (United States)

    Kolhe, Parag; Badkar, Advait

    2011-01-01

    Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  8. A Soluble, Folded Protein without Charged Amino Acid Residues

    DEFF Research Database (Denmark)

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall

    2016-01-01

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find...

  9. Origin and Evolution of Protein Fold Designs Inferred from Phylogenomic Analysis of CATH Domain Structures in Proteomes

    Science.gov (United States)

    Bukhari, Syed Abbas; Caetano-Anollés, Gustavo

    2013-01-01

    The spatial arrangements of secondary structures in proteins, irrespective of their connectivity, depict the overall shape and organization of protein domains. These features have been used in the CATH and SCOP classifications to hierarchically partition fold space and define the architectural make up of proteins. Here we use phylogenomic methods and a census of CATH structures in hundreds of genomes to study the origin and diversification of protein architectures (A) and their associated topologies (T) and superfamilies (H). Phylogenies that describe the evolution of domain structures and proteomes were reconstructed from the structural census and used to generate timelines of domain discovery. Phylogenies of CATH domains at T and H levels of structural abstraction and associated chronologies revealed patterns of reductive evolution, the early rise of Archaea, three epochs in the evolution of the protein world, and patterns of structural sharing between superkingdoms. Phylogenies of proteomes confirmed the early appearance of Archaea. While these findings are in agreement with previous phylogenomic studies based on the SCOP classification, phylogenies unveiled sharing patterns between Archaea and Eukarya that are recent and can explain the canonical bacterial rooting typically recovered from sequence analysis. Phylogenies of CATH domains at A level uncovered general patterns of architectural origin and diversification. The tree of A structures showed that ancient structural designs such as the 3-layer (αβα) sandwich (3.40) or the orthogonal bundle (1.10) are comparatively simpler in their makeup and are involved in basic cellular functions. In contrast, modern structural designs such as prisms, propellers, 2-solenoid, super-roll, clam, trefoil and box are not widely distributed and were probably adopted to perform specialized functions. Our timelines therefore uncover a universal tendency towards protein structural complexity that is remarkable. PMID:23555236

  10. Order through disorder: hyper-mobile C-terminal residues stabilize the folded state of a helical peptide. a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Kalliopi K Patapati

    Full Text Available Conventional wisdom has it that the presence of disordered regions in the three-dimensional structures of polypeptides not only does not contribute significantly to the thermodynamic stability of their folded state, but, on the contrary, that the presence of disorder leads to a decrease of the corresponding proteins' stability. We have performed extensive 3.4 µs long folding simulations (in explicit solvent and with full electrostatics of an undecamer peptide of experimentally known helical structure, both with and without its disordered (four residue long C-terminal tail. Our simulations clearly indicate that the presence of the apparently disordered (in structural terms C-terminal tail, increases the thermodynamic stability of the peptide's folded (helical state. These results show that at least for the case of relatively short peptides, the interplay between thermodynamic stability and the apparent structural stability can be rather subtle, with even disordered regions contributing significantly to the stability of the folded state. Our results have clear implications for the understanding of peptide energetics and the design of foldable peptides.

  11. Histopathologic study of human vocal fold mucosa unphonated over a decade.

    Science.gov (United States)

    Sato, Kiminori; Umeno, Hirohito; Ono, Takeharu; Nakashima, Tadashi

    2011-12-01

    Mechanotransduction caused by vocal fold vibration could possibly be an important factor in the maintenance of extracellular matrices and layered structure of the human adult vocal fold mucosa as a vibrating tissue after the layered structure has been completed. Vocal fold stellate cells (VFSCs) in the human maculae flavae of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Maculae flavae are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate the VFSCs to accelerate production of extracellular matrices. A human adult vocal fold mucosa unphonated over a decade was investigated histopathologically. Vocal fold mucosa unphonated for 11 years and 2 months of a 64-year-old male with cerebral hemorrhage was investigated by light and electron microscopy. The vocal fold mucosae (including maculae flavae) were atrophic. The vocal fold mucosa did not have a vocal ligament, Reinke's space or a layered structure. The lamina propria appeared as a uniform structure. Morphologically, the VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. Consequently, VFSCs appeared to decrease their level of activity.

  12. RNAslider: a faster engine for consecutive windows folding and its application to the analysis of genomic folding asymmetry

    Directory of Open Access Journals (Sweden)

    Ziv-Ukelson Michal

    2009-03-01

    Full Text Available Abstract Background Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L 3 by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2 solution for this problem has been described. Results Here, we describe and implement an O(NLψ(L engine for the consecutive windows folding problem, where ψ(L is shown to converge to O(1 under the assumption of a standard probabilistic polymer folding model, yielding an O(L speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5' folding bias, i.e. that the minimal free energy (MFE of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. Conclusion The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.

  13. Folding and activity of hybrid sequence, disulfide-stabilized peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))

    1990-08-01

    Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.

  14. Folding and activity of hybrid sequence, disulfide-stabilized peptides

    International Nuclear Information System (INIS)

    Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.

    1990-01-01

    Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences

  15. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

    Directory of Open Access Journals (Sweden)

    Dominik R Schmitt

    Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

  16. The human PDI family: Versatility packed into a single fold

    DEFF Research Database (Denmark)

    Appenzeller-Herzog, Christian; Ellgaard, Lars

    2007-01-01

    in promoting oxidative protein folding in the ER has been extended in recent years to include roles in other processes such as ER-associated degradation (ERAD), trafficking, calcium homeostasis, antigen presentation and virus entry. Some of these functions are performed by non-catalytic members of the family...... that lack the active-site cysteines. Regardless of their function, all human PDIs contain at least one domain of approximately 100 amino acid residues with structural homology to thioredoxin. As we learn more about the individual proteins of the family, a complex picture is emerging that emphasizes as much...... their differences as their similarities, and underlines the versatility of the thioredoxin fold. Here, we primarily explore the diversity of cellular functions described for the human PDIs. Udgivelsesdato: 2007-Dec-3...

  17. Understanding the mechanical properties of DNA origami tiles and controlling the kinetics of their folding and unfolding reconfiguration.

    Science.gov (United States)

    Chen, Haorong; Weng, Te-Wei; Riccitelli, Molly M; Cui, Yi; Irudayaraj, Joseph; Choi, Jong Hyun

    2014-05-14

    DNA origami represents a class of highly programmable macromolecules that can go through conformational changes in response to external signals. Here we show that a two-dimensional origami rectangle can be effectively folded into a short, cylindrical tube by connecting the two opposite edges through the hybridization of linker strands and that this process can be efficiently reversed via toehold-mediated strand displacement. The reconfiguration kinetics was experimentally studied as a function of incubation temperature, initial origami concentration, missing staples, and origami geometry. A kinetic model was developed by introducing the j factor to describe the reaction rates in the cyclization process. We found that the cyclization efficiency (j factor) increases sharply with temperature and depends strongly on the structural flexibility and geometry. A simple mechanical model was used to correlate the observed cyclization efficiency with origami structure details. The mechanical analysis suggests two sources of the energy barrier for DNA origami folding: overcoming global twisting and bending the structure into a circular conformation. It also provides the first semiquantitative estimation of the rigidity of DNA interhelix crossovers, an essential element in structural DNA nanotechnology. This work demonstrates efficient DNA origami reconfiguration, advances our understanding of the dynamics and mechanical properties of self-assembled DNA structures, and should be valuable to the field of DNA nanotechnology.

  18. Structures of two Arabidopsis thaliana major latex proteins represent novel helix-grip folds

    Energy Technology Data Exchange (ETDEWEB)

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, Jr., George N.; Volkman, Brian F.; (MCW); (UW)

    2009-06-02

    Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.

  19. Detecting Repetitions and Periodicities in Proteins by Tiling the Structural Space

    OpenAIRE

    Parra, R. Gonzalo; Espada, Rocío; Sánchez, Ignacio E.; Sippl, Manfred J.; Ferreiro, Diego U.

    2013-01-01

    The notion of energy landscapes provides conceptual tools for understanding the complexities of protein folding and function. Energy landscape theory indicates that it is much easier to find sequences that satisfy the “Principle of Minimal Frustration” when the folded structure is symmetric (Wolynes, P. G. Symmetry and the Energy Landscapes of Biomolecules. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14249–14255). Similarly, repeats and structural mosaics may be fundamentally related to landscape...

  20. Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

    OpenAIRE

    Li, Yaohang

    2013-01-01

    Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increas...

  1. Reconciling the understanding of 'hydrophobicity' with physics-based models of proteins.

    Science.gov (United States)

    Harris, Robert C; Pettitt, B Montgomery

    2016-03-02

    The idea that a 'hydrophobic energy' drives protein folding, aggregation, and binding by favoring the sequestration of bulky residues from water into the protein interior is widespread. The solvation free energies (ΔGsolv) of small nonpolar solutes increase with surface area (A), and the free energies of creating macroscopic cavities in water increase linearly with A. These observations seem to imply that there is a hydrophobic component (ΔGhyd) of ΔGsolv that increases linearly with A, and this assumption is widely used in implicit solvent models. However, some explicit-solvent molecular dynamics studies appear to contradict these ideas. For example, one definition (ΔG(LJ)) of ΔGhyd is that it is the free energy of turning on the Lennard-Jones (LJ) interactions between the solute and solvent. However, ΔG(LJ) decreases with A for alanine and glycine peptides. Here we argue that these apparent contradictions can be reconciled by defining ΔGhyd to be a near hard core insertion energy (ΔGrep), as in the partitioning proposed by Weeks, Chandler, and Andersen. However, recent results have shown that ΔGrep is not a simple function of geometric properties of the molecule, such as A and the molecular volume, and that the free energy of turning on the attractive part of the LJ potential cannot be computed from first-order perturbation theory for proteins. The theories that have been developed from these assumptions to predict ΔGhyd are therefore inadequate for proteins.

  2. Exploring the universe of protein structures beyond the Protein Data Bank.

    Science.gov (United States)

    Cossio, Pilar; Trovato, Antonio; Pietrucci, Fabio; Seno, Flavio; Maritan, Amos; Laio, Alessandro

    2010-11-04

    It is currently believed that the atlas of existing protein structures is faithfully represented in the Protein Data Bank. However, whether this atlas covers the full universe of all possible protein structures is still a highly debated issue. By using a sophisticated numerical approach, we performed an exhaustive exploration of the conformational space of a 60 amino acid polypeptide chain described with an accurate all-atom interaction potential. We generated a database of around 30,000 compact folds with at least of secondary structure corresponding to local minima of the potential energy. This ensemble plausibly represents the universe of protein folds of similar length; indeed, all the known folds are represented in the set with good accuracy. However, we discover that the known folds form a rather small subset, which cannot be reproduced by choosing random structures in the database. Rather, natural and possible folds differ by the contact order, on average significantly smaller in the former. This suggests the presence of an evolutionary bias, possibly related to kinetic accessibility, towards structures with shorter loops between contacting residues. Beside their conceptual relevance, the new structures open a range of practical applications such as the development of accurate structure prediction strategies, the optimization of force fields, and the identification and design of novel folds.

  3. Adjustable thermal resistor by reversibly folding a graphene sheet.

    Science.gov (United States)

    Song, Qichen; An, Meng; Chen, Xiandong; Peng, Zhan; Zang, Jianfeng; Yang, Nuo

    2016-08-11

    Phononic (thermal) devices such as thermal diodes, thermal transistors, thermal logic gates, and thermal memories have been studied intensively. However, tunable thermal resistors have not been demonstrated yet. Here, we propose an instantaneously adjustable thermal resistor based on folded graphene. Through theoretical analysis and molecular dynamics simulations, we study the phonon-folding scattering effect and the dependence of thermal resistivity on the length between two folds and the overall length. Furthermore, we discuss the possibility of realizing instantaneously adjustable thermal resistors in experiment. Our studies bring new insights into designing thermal resistors and understanding the thermal modulation of 2D materials by adjusting basic structure parameters.

  4. Folding of DsbB in mixed micelles

    DEFF Research Database (Denmark)

    Otzen, Daniel

    2003-01-01

    state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic...... is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS...

  5. A nonadaptive origin of a beneficial trait: in silico selection for free energy of folding leads to the neutral emergence of mutational robustness in single domain proteins.

    Science.gov (United States)

    Pagan, Rafael F; Massey, Steven E

    2014-02-01

    Proteins are regarded as being robust to the deleterious effects of mutations. Here, the neutral emergence of mutational robustness in a population of single domain proteins is explored using computer simulations. A pairwise contact model was used to calculate the ΔG of folding (ΔG folding) using the three dimensional protein structure of leech eglin C. A random amino acid sequence with low mutational robustness, defined as the average ΔΔG resulting from a point mutation (ΔΔG average), was threaded onto the structure. A population of 1,000 threaded sequences was evolved under selection for stability, using an upper and lower energy threshold. Under these conditions, mutational robustness increased over time in the most common sequence in the population. In contrast, when the wild type sequence was used it did not show an increase in robustness. This implies that the emergence of mutational robustness is sequence specific and that wild type sequences may be close to maximal robustness. In addition, an inverse relationship between ∆∆G average and protein stability is shown, resulting partly from a larger average effect of point mutations in more stable proteins. The emergence of mutational robustness was also observed in the Escherichia coli colE1 Rop and human CD59 proteins, implying that the property may be common in single domain proteins under certain simulation conditions. The results indicate that at least a portion of mutational robustness in small globular proteins might have arisen by a process of neutral emergence, and could be an example of a beneficial trait that has not been directly selected for, termed a "pseudaptation."

  6. Expression of tenascin-C in a rat vocal fold injury model and its regulation of fibroblasts.

    Science.gov (United States)

    Li, Juan; Liu, Yiqiong; Wang, Yiming; Xu, Wen

    2018-03-23

    Tenascin-C (Tnc) is an extracellular matrix (ECM) glycoprotein that plays a vital role in wound healing and fibrotic disease. Tnc is highly upregulated soon after vocal fold injury, but its function in the vocal fold has not yet been defined. In this study, we investigated Tnc expression in a rat vocal fold injury model in vivo and its roles in fibroblasts in vitro. In vivo and in vitro. Tnc mRNA and protein expression levels were quantified on days 3, 7, 14, 28, and 56 after vocal fold injury in Sprague-Dawley rats. In vitro, immunocytochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed in primary rat vocal fold fibroblasts following Tnc or transforming growth factor (TGF)-β1 stimulation to investigate the phenotypic effects. Tnc mRNA and protein expression was upregulated dramatically on days 3 and 7 after injury, and significant differences were observed by qRT-PCR (P vocal fold fibroblasts. Following incubation with Tnc for 72 hours, α-smooth muscle actin, collagen I, and fibronectin expression was significantly upregulated (P vocal fold fibroblast migration, transdifferentiation, and ECM protein synthesis in vitro. Tnc was induced by TGF-β1 in a SMAD3-dependent manner. Transient expression of Tnc is likely to promote regeneration, but its potential role in fibrosis requires further study. NA Laryngoscope, 2018. © 2018 The American Laryngological, Rhinological and Otological Society, Inc.

  7. Aromatic claw: A new fold with high aromatic content that evades structural prediction: Aromatic Claw

    Energy Technology Data Exchange (ETDEWEB)

    Sachleben, Joseph R. [Biomolecular NMR Core Facility, University of Chicago, Chicago Illinois; Adhikari, Aashish N. [Department of Chemistry, University of Chicago, Chicago Illinois; Gawlak, Grzegorz [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois; Hoey, Robert J. [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois; Liu, Gaohua [Northeast Structural Genomics Consortium (NESG), Department of Molecular Biology and Biochemistry, School of Arts and Sciences, and Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, and Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey; Joachimiak, Andrzej [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois; Biological Sciences Division, Argonne National Laboratory, Argonne Illinois; Montelione, Gaetano T. [Northeast Structural Genomics Consortium (NESG), Department of Molecular Biology and Biochemistry, School of Arts and Sciences, and Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, and Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey; Sosnick, Tobin R. [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois; Koide, Shohei [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois; Department of Biochemistry and Molecular Pharmacology and the Perlmutter Cancer Center, New York University School of Medicine, New York New York

    2016-11-10

    We determined the NMR structure of a highly aromatic (13%) protein of unknown function, Aq1974 from Aquifex aeolicus (PDB ID: 5SYQ). The unusual sequence of this protein has a tryptophan content five times the normal (six tryptophan residues of 114 or 5.2% while the average tryptophan content is 1.0%) with the tryptophans occurring in a WXW motif. It has no detectable sequence homology with known protein structures. Although its NMR spectrum suggested that the protein was rich in β-sheet, upon resonance assignment and solution structure determination, the protein was found to be primarily α-helical with a small two-stranded β-sheet with a novel fold that we have termed an Aromatic Claw. As this fold was previously unknown and the sequence unique, we submitted the sequence to CASP10 as a target for blind structural prediction. At the end of the competition, the sequence was classified a hard template based model; the structural relationship between the template and the experimental structure was small and the predictions all failed to predict the structure. CSRosetta was found to predict the secondary structure and its packing; however, it was found that there was little correlation between CSRosetta score and the RMSD between the CSRosetta structure and the NMR determined one. This work demonstrates that even in relatively small proteins, we do not yet have the capacity to accurately predict the fold for all primary sequences. The experimental discovery of new folds helps guide the improvement of structural prediction methods.

  8. Annotating the protein-RNA interaction sites in proteins using evolutionary information and protein backbone structure.

    Science.gov (United States)

    Li, Tao; Li, Qian-Zhong

    2012-11-07

    RNA-protein interactions play important roles in various biological processes. The precise detection of RNA-protein interaction sites is very important for understanding essential biological processes and annotating the function of the proteins. In this study, based on various features from amino acid sequence and structure, including evolutionary information, solvent accessible surface area and torsion angles (φ, ψ) in the backbone structure of the polypeptide chain, a computational method for predicting RNA-binding sites in proteins is proposed. When the method is applied to predict RNA-binding sites in three datasets: RBP86 containing 86 protein chains, RBP107 containing 107 proteins chains and RBP109 containing 109 proteins chains, better sensitivities and specificities are obtained compared to previously published methods in five-fold cross-validation tests. In order to make further examination for the efficiency of our method, the RBP107 dataset is used as training set, RBP86 and RBP109 datasets are used as the independent test sets. In addition, as examples of our prediction, RNA-binding sites in a few proteins are presented. The annotated results are consistent with the PDB annotation. These results show that our method is useful for annotating RNA binding sites of novel proteins.

  9. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    Science.gov (United States)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  10. A Stevedore's protein knot.

    Directory of Open Access Journals (Sweden)

    Daniel Bölinger

    2010-04-01

    Full Text Available Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered alpha-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip.

  11. Side chain and backbone contributions of Phe508 to CFTR folding

    Energy Technology Data Exchange (ETDEWEB)

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  12. Temperature-induced transitions in disordered proteins probed by NMR spectroscopy

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Poulsen, Flemming Martin; Kragelund, Birthe Brandt

    2012-01-01

    Intrinsically disordered proteins are abundant in nature and perform many important physiological functions. Multidimensional NMR spectroscopy has been crucial for the understanding of the conformational properties of disordered proteins and is increasingly used to probe their conformational...... ensembles. Compared to folded proteins, disordered proteins are more malleable and more easily perturbed by environmental factors. Accordingly, the experimental conditions and especially the temperature modify the structural and functional properties of disordered proteins. NMR spectroscopy allows analysis...... of temperature-induced structural changes at residue resolution using secondary chemical shift analysis, paramagnetic relaxation enhancement, and residual dipolar couplings. This chapter discusses practical aspects of NMR studies of temperature-induced structural changes in disordered proteins....

  13. Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801.

    Science.gov (United States)

    Gonzalez, Claudio F; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Evdokimova, Elena; Xu, Xiaohui; Osipiuk, Jerzy; Cuff, Marianne E; Lynch, Susan; Joachimiak, Andrzej; Savchenko, Alexei; Yakunin, Alexander F

    2012-06-15

    The hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (at 1.70 and 1.75 Å for PA5202 and PA2801 respectively) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long α-helix (Asp(57) in PA5202 and Glu(35) in PA2801). Alanine residue replacement mutagenesis of PA5202 identified four residues (Asn(42), Arg(43), Asp(57) and Thr(76)) that are critical for its activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between PA5202 activity and pyocyanin production. Thus the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.

  14. Effect of the unfolded protein response on ER protein export: a potential new mechanism to relieve ER stress.

    Science.gov (United States)

    Shaheen, Alaa

    2018-05-05

    The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.

  15. Covering folded shapes

    Directory of Open Access Journals (Sweden)

    Oswin Aichholzer

    2014-05-01

    Full Text Available Can folding a piece of paper flat make it larger? We explore whether a shape S must be scaled to cover a flat-folded copy of itself. We consider both single folds and arbitrary folds (continuous piecewise isometries \\(S\\to\\mathbb{R}^2\\. The underlying problem is motivated by computational origami, and is related to other covering and fixturing problems, such as Lebesgue's universal cover problem and force closure grasps. In addition to considering special shapes (squares, equilateral triangles, polygons and disks, we give upper and lower bounds on scale factors for single folds of convex objects and arbitrary folds of simply connected objects.

  16. The porous borders of the protein world.

    Science.gov (United States)

    Cordes, Matthew H J; Stewart, Katie L

    2012-02-08

    Fold switching may play a role in the evolution of new protein folds and functions. He et al., in this issue of Structure, use protein design to illustrate that the same drastic change in a protein fold can occur via multiple different mutational pathways. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

  18. Therapeutic potential of gel-based injectables for vocal fold regeneration

    Science.gov (United States)

    Bartlett, Rebecca S.; Thibeault, Susan L.; Prestwich, Glenn D.

    2012-01-01

    Vocal folds are anatomically and biomechanically unique, thus complicating the design and implementation of tissue engineering strategies for repair and regeneration. Integration of an enhanced understanding of tissue biomechanics, wound healing dynamics and innovative gel-based therapeutics has generated enthusiasm for the notion that an efficacious treatment for vocal fold scarring could be clinically attainable within several years. Fibroblast phenotype and gene expression are mediated by the three-dimensional mechanical and chemical microenvironment at an injury site. Thus, therapeutic approaches need to coordinate spatial and temporal aspects of the wound healing response in an injured vocal tissue to achieve an optimal clinical outcome. Successful gel-based injectables for vocal fold scarring will require a keen understanding of how the native inflammatory response sets into motion the later extracellular matrix remodeling, which in turn will determine the ultimate biomechanical properties of the tissue. We present an overview of the challenges associated with this translation as well as the proposed gel-based injectable solutions. PMID:22456756

  19. Therapeutic potential of gel-based injectables for vocal fold regeneration

    International Nuclear Information System (INIS)

    Bartlett, Rebecca S; Thibeault, Susan L; Prestwich, Glenn D

    2012-01-01

    Vocal folds are anatomically and biomechanically unique, thus complicating the design and implementation of tissue engineering strategies for repair and regeneration. Integration of an enhanced understanding of tissue biomechanics, wound healing dynamics and innovative gel-based therapeutics has generated enthusiasm for the notion that an efficacious treatment for vocal fold scarring could be clinically attainable within several years. Fibroblast phenotype and gene expression are mediated by the three-dimensional mechanical and chemical microenvironment at an injury site. Thus, therapeutic approaches need to coordinate spatial and temporal aspects of the wound healing response in an injured vocal tissue to achieve an optimal clinical outcome. Successful gel-based injectables for vocal fold scarring will require a keen understanding of how the native inflammatory response sets into motion the later extracellular matrix remodeling, which in turn will determine the ultimate biomechanical properties of the tissue. We present an overview of the challenges associated with this translation as well as the proposed gel-based injectable solutions. (paper)

  20. Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations

    NARCIS (Netherlands)

    Meuzelaar, H.; Marino, K.A.; Huerta-Viga, A.; Panman, M.R.; Smeenk, L.E.J.; Kettelarij, A.J.; van Maarseveen, J.H.; Timmerman, P.; Bolhuis, P.G.; Woutersen, S.

    2013-01-01

    Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding

  1. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  2. Exploring the universe of protein structures beyond the Protein Data Bank.

    Directory of Open Access Journals (Sweden)

    Pilar Cossio

    Full Text Available It is currently believed that the atlas of existing protein structures is faithfully represented in the Protein Data Bank. However, whether this atlas covers the full universe of all possible protein structures is still a highly debated issue. By using a sophisticated numerical approach, we performed an exhaustive exploration of the conformational space of a 60 amino acid polypeptide chain described with an accurate all-atom interaction potential. We generated a database of around 30,000 compact folds with at least of secondary structure corresponding to local minima of the potential energy. This ensemble plausibly represents the universe of protein folds of similar length; indeed, all the known folds are represented in the set with good accuracy. However, we discover that the known folds form a rather small subset, which cannot be reproduced by choosing random structures in the database. Rather, natural and possible folds differ by the contact order, on average significantly smaller in the former. This suggests the presence of an evolutionary bias, possibly related to kinetic accessibility, towards structures with shorter loops between contacting residues. Beside their conceptual relevance, the new structures open a range of practical applications such as the development of accurate structure prediction strategies, the optimization of force fields, and the identification and design of novel folds.

  3. Structural basis of the 9-fold symmetry of centrioles.

    Science.gov (United States)

    Kitagawa, Daiju; Vakonakis, Ioannis; Olieric, Natacha; Hilbert, Manuel; Keller, Debora; Olieric, Vincent; Bortfeld, Miriam; Erat, Michèle C; Flückiger, Isabelle; Gönczy, Pierre; Steinmetz, Michel O

    2011-02-04

    The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Computational design of proteins with novel structure and functions

    International Nuclear Information System (INIS)

    Yang Wei; Lai Lu-Hua

    2016-01-01

    Computational design of proteins is a relatively new field, where scientists search the enormous sequence space for sequences that can fold into desired structure and perform desired functions. With the computational approach, proteins can be designed, for example, as regulators of biological processes, novel enzymes, or as biotherapeutics. These approaches not only provide valuable information for understanding of sequence–structure–function relations in proteins, but also hold promise for applications to protein engineering and biomedical research. In this review, we briefly introduce the rationale for computational protein design, then summarize the recent progress in this field, including de novo protein design, enzyme design, and design of protein–protein interactions. Challenges and future prospects of this field are also discussed. (topical review)

  5. Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.

    Directory of Open Access Journals (Sweden)

    Rashmi Tripathi

    Full Text Available The cystic fibrosis transmembrane regulator (CFTR is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs. We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with

  6. Kinks, loops, and protein folding, with protein A as an example

    International Nuclear Information System (INIS)

    Krokhotin, Andrey; Liwo, Adam; Maisuradze, Gia G.; Scheraga, Harold A.; Niemi, Antti J.

    2014-01-01

    The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a C α -trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the C α backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain–side chain interactions are responsible for initiation of loop formation. It was also found that the individual kinks are

  7. Non-equilibrium coupling of protein structure and function to translation-elongation kinetics.

    Science.gov (United States)

    Sharma, Ajeet K; O'Brien, Edward P

    2018-04-01

    Protein folding research has been dominated by the assumption that thermodynamics determines protein structure and function. And that when the folding process is compromised in vivo the proteostasis machinery-chaperones, deaggregases, the proteasome-work to restore proteins to their soluble, functional form or degrade them to maintain the cellular pool of proteins in a quasi-equilibrium state. During the past decade, however, more and more proteins have been identified for which altering only their speed of synthesis alters their structure and function, the efficiency of the down-stream processes they take part in, and cellular phenotype. Indeed, evidence has emerged that evolutionary selection pressures have encoded translation-rate information into mRNA molecules to coordinate diverse co-translational processes. Thus, non-equilibrium physics can play a fundamental role in influencing nascent protein behavior, mRNA sequence evolution, and disease. Here, we discuss how our understanding of this phenomenon is being advanced by the application of theoretical tools from the physical sciences. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. One Peptide Reveals the Two Faces of α-Helix Unfolding-Folding Dynamics.

    Science.gov (United States)

    Jesus, Catarina S H; Cruz, Pedro F; Arnaut, Luis G; Brito, Rui M M; Serpa, Carlos

    2018-04-12

    The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. 1 H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol -1 , 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol -1 , ∼1.7 μs). The essential role of two

  9. Protein folding and translocation : single-molecule investigations

    NARCIS (Netherlands)

    Leeuwen, Rudolphus Gerardus Henricus van

    2006-01-01

    This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly

  10. Vocal Fold Paralysis

    Science.gov (United States)

    ... here Home » Health Info » Voice, Speech, and Language Vocal Fold Paralysis On this page: What is vocal fold ... Where can I get additional information? What is vocal fold paralysis? Structures involved in speech and voice production ...

  11. Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides.

    Science.gov (United States)

    Undheim, Eivind A B; Mobli, Mehdi; King, Glenn F

    2016-06-01

    Three-dimensional (3D) structures have been used to explore the evolution of proteins for decades, yet they have rarely been utilized to study the molecular evolution of peptides. Here, we highlight areas in which 3D structures can be particularly useful for studying the molecular evolution of peptide toxins. Although we focus our discussion on animal toxins, including one of the most widespread disulfide-rich peptide folds known, the inhibitor cystine knot, our conclusions should be widely applicable to studies of the evolution of disulfide-constrained peptides. We show that conserved 3D folds can be used to identify evolutionary links and test hypotheses regarding the evolutionary origin of peptides with extremely low sequence identity; construct accurate multiple sequence alignments; and better understand the evolutionary forces that drive the molecular evolution of peptides. Also watch the video abstract. © 2016 WILEY Periodicals, Inc.

  12. How are proteins reduced in the endoplasmic reticulum?

    DEFF Research Database (Denmark)

    Ellgaard, Lars; Sevier, Carolyn S.; Bulleid, Neil J.

    2018-01-01

    The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction...... of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here...... why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway....

  13. A thermodynamic definition of protein domains.

    Science.gov (United States)

    Porter, Lauren L; Rose, George D

    2012-06-12

    Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

  14. Folding and insertion thermodynamics of the transmembrane WALP peptide

    International Nuclear Information System (INIS)

    Bereau, Tristan; Bennett, W. F. Drew; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko

    2015-01-01

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA) n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide’s insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum—in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence

  15. Folding and insertion thermodynamics of the transmembrane WALP peptide

    Energy Technology Data Exchange (ETDEWEB)

    Bereau, Tristan, E-mail: bereau@mpip-mainz.mpg.de [Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz (Germany); Bennett, W. F. Drew [Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Pfaendtner, Jim [Department of Chemical Engineering, University of Washington, Seattle, Washington 98195 (United States); Deserno, Markus [Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Karttunen, Mikko [Department of Mathematics and Computer Science & Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, MetaForum, 5600 MB Eindhoven (Netherlands)

    2015-12-28

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA){sub n} (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide’s insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum—in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

  16. FOLD-EM: automated fold recognition in medium- and low-resolution (4-15 Å) electron density maps.

    Science.gov (United States)

    Saha, Mitul; Morais, Marc C

    2012-12-15

    Owing to the size and complexity of large multi-component biological assemblies, the most tractable approach to determining their atomic structure is often to fit high-resolution radiographic or nuclear magnetic resonance structures of isolated components into lower resolution electron density maps of the larger assembly obtained using cryo-electron microscopy (cryo-EM). This hybrid approach to structure determination requires that an atomic resolution structure of each component, or a suitable homolog, is available. If neither is available, then the amount of structural information regarding that component is limited by the resolution of the cryo-EM map. However, even if a suitable homolog cannot be identified using sequence analysis, a search for structural homologs should still be performed because structural homology often persists throughout evolution even when sequence homology is undetectable, As macromolecules can often be described as a collection of independently folded domains, one way of searching for structural homologs would be to systematically fit representative domain structures from a protein domain database into the medium/low resolution cryo-EM map and return the best fits. Taken together, the best fitting non-overlapping structures would constitute a 'mosaic' backbone model of the assembly that could aid map interpretation and illuminate biological function. Using the computational principles of the Scale-Invariant Feature Transform (SIFT), we have developed FOLD-EM-a computational tool that can identify folded macromolecular domains in medium to low resolution (4-15 Å) electron density maps and return a model of the constituent polypeptides in a fully automated fashion. As a by-product, FOLD-EM can also do flexible multi-domain fitting that may provide insight into conformational changes that occur in macromolecular assemblies.

  17. The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

    Science.gov (United States)

    Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

    2014-01-01

    Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

  18. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    International Nuclear Information System (INIS)

    Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

    2013-01-01

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  19. Five-fold local symmetry in metallic liquids and glasses

    International Nuclear Information System (INIS)

    Li M Z; Li F X; Zhang H P; Peng H L; Hu Y C; Wang W H

    2017-01-01

    The structure of metallic glasses has been a long-standing mystery. Owing to the disordered nature of atomic structures in metallic glasses, it is a great challenge to find a simple structural description, such as periodicity for crystals, for establishing the structure–property relationship in amorphous materials. In this paper, we briefly review the recent developments of the five-fold local symmetry in metallic liquids and glasses and the understanding of the structure–property relationship based on this parameter. Experimental evidence demonstrates that five-fold local symmetry is found to be general in metallic liquids and glasses. Comprehensive molecular dynamics simulations show that the temperature evolution of five-fold local symmetry reflects the structural evolution in glass transition in cooling process, and the structure–property relationship such as relaxation dynamics, dynamic crossover phenomena, glass transition, and mechanical deformation in metallic liquids and glasses can be well understood base on the simple and general structure parameter of five-fold local symmetry. (paper)

  20. ProteinShop: A tool for interactive protein manipulation and steering

    Energy Technology Data Exchange (ETDEWEB)

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  1. Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function.

    Science.gov (United States)

    Liu, Xinxin; Zheng, Wei; Sivasankar, M Preeti

    2016-01-01

    Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3) expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001). Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (pacrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (pacrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium.

  2. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  3. Recovery of Vocal Fold Epithelium after Acute Phonotrauma.

    Science.gov (United States)

    Rousseau, Bernard; Kojima, Tsuyoshi; Novaleski, Carolyn K; Kimball, Emily E; Valenzuela, Carla V; Mizuta, Masanobu; Daniero, James J; Garrett, C Gaelyn; Sivasankar, M Preeti

    2017-01-01

    We investigated the timeline of tissue repair of vocal fold epithelium after acute vibration exposure using an in vivo rabbit model. Sixty-five New Zealand white breeder rabbits were randomized to 120 min of modal- or raised-intensity phonation. After the larynges were harvested at 0, 4, 8, and 24 h, and at 3 and 7 days, the vocal fold tissue was evaluated using electron microscopy and quantitative real-time polymerase chain reaction. There was an immediate decrease in the microprojection depth and height following raised-intensity phonation, paired with upregulation of cyclooxygenase-2. This initial 24-h period was also characterized by the significant downregulation of junction proteins. Interleukin 1β and transforming growth factor β1 were upregulated for 3 and 7 days, respectively, followed by an increase in epithelial cell surface depth at 3 and 7 days. These data appear to demonstrate a shift from inflammatory response to the initiation of a restorative process in the vocal fold epithelium between 24 h and 3 days. Despite the initial damage from raised-intensity phonation, the vocal fold epithelium demonstrates a remarkable capacity for the expeditious recovery of structural changes from transient episodes of acute phonotrauma. While structurally intact, the return of functional barrier integrity may be delayed by repeated episodes of phonotrauma and may also play an important role in the pathophysiology of vocal fold lesions. © 2017 S. Karger AG, Basel.

  4. Heavy metal ions are potent inhibitors of protein folding

    International Nuclear Information System (INIS)

    Sharma, Sandeep K.; Goloubinoff, Pierre; Christen, Philipp

    2008-01-01

    Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd 2+ , Hg 2+ and Pb 2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC 50 in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far

  5. Single-molecule studies of the Im7 folding landscape.

    Science.gov (United States)

    Pugh, Sara D; Gell, Christopher; Smith, D Alastair; Radford, Sheena E; Brockwell, David J

    2010-04-23

    Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. (c) 2010 Elsevier Ltd. All rights reserved.

  6. The influence of thyroarytenoid and cricothyroid muscle activation on vocal fold stiffness and eigenfrequencies

    Science.gov (United States)

    Yin, Jun; Zhang, Zhaoyan

    2013-01-01

    The influence of the thyroarytenoid (TA) and cricothyroid (CT) muscle activation on vocal fold stiffness and eigenfrequencies was investigated in a muscularly controlled continuum model of the vocal folds. Unlike the general understanding that vocal fold fundamental frequency was determined by vocal fold tension, this study showed that vocal fold eigenfrequencies were primarily determined by vocal fold stiffness. This study further showed that, with reference to the resting state of zero strain, vocal fold stiffness in both body and cover layers increased with either vocal fold elongation or shortening. As a result, whether vocal fold eigenfrequencies increased or decreased with CT/TA activation depended on how the CT/TA interaction influenced vocal fold deformation. For conditions of strong CT activation and thus an elongated vocal fold, increasing TA contraction reduced the degree of vocal fold elongation and thus reduced vocal fold eigenfrequencies. For conditions of no CT activation and thus a resting or slightly shortened vocal fold, increasing TA contraction increased the degree of vocal fold shortening and thus increased vocal fold eigenfrequencies. In the transition region of a slightly elongated vocal fold, increasing TA contraction first decreased and then increased vocal fold eigenfrequencies. PMID:23654401

  7. Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A; Boudko, Sergei P; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R; Rashmir-Raven, Ann M; Nagata, Kazuhiro; Winand, Nena J; Bächinger, Hans Peter

    2012-06-22

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

  8. Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

    2012-01-01

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

  9. Vocal fold contact patterns based on normal modes of vibration.

    Science.gov (United States)

    Smith, Simeon L; Titze, Ingo R

    2018-05-17

    The fluid-structure interaction and energy transfer from respiratory airflow to self-sustained vocal fold oscillation continues to be a topic of interest in vocal fold research. Vocal fold vibration is driven by pressures on the vocal fold surface, which are determined by the shape of the glottis and the contact between vocal folds. Characterization of three-dimensional glottal shapes and contact patterns can lead to increased understanding of normal and abnormal physiology of the voice, as well as to development of improved vocal fold models, but a large inventory of shapes has not been directly studied previously. This study aimed to take an initial step toward characterizing vocal fold contact patterns systematically. Vocal fold motion and contact was modeled based on normal mode vibration, as it has been shown that vocal fold vibration can be almost entirely described by only the few lowest order vibrational modes. Symmetric and asymmetric combinations of the four lowest normal modes of vibration were superimposed on left and right vocal fold medial surfaces, for each of three prephonatory glottal configurations, according to a surface wave approach. Contact patterns were generated from the interaction of modal shapes at 16 normalized phases during the vibratory cycle. Eight major contact patterns were identified and characterized by the shape of the flow channel, with the following descriptors assigned: convergent, divergent, convergent-divergent, uniform, split, merged, island, and multichannel. Each of the contact patterns and its variation are described, and future work and applications are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin.

    Science.gov (United States)

    Kumar, Raj; Kukreja, Roshan V; Cai, Shuowei; Singh, Bal R

    2014-06-01

    Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37°C. LCE, on the other hand, was although optimally active at 37°C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Interplay between chaperones and protein disorder promotes the evolution of protein networks.

    Directory of Open Access Journals (Sweden)

    Sebastian Pechmann

    2014-06-01

    Full Text Available Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the

  12. Free energy landscape and multiple folding pathways of an H-type RNA pseudoknot.

    Directory of Open Access Journals (Sweden)

    Yunqiang Bian

    Full Text Available How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA.

  13. Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.

    Science.gov (United States)

    Wang, Ning; Seko, Akira; Takeda, Yoichi; Kikuma, Takashi; Ito, Yukishige

    2015-10-01

    Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. First passage analysis of the folding of a β-sheet miniprotein: is it more realistic than the standard equilibrium approach?

    Science.gov (United States)

    Kalgin, Igor V; Chekmarev, Sergei F; Karplus, Martin

    2014-04-24

    Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed "streamlines", the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations.

  15. First Passage Analysis of the Folding of a β-Sheet Miniprotein: Is it More Realistic Than the Standard Equilibrium Approach?

    Science.gov (United States)

    2015-01-01

    Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed ”streamlines”, the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations. PMID:24669953

  16. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

    International Nuclear Information System (INIS)

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C. K.

    2010-01-01

    The crystal structure of phenolic acid decarboxylase from B. pumilus strain UI-670 has been determined and refined at 1.69 Å resolution. The enzyme is a dimer, with each subunit adopting a β-barrel structure belonging to the lipocalin fold. The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site

  17. Peroxisome protein import: a complex journey.

    Science.gov (United States)

    Baker, Alison; Lanyon-Hogg, Thomas; Warriner, Stuart L

    2016-06-15

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. © 2016 The Author(s).

  18. Rapid fold and structure determination of the archaeal translation elongation factor 1{beta} from Methanobacterium thermoautotrophicum

    Energy Technology Data Exchange (ETDEWEB)

    Kozlov, Guennadi [McGill University, Department of Biochemistry (Canada); Ekiel, Irena [National Research Council of Canada, Biomolecular NMR Group, Sector of Pharmaceutical Biotechnology, Biotechnology Research Institute (Canada); Beglova, Natalia [McGill University, Department of Biochemistry (Canada); Yee, Adelinda; Dharamsi, Akil; Engel, Asaph [University of Toronto, Department of Medical Biophysics (Canada); Siddiqui, Nadeem; Nong, Andrew; Gehring, Kalle [McGill University, Department of Biochemistry (Canada)

    2000-07-15

    The tertiary fold of the elongation factor, aEF-1{beta}, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1{beta} was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1{beta} structure revealed close similarity to its human analogue, eEF-1{beta}. In agreement with studies on EF-Ts and human EF-1{beta}, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1{alpha}. aEF-1{beta} was also found to bind calcium in the groove between helix {alpha}2 and strand {beta}4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.

  19. Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    Kozlov, Guennadi; Ekiel, Irena; Beglova, Natalia; Yee, Adelinda; Dharamsi, Akil; Engel, Asaph; Siddiqui, Nadeem; Nong, Andrew; Gehring, Kalle

    2000-01-01

    The tertiary fold of the elongation factor, aEF-1β, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1β was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1β structure revealed close similarity to its human analogue, eEF-1β. In agreement with studies on EF-Ts and human EF-1β, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1α. aEF-1β was also found to bind calcium in the groove between helix α2 and strand β4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation

  20. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

    2013-01-01

    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...... to score the likelihood of the interaction between two proteins and to develop a method for the prediction of PPIs. We have tested our method on several sets with unbalanced ratios of interactions and non-interactions to simulate real conditions, obtaining accuracies higher than 25% in the most unfavorable...

  1. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    Energy Technology Data Exchange (ETDEWEB)

    Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

    2013-04-15

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  2. Positive therapy of andrographolide in vocal fold leukoplakia.

    Science.gov (United States)

    Xu, Jue; Xue, Tao; Bao, Ying; Wang, Dong-Hai; Ma, Bing-Liang; Yin, Chen-Yi; Yang, Guang-Hui; Ren, Gang; Lan, Long-Jiang; Wang, Jian-Qiu; Zhang, Xiao-Lan; Zhao, Yu-Qin

    2014-01-01

    Vocal fold leukoplakia is a premalignant precursor of squamous cell carcinoma. Although many efforts have been contributed to therapy of this disease, none exhibits a satisfactory result. The aims of this study were to investigate the effectiveness and feasibility of andrographolide therapy in vocal fold leukoplakia and to explore the preliminary mechanism underlying. Forty-one eligible patients were enrolled in the study. The patients were treated for 10-minute exposures of 5 ml (25mg/ml) andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Electronic laryngoscope was used to observe the condition of vocal fold leukoplakia during the treatment. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months. Toxic reactions of treatments were evaluated on the basis of the standards of the United States MD Anderson Cancer Center. Moreover, laryngeal carcinoma cell line Hep2 was applied to explore the mechanism of effect of andrographolide. Anti-proliferative effect on Hep2, cell nuclear morphology, express of mitogen-activated protein kinases (MAPK) and pro-apoptotic protein were detected after andrographolide treatment. We found that andrographolide exhibited significant curative effects on treatments, which were accompanied by thinning of the lesion of leukoplakia, reduction in the whitish surface area, and return of pink or red epithelium. A complete response up to 85% was observed, and no toxic side effect events occurred during the study. No patient with a complete response had a recurrence in the follow-up. Moreover, cellular experiments in Hep2 indicated that andrographolide activated MAPK pathway and caspase cascade, and finally induced apoptosis in laryngeal carcinoma cell. The advantages of andrographolide are connected with minimally invasive and localized character of the treatment and no damage of collagenous tissue structures, which are more convenient and less painful

  3. Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange: THERMODYNAMIC STABILITY CORRELATES WITH RATES OF UNFOLDING RATHER THAN FOLDING.

    Science.gov (United States)

    Shah, Riddhi; Ohashi, Tomoo; Erickson, Harold P; Oas, Terrence G

    2017-01-20

    Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s -1 , for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Casein and soya-bean protein have different effects on whole body protein turnover at the same nitrogen balance

    DEFF Research Database (Denmark)

    Nielsen, K; Kondrup, J; Elsner, Petteri

    1994-01-01

    , or hydrolysed soya-bean protein at a level of 9.1 g/kg BW per d. The diets, which were isoenergetic with the same carbohydrate: fat ratio, were given as a continuous intragastric infusion for at least 4 d. During the last 19 h 15N-glycine (a primed continuous infusion) was given intragastrically and 15N...... synthesis. The protein diets produced a positive N balance which was independent of the protein source. Intact and hydrolysed casein increased protein synthesis 2.6- and 2.0-fold respectively, compared with the protein-free diet. Protein degradation increased 1.4- and 1.2-fold respectively. Hydrolysed soya-bean...... protein did not increase protein synthesis but decreased protein degradation by 35% compared with the protein-free diet. Compared with the hydrolysed soya-bean protein, intact casein resulted in 2.2- and 2.8-fold higher rates of protein synthesis and degradation respectively. These results are not easily...

  5. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

    Directory of Open Access Journals (Sweden)

    Sinjan Choudhary

    Full Text Available We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  6. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

    Science.gov (United States)

    Choudhary, Sinjan; Save, Shreyada N; Kishore, Nand; Hosur, Ramakrishna V

    2016-01-01

    We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  7. Process of tight junction recovery in the injured vocal fold epithelium: Morphological and paracellular permeability analysis.

    Science.gov (United States)

    Suzuki, Ryo; Katsuno, Tatsuya; Kishimoto, Yo; Nakamura, Ryosuke; Mizuta, Masanobu; Suehiro, Atsushi; Yamashita, Masaru; Nakamura, Tatsuo; Tateya, Ichiro; Omori, Koichi

    2018-04-01

    The vocal fold epithelium that includes tight junction (TJ)-based barrier function protects underlying connective tissues from external insults. TJs play an important role to control paracellular permeability of not only solutes but also ions, and preserve the vocal fold homeostasis. However, the distribution of TJs and paracellular diffusion barrier across the entire vocal fold epithelium are still unknown. The aim of this study was to identify the distribution of TJs in the vocal fold epithelium and to characterize the recovery process of TJ-based paracellular diffusion barrier in a rat model of vocal fold injury. Animal experiments with controls. Normal and vocal fold-injured rats were used. Larynges were harvested for immunohistochemical examination of TJ proteins. For functional analysis, a tracer permeability assay was performed using EZ-Link Sulfo-NHS-LC-Biotin. TJ proteins occludin and zonula occludens 1 signals were localized to the junctional regions of the most luminal cell layers of the vocal fold epithelium. The injured region had been recovered with epithelium at 5 days postinjury, but the paracellular diffusion barrier assays revealed that biotinylation reagents diffused into the lamina propria at 5 days postinjury, and were blocked at the epithelium at 14 and 28 days postinjury. It was strongly suggested that TJs in the vocal fold epithelium exist at the junctional regions of the first layer of stratified squamous epithelium. TJ-based paracellular diffusion barrier following vocal fold injury is recovered by 14 days postinjury, and this period corresponds with the time course of structural changes in the regenerating epithelium layer. NA. Laryngoscope, 128:E150-E156, 2018. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  8. From Sequence and Forces to Structure, Function and Evolution of Intrinsically Disordered Proteins

    Science.gov (United States)

    Forman-Kay, Julie D.; Mittag, Tanja

    2015-01-01

    Intrinsically disordered proteins (IDPs), which lack persistent structure, are a challenge to structural biology due to the inapplicability of standard methods for characterization of folded proteins as well as their deviation from the dominant structure/function paradigm. Their widespread presence and involvement in biological function, however, has spurred the growing acceptance of the importance of IDPs and the development of new tools for studying their structure, dynamics and function. The interplay of folded and disordered domains or regions for function and the existence of a continuum of protein states with respect to conformational energetics, motional timescales and compactness is shaping a unified understanding of structure-dynamics-disorder/function relationships. On the 20th anniversary of this journal, Structure, we provide a historical perspective on the investigation of IDPs and summarize the sequence features and physical forces that underlie their unique structural, functional and evolutionary properties. PMID:24010708

  9. How Four Scientists Integrate Thermodynamic and Kinetic Theory, Context, Analogies, and Methods in Protein-Folding and Dynamics Research: Implications for Biochemistry Instruction.

    Science.gov (United States)

    Jeffery, Kathleen A; Pelaez, Nancy; Anderson, Trevor R

    2018-01-01

    To keep biochemistry instruction current and relevant, it is crucial to expose students to cutting-edge scientific research and how experts reason about processes governed by thermodynamics and kinetics such as protein folding and dynamics. This study focuses on how experts explain their research into this topic with the intention of informing instruction. Previous research has modeled how expert biologists incorporate research methods, social or biological context, and analogies when they talk about their research on mechanisms. We used this model as a guiding framework to collect and analyze interview data from four experts. The similarities and differences that emerged from analysis indicate that all experts integrated theoretical knowledge with their research context, methods, and analogies when they explained how phenomena operate, in particular by mapping phenomena to mathematical models; they explored different processes depending on their explanatory aims, but readily transitioned between different perspectives and explanatory models; and they explained thermodynamic and kinetic concepts of relevance to protein folding in different ways that aligned with their particular research methods. We discuss how these findings have important implications for teaching and future educational research. © 2018 K. A. Jeffery et al. CBE—Life Sciences Education © 2018 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. Phi-value analysis of apo-azurin folding: comparison between experiment and theory.

    Science.gov (United States)

    Zong, Chenghang; Wilson, Corey J; Shen, Tongye; Wolynes, Peter G; Wittung-Stafshede, Pernilla

    2006-05-23

    Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.

  11. Scale-free behaviour of amino acid pair interactions in folded proteins

    DEFF Research Database (Denmark)

    Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Mortensen, Rasmus J.

    2012-01-01

    The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the ‘‘protein structure code’’ has not been fully identified. Our manuscript...... presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence...... which amino acid paired residues contributed to the cells with a population above 50, pairs of Ala, Ile, Leu and Val dominate the results. This result is statistically highly significant. We postulate that such pairs form ‘‘structural stability points’’ in the protein structure. Our data shows...

  12. Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study.

    Science.gov (United States)

    Strickland, Mallory L; Boylan, Helen M

    2010-07-01

    The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.

  13. Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

    Directory of Open Access Journals (Sweden)

    Angel L. Pey

    2013-01-01

    Full Text Available Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis.

  14. Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul [Department of Physics, University at Buffalo, Buffalo, New York 14260 (United States)

    2015-01-21

    The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.

  15. Sequence-based prediction of protein protein interaction using a deep-learning algorithm.

    Science.gov (United States)

    Sun, Tanlin; Zhou, Bo; Lai, Luhua; Pei, Jianfeng

    2017-05-25

    Protein-protein interactions (PPIs) are critical for many biological processes. It is therefore important to develop accurate high-throughput methods for identifying PPI to better understand protein function, disease occurrence, and therapy design. Though various computational methods for predicting PPI have been developed, their robustness for prediction with external datasets is unknown. Deep-learning algorithms have achieved successful results in diverse areas, but their effectiveness for PPI prediction has not been tested. We used a stacked autoencoder, a type of deep-learning algorithm, to study the sequence-based PPI prediction. The best model achieved an average accuracy of 97.19% with 10-fold cross-validation. The prediction accuracies for various external datasets ranged from 87.99% to 99.21%, which are superior to those achieved with previous methods. To our knowledge, this research is the first to apply a deep-learning algorithm to sequence-based PPI prediction, and the results demonstrate its potential in this field.

  16. Signal Transduction by a Fungal NOD-Like Receptor Based on Propagation of a Prion Amyloid Fold

    NARCIS (Netherlands)

    Daskalov, A.; Habenstein, B.; Martinez, D.; Debets, A.J.M.; Sabate, R.; Loquet, A.; Saupe, S.J.

    2015-01-01

    In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s ß-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S

  17. The structure of KPN03535 (gi|152972051), a novel putative lipoprotein from Klebsiella pneumoniae, reveals an OB-fold

    International Nuclear Information System (INIS)

    Das, Debanu; Kozbial, Piotr; Han, Gye Won; Carlton, Dennis; Jaroszewski, Lukasz; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Elsliger, Marc-André; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grzechnik, Anna; Grant, Joanna C.; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    KPN03535 is a protein unique to K. pneumoniae. The crystal structure reveals that KPN03535 represents a novel variant of the OB-fold and is likely to be a DNA-binding lipoprotein. KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family

  18. Unusual armadillo fold in the human general vesicular transport factor p115.

    Directory of Open Access Journals (Sweden)

    Harald Striegl

    Full Text Available The golgin family gives identity and structure to the Golgi apparatus and is part of a complex protein network at the Golgi membrane. The golgin p115 is targeted by the GTPase Rab1a, contains a large globular head region and a long region of coiled-coil which forms an extended rod-like structure. p115 serves as vesicle tethering factor and plays an important role at different steps of vesicular transport. Here we present the 2.2 A-resolution X-ray structure of the globular head region of p115. The structure exhibits an armadillo fold that is decorated by elongated loops and carries a C-terminal non-canonical repeat. This terminal repeat folds into the armadillo superhelical groove and allows homodimeric association with important implications for p115 mediated multiple protein interactions and tethering.

  19. Impaired folding and subunit assembly as disease mechanism

    DEFF Research Database (Denmark)

    Bross, P; Andresen, B S; Gregersen, N

    1998-01-01

    folding is a common effect of missense mutations occurring in genetic diseases, (ii) increasing the level of available chaperones may augment the level of functional mutant protein in vivo, and (iii) one mutation may have multiple effects. The interplay between the chaperones assisting folding......Rapid progress in DNA technology has entailed the possibility of readily detecting mutations in disease genes. In contrast to this, techniques to characterize the effects of mutations are still very time consuming. It has turned out that many of the mutations detected in disease genes are missense...... mutations. Characterization of the effect of these mutations is particularly important in order to establish that they are disease causing and to estimate their severity. We use the experiences with investigation of medium-chain acyl-CoA dehydrogenase deficiency as an example to illustrate that (i) impaired...

  20. Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

    Science.gov (United States)

    Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

    2017-05-15

    Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.