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Sample records for understanding protein folding

  1. MODELS OF PROTEIN FOLDING

    Directory of Open Access Journals (Sweden)

    Unnati Ahluwalia

    2012-12-01

    Full Text Available In an attempt to explore the understanding of protein folding mechanism, various models have been proposed in the literature. Advances in recent experimental and computational techniques rationalized our understanding on some of the fundamental features of the protein folding pathways. The goal of this review is to revisit the various models and outline the essential aspects of the folding reaction.

  2. Unified understanding of folding and binding mechanisms of globular and intrinsically disordered proteins.

    Science.gov (United States)

    Arai, Munehito

    2018-01-06

    Extensive experimental and theoretical studies have advanced our understanding of the mechanisms of folding and binding of globular proteins, and coupled folding and binding of intrinsically disordered proteins (IDPs). The forces responsible for conformational changes and binding are common in both proteins; however, these mechanisms have been separately discussed. Here, we attempt to integrate the mechanisms of coupled folding and binding of IDPs, folding of small and multi-subdomain proteins, folding of multimeric proteins, and ligand binding of globular proteins in terms of conformational selection and induced-fit mechanisms as well as the nucleation-condensation mechanism that is intermediate between them. Accumulating evidence has shown that both the rate of conformational change and apparent rate of binding between interacting elements can determine reaction mechanisms. Coupled folding and binding of IDPs occurs mainly by induced-fit because of the slow folding in the free form, while ligand binding of globular proteins occurs mainly by conformational selection because of rapid conformational change. Protein folding can be regarded as the binding of intramolecular segments accompanied by secondary structure formation. Multi-subdomain proteins fold mainly by the induced-fit (hydrophobic collapse) mechanism, as the connection of interacting segments enhances the binding (compaction) rate. Fewer hydrophobic residues in small proteins reduce the intramolecular binding rate, resulting in the nucleation-condensation mechanism. Thus, the folding and binding of globular proteins and IDPs obey the same general principle, suggesting that the coarse-grained, statistical mechanical model of protein folding is promising for a unified theoretical description of all mechanisms.

  3. 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...

  4. Protein folding: understanding the role of water and the low Reynolds number environment as the peptide chain emerges from the ribosome and folds.

    Science.gov (United States)

    Sen, Siddhartha; Voorheis, H Paul

    2014-12-21

    The mechanism of protein folding during early stages of the process has three determinants. First, moving water molecules obey the rules of low Reynolds number physics without an inertial component. Molecular movement is instantaneous and size insensitive. Proteins emerging from the ribosome move and rotate without an external force if they change shape, forming and propagating helical structures that increases translocational efficiency. Forward motion ceases when the shape change or propelling force ceases. Second, application of quantum field theory to water structure predicts the spontaneous formation of low density coherent units of fixed size that expel dissolved atmospheric gases. Structured water layers with both coherent and non-coherent domains, form a sheath around the new protein. The surface of exposed hydrophobic amino acids is protected from water contact by small nanobubbles of dissolved atmospheric gases, 5 or 6 molecules on average, that vibrate, attracting even widely separated resonating nanobubbles. This force results from quantum effects, appearing only when the system is within and interacts with an oscillating electromagnetic field. The newly recognized quantum force sharply bends the peptide and is part of a dynamic field determining the pathway of protein folding. Third, the force initiating the tertiary folding of proteins arises from twists at the position of each hydrophobic amino acid, that minimizes surface exposure of the hydrophobic amino acids and propagates along the protein. When the total bend reaches 360°, the leading segment of water sheath intersects the trailing segment. This steric self-intersection expels water from overlapping segments of the sheath and by Newton׳s second law moves the polypeptide chain in an opposite direction. Consequently, with very few exceptions that we enumerate and discuss, tertiary structures are absent from proteins without hydrophobic amino acids, which control the early stages of protein

  5. Evolutionary optimization of protein folding.

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    Cédric Debès

    Full Text Available Nature has shaped the make up of proteins since their appearance, [Formula: see text]3.8 billion years ago. However, the fundamental drivers of structural change responsible for the extraordinary diversity of proteins have yet to be elucidated. Here we explore if protein evolution affects folding speed. We estimated folding times for the present-day catalog of protein domains directly from their size-modified contact order. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of protein domains in 989 fully-sequenced genomes. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase during the last [Formula: see text]1.5 billion years that began during the "big bang" of domain combinations. As a consequence, these domain structures are on average slow folders today. Our results suggest that fast and efficient folding of domains shaped the universe of protein structure. This finding supports the hypothesis that optimization of the kinetic and thermodynamic accessibility of the native fold reduces protein aggregation propensities that hamper cellular functions.

  6. 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...

  7. 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.

  8. 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 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...... of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode....

  9. 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...

  10. 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.

  11. 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

  12. Protein-Folding Landscapes in Multi-Chain Systems

    Energy Technology Data Exchange (ETDEWEB)

    Cellmer, Troy; Bratko, Dusan; Prausnitz, John M.; Blanch, Harvey

    2005-06-20

    Computational studies of proteins have significantly improved our understanding of protein folding. These studies are normally carried out using chains in isolation. However, in many systems of practical interest, proteins fold in the presence of other molecules. To obtain insight into folding in such situations, we compare the thermodynamics of folding for a Miyazawa-Jernigan model 64-mer in isolation to results obtained in the presence of additional chains. The melting temperature falls as the chain concentration increases. In multi-chain systems, free-energy landscapes for folding show an increased preference for misfolded states. Misfolding is accompanied by an increase in inter-protein interactions; however, near the folding temperature, the transition from folded chains to misfolded and associated chains isentropically driven. A majority of the most probable inter-protein contacts are also native contacts, suggesting that native topology plays a role in early stages of aggregation.

  13. Accelerated molecular dynamics simulations of protein folding.

    Science.gov (United States)

    Miao, Yinglong; Feixas, Ferran; Eun, Changsun; McCammon, J Andrew

    2015-07-30

    Folding of four fast-folding proteins, including chignolin, Trp-cage, villin headpiece and WW domain, was simulated via accelerated molecular dynamics (aMD). In comparison with hundred-of-microsecond timescale conventional molecular dynamics (cMD) simulations performed on the Anton supercomputer, aMD captured complete folding of the four proteins in significantly shorter simulation time. The folded protein conformations were found within 0.2-2.1 Å of the native NMR or X-ray crystal structures. Free energy profiles calculated through improved reweighting of the aMD simulations using cumulant expansion to the second-order are in good agreement with those obtained from cMD simulations. This allows us to identify distinct conformational states (e.g., unfolded and intermediate) other than the native structure and the protein folding energy barriers. Detailed analysis of protein secondary structures and local key residue interactions provided important insights into the protein folding pathways. Furthermore, the selections of force fields and aMD simulation parameters are discussed in detail. Our work shows usefulness and accuracy of aMD in studying protein folding, providing basic references in using aMD in future protein-folding studies. © 2015 Wiley Periodicals, Inc.

  14. 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)

  15. Analyzing the effect of homogeneous frustration in protein folding.

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    Contessoto, Vinícius G; Lima, Debora T; Oliveira, Ronaldo J; Bruni, Aline T; Chahine, Jorge; Leite, Vitor B P

    2013-10-01

    The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. Copyright © 2013 Wiley Periodicals, Inc.

  16. 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.

  17. Thermostable exoshells fold and stabilize recombinant proteins.

    Science.gov (United States)

    Deshpande, Siddharth; Masurkar, Nihar D; Girish, Vallerinteavide Mavelli; Desai, Malan; Chakraborty, Goutam; Chan, Juliana M; Drum, Chester L

    2017-11-13

    The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

  18. Restrictions to protein folding determined by the protein size.

    Science.gov (United States)

    Finkelstein, Alexei V; Bogatyreva, Natalya S; Garbuzynskiy, Sergiy O

    2013-06-27

    Experimentally measured rates of spontaneous folding of single-domain globular proteins range from microseconds to hours: the difference (11 orders of magnitude!) is akin to the difference between the life span of a mosquito and the age of the Universe. We show that physical theory with biological constraints outlines the possible range of folding rates for single-domain globular proteins of various size and stability, and that the experimentally measured folding rates fall within this narrow "golden triangle" built without any adjustable parameters, filling it almost completely. This "golden triangle" also successfully predicts the maximal allowed size of the "foldable" protein domains, as well as the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control. In conclusion, we give a phenomenological formula for dependence of the folding rate on the size, shape and stability of the protein fold. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. 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.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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......The mechanism by which proteins fold to their native states has been the focus of intense research in recent years. The rate-limiting event in the folding reaction is the formation of a conformation in a set known as the transition-state ensemble. The structural features present within...

  1. 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.

  2. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    Proteins are important biomolecules, which perform diverse structural and functional roles in living systems. Starting from a linear chain of amino acids, proteins fold to different secondary structures, which then fold through short- and long-range interactions to give rise to the final three-dimensional shapes useful to carry out ...

  3. 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...... magic number of secondary structures. Thermodynamic arguments for the increased abundance and a phase diagram for the folding scenario are given. This includes an intermediate high symmetry phase, the parent structures, between the molten globule and the native states. We have made an exhaustive...

  4. 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.

  5. Laser induced temperature jump investigations of fast protein folding dynamics

    Science.gov (United States)

    Qiu, Linlin

    Protein folding has a large parameter space, diverse mechanism, and multipath kinetics. However, there are some common features many proteins share in their folding processes: all seem to fold at the rates much faster than the random conformation search, and all fold into the structures which have the highly regular motifs like alpha-helices, beta-sheets and turns. Understanding how fast proteins can fold is one of the central issues in solving the protein folding problem. Ultrafast folding kinetics had not been accessible until a few sub-millisecond probes were invented and applied lately. We constructed a laser induced temperature jump spectrometer which is a great utility in identifying the local structure and tertiary contact formation of proteins on the time scale from 10 -8 to 10-3 s with time resolution of 10 -9 s. With this spectrometer we studied the fast folding mini-protein, TrpCage and a few short stable beta-hairpins, the TrpZip series. Studying TrpCage was a major breakthrough it was a pioneer protein model which brought experiment and simulation very close: its structures measured by NMR and predicted by the molecular dynamics were amazingly alike. Our kinetic results showed that it folds in 4 mus at room temperature which turned out to be the fastest ever known for protein-like molecules. Also this folding time constant is consistent with what was later on simulated by distributed computation. TrpZips are among the smallest and stablest polypeptide chains which form secondary structures. They are slightly different from each other based on structural stability and by forming various types of beta-hairpins which are the minimum units of beta tertiary structure. The beta-hairpins form in the time range of 1--10 mus that confirms the theory that loop formation is controlled by the diffusion process (˜mus). We also investigated the kinetics of the protein chain collapse, a very controversial problem. By comparing the collapse of the foldable 104

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Understanding the folding process of synthetic polymers by small ...

    Indian Academy of Sciences (India)

    WINTEC

    Understanding the folding process of synthetic polymers by small-molecule folding agents. S G RAMKUMAR and S RAMAKRISHNAN*. Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012 e-mail: raman@ipc.iisc.ernet.in. Abstract. Two acceptor containing polyimides PDI and NDI ...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    such ensembles have now been analysed for a series of proteins using data from a combination of biochemical and biophysical experiments together with computer-simulation methods. These studies show that the topology of the transition state is determined by a set of interactions involving a small number of key...... 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...

  13. Hydrophobic-hydrophilic forces in protein folding.

    Science.gov (United States)

    Durell, Stewart R; Ben-Naim, Arieh

    2017-08-01

    The process of protein folding is obviously driven by forces exerted on the atoms of the amino-acid chain. These forces arise from interactions with other parts of the protein itself (direct forces), as well as from interactions with the solvent (solvent-induced forces). We present a statistical-mechanical formalism that describes both these direct and indirect, solvent-induced thermodynamic forces on groups of the protein. We focus on 2 kinds of protein groups, commonly referred to as hydrophobic and hydrophilic. Analysis of this result leads to the conclusion that the forces on hydrophilic groups are in general stronger than on hydrophobic groups. This is then tested and verified by a series of molecular dynamics simulations, examining both hydrophobic alkanes of different sizes and hydrophilic moieties represented by polar-neutral hydroxyl groups. The magnitude of the force on assemblies of hydrophilic groups is dependent on their relative orientation: with 2 to 4 times larger forces on groups that are able to form one or more direct hydrogen bonds. © 2017 Wiley Periodicals, Inc.

  14. Probing folding free energy landscape of small proteins through ...

    Indian Academy of Sciences (India)

    Unknown

    lattice and off-lattice models of proteins have been used to study the statistical and dynamical aspects of folding.12,13 Levitt pioneered in the computational studies of protein folding using off-lattice protein models.14 A recent off-lattice model study of HP-36 based on hydrophobicity tried to correlate the folding with many ...

  15. Novel Protein Folding Pathways for Protein Salvage and Recycling

    Science.gov (United States)

    2013-08-26

    Chaperones are protein complexes that facilitate protein folding in both eukaryotes and prokaryotes . From a study of the C-terminal domains of...Igor Lednev, CoPI University of New York at Albany Summary Proteostasis is the term used to describe the combined mechanisms by which cells ...insulin filaments have no cellular toxicity, whereas mature fibrils are toxic to pheochromocytoma (PC 12) cells (10). An MTT assay was used to assess

  16. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    range in- teractions to give rise to the final three-dimensional ... data. As defined by SCOP, there exist several hierarchies. The principal levels are family, superfamily, fold and class. According to SCOP, proteins clustered together into families are ...

  17. Understanding the folding process of synthetic polymers by small ...

    Indian Academy of Sciences (India)

    WINTEC

    *For correspondence. Understanding the folding process of synthetic polymers by ... Conformational control in biological macromole- cules depends largely ... context of sensors. 11–13 and more recently with regard to foldamers. 14–17. In these systems, the com- plexation of the OE segment by a metal-ion leads to either a ...

  18. 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.

  19. 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.

  20. Real-time protein NMR spectroscopy and investigation of assisted protein folding.

    Science.gov (United States)

    Kumar, Amit; Balbach, Jochen

    2015-10-01

    During protein-folding reactions toward the native structure, short-lived intermediate states can be populated. Such intermediates expose hydrophobic patches and can self-associate leading to non-productive protein misfolding. A major focus of current research is the characterization of short-lived intermediates and how molecular chaperones enable productive folding. Real-time NMR spectroscopy, together with the development of advanced methods, is reviewed here and the potential these methods have to characterize intermediate states as well as interactions with molecular chaperone proteins at single-residue resolution is highlighted. Various chaperone interactions can guide the protein-folding reaction and thus are important for protein structure formation, stability, and activity of their substrates. Chaperone-assisted protein folding, characterization of intermediates, and their molecular interactions using real-time NMR spectroscopy will be discussed. Additionally, recent advances in NMR methods employed for characterization of high-energy intermediates will be discussed. Real-time NMR combines high resolution with kinetic information of protein reactions, which can be employed not only for protein-folding studies and the characterization of folding intermediates but also to investigate the molecular mechanisms of assisted protein folding. Real-time NMR spectroscopy remains an effective tool to reveal structural details about the interaction between chaperones and transient intermediates. Methodologically, it provides in-depth understanding of how kinetic intermediates and their thermodynamics contribute to the protein-folding reaction. This review summarizes the most recent advances in this field. This article is part of a Special Issue titled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Theoretical and computational studies in protein folding, design, and function

    Science.gov (United States)

    Morrissey, Michael Patrick

    2000-10-01

    In this work, simplified statistical models are used to understand an array of processes related to protein folding and design. In Part I, lattice models are utilized to test several theories about the statistical properties of protein-like systems. In Part II, sequence analysis and all-atom simulations are used to advance a novel theory for the behavior of a particular protein. Part I is divided into five chapters. In Chapter 2, a method of sequence design for model proteins, based on statistical mechanical first-principles, is developed. The cumulant design method uses a mean-field approximation to expand the free energy of a sequence in temperature. The method successfully designs sequences which fold to a target lattice structure at a specific temperature, a feat which was not possible using previous design methods. The next three chapters are computational studies of the double mutant cycle, which has been used experimentally to predict intra-protein interactions. Complete structure prediction is demonstrated for a model system using exhaustive, and also sub-exhaustive, double mutants. Nonadditivity of enthalpy, rather than of free energy, is proposed and demonstrated to be a superior marker for inter-residue contact. Next, a new double mutant protocol, called exchange mutation, is introduced. Although simple statistical arguments predict exchange mutation to be a more accurate contact predictor than standard mutant cycles, this hypothesis was not upheld in lattice simulations. Reasons for this inconsistency will be discussed. Finally, a multi-chain folding algorithm is introduced. Known as LINKS, this algorithm was developed to test a method of structure prediction which utilizes chain-break mutants. While structure prediction was not successful, LINKS should nevertheless be a useful tool for the study of protein-protein and protein-ligand interactions. The last chapter of Part I utilizes the lattice to explore the differences between standard folding, from

  2. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    Directory of Open Access Journals (Sweden)

    Ola B. Nilsson

    2015-09-01

    Full Text Available At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

  3. 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.

  4. Fluorescence of Alexa Fluor dye tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Borst, J.W.; Visser, A.J.W.G.; Mierlo, van C.P.M.

    2012-01-01

    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

  5. Co- and post-translational protein folding in the ER

    DEFF Research Database (Denmark)

    Ellgaard, Lars; McCaul, Nicholas; Chatsisvili, Anna

    2016-01-01

    The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic...... and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers...... to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding....

  6. A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis.

    Science.gov (United States)

    Horne, Jim E; Radford, Sheena E

    2016-06-15

    Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed. © 2016 The Author(s).

  7. 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.

  8. Folding and Biogenesis of Mitochondrial Small Tim Proteins

    Directory of Open Access Journals (Sweden)

    Efrain Ceh-Pavia

    2013-08-01

    Full Text Available Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.

  9. Neutron Protein Crystallography: Beyond the Folding Structure

    International Nuclear Information System (INIS)

    Niimura, N.

    2008-01-01

    Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, a technique complementary to ultra-high-resolution X-ray diffraction. A neutron diffractometers for biological macromolecules has been constructed in Japan, and it has been used to determine the crystal structures of proteins up to resolution limits of 1.5-2.5 A. Results relating to hydrogen positions and hydration patterns in proteins have been obtained from these studies. Examples include the geometrical details of hydrogen bonds, the role of hydrogen atoms in enzymatic activity, CH 3 configuration, H/D exchange in proteins and oligonucleotides, and the dynamical behavior of hydration structures, all of which have been extracted from these structural results and reviewed

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 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.

  12. Fluorescence of Alexa fluor dye tracks protein folding.

    Science.gov (United States)

    Lindhoud, Simon; Westphal, Adrie H; Visser, Antonie J W G; Borst, Jan Willem; van Mierlo, Carlo P M

    2012-01-01

    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.

  13. 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

  14. 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.

  15. 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

  16. The Complex Kinetics of Protein Folding in Wide Temperature Ranges

    OpenAIRE

    Wang, Jin

    2004-01-01

    The complex protein folding kinetics in wide temperature ranges is studied through diffusive dynamics on the underlying energy landscape. The well-known kinetic chevron rollover behavior is recovered from the mean first passage time, with the U-shape dependence on temperature. The fastest folding temperature T0 is found to be smaller than the folding transition temperature Tf. We found that the fluctuations of the kinetics through the distribution of first passage time show rather universal b...

  17. Molecular dynamics studies of protein folding and aggregation

    Science.gov (United States)

    Ding, Feng

    This thesis applies molecular dynamics simulations and statistical mechanics to study: (i) protein folding; and (ii) protein aggregation. Most small proteins fold into their native states via a first-order-like phase transition with a major free energy barrier between the folded and unfolded states. A set of protein conformations corresponding to the free energy barrier, Delta G >> kBT, are the folding transition state ensemble (TSE). Due to their evasive nature, TSE conformations are hard to capture (probability ∝ exp(-DeltaG/k BT)) and characterize. A coarse-grained discrete molecular dynamics model with realistic steric constraints is constructed to reproduce the experimentally observed two-state folding thermodynamics. A kinetic approach is proposed to identify the folding TSE. A specific set of contacts, common to the TSE conformations, is identified as the folding nuclei which are necessary to be formed in order for the protein to fold. Interestingly, the amino acids at the site of the identified folding nuclei are highly conserved for homologous proteins sharing the same structures. Such conservation suggests that amino acids that are important for folding kinetics are under selective pressure to be preserved during the course of molecular evolution. In addition, studies of the conformations close to the transition states uncover the importance of topology in the construction of order parameter for protein folding transition. Misfolded proteins often form insoluble aggregates, amyloid fibrils, that deposit in the extracellular space and lead to a type of disease known as amyloidosis. Due to its insoluble and non-crystalline nature, the aggregation structure and, thus the aggregation mechanism, has yet to be uncovered. Discrete molecular dynamics studies reveal an aggregate structure with the same structural signatures as in experimental observations and show a nucleation aggregation scenario. The simulations also suggest a generic aggregation mechanism

  18. Learning generative models for protein fold families.

    Science.gov (United States)

    Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G; Lee, Su-In; Langmead, Christopher James

    2011-04-01

    We introduce a new approach to learning statistical models from multiple sequence alignments (MSA) of proteins. Our method, called GREMLIN (Generative REgularized ModeLs of proteINs), learns an undirected probabilistic graphical model of the amino acid composition within the MSA. The resulting model encodes both the position-specific conservation statistics and the correlated mutation statistics between sequential and long-range pairs of residues. Existing techniques for learning graphical models from MSA either make strong, and often inappropriate assumptions about the conditional independencies within the MSA (e.g., Hidden Markov Models), or else use suboptimal algorithms to learn the parameters of the model. In contrast, GREMLIN makes no a priori assumptions about the conditional independencies within the MSA. We formulate and solve a convex optimization problem, thus guaranteeing that we find a globally optimal model at convergence. The resulting model is also generative, allowing for the design of new protein sequences that have the same statistical properties as those in the MSA. We perform a detailed analysis of covariation statistics on the extensively studied WW and PDZ domains and show that our method out-performs an existing algorithm for learning undirected probabilistic graphical models from MSA. We then apply our approach to 71 additional families from the PFAM database and demonstrate that the resulting models significantly out-perform Hidden Markov Models in terms of predictive accuracy. Copyright © 2011 Wiley-Liss, Inc.

  19. Understanding the folding process of synthetic polymers by small ...

    Indian Academy of Sciences (India)

    This two-point interaction between the folding agent and the polymer backbone leads to a folding of the polymer chain, which was readily monitored by NMR titrations. The effect of various parameters, such as structures of the folding agent and polymer, and the solvent composition, on the folding propensities of the polymer ...

  20. Metal ion coupled protein folding and allosteric motions

    Science.gov (United States)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  1. 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…

  2. Lymphotactin: How a protein can adopt two folds

    Science.gov (United States)

    Camilloni, Carlo; Sutto, Ludovico

    2009-12-01

    Metamorphic proteins such as lymphotactin are a notable exception of the empirical principle that structured natural proteins possess a unique three-dimensional structure. In particular, the human chemokine lymphotactin protein exists in two distinct conformations (one monomeric and one dimeric) under physiological conditions. In this work, we use a Cα Go¯ model to show how this very peculiar behavior can be reproduced. From the study of the thermodynamics and of the kinetics, we characterize the interconversion mechanism. In particular, this takes place through the docking of the two chains living in a third monomeric, partially unfolded, state which shows a residual structure involving a set of local contacts common to the two native conformations. The main feature of two fold proteins appears to be the sharing of a common set of local contacts between the two distinct folds as confirmed by the study of two designed two fold proteins. Metamorphic proteins may be more common than expected.

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

    DEFF Research Database (Denmark)

    Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel

    2010-01-01

    that provides such an overview. GPS NMR exploits the unique ability of NMR to simultaneously record signals from individual hydrogen atoms in complex macromolecular systems and of multivariate analysis to describe spectral variations from these by a few variables for establishment of, and positioning in......, protein-folding state maps. The method is fast, sensitive, and robust, and it works without isotope-labelling. The unique capabilities of GPS NMR to identify different folding states and to compare different unfolding processes are demonstrated by mapping of the equilibrium folding space of bovine alpha......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...

  4. Golden triangle for folding rates of globular proteins.

    Science.gov (United States)

    Garbuzynskiy, Sergiy O; Ivankov, Dmitry N; Bogatyreva, Natalya S; Finkelstein, Alexei V

    2013-01-02

    The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured rates of spontaneous folding of single-domain globular proteins range from microseconds to hours: the difference (11 orders of magnitude) is akin to the difference between the life span of a mosquito and the age of the universe. Here, we show that physical theory with biological constraints outlines a "golden triangle" limiting the possible range of folding rates for single-domain globular proteins of various size and stability, and that the experimentally measured folding rates fall within this narrow triangle built without any adjustable parameters, filling it almost completely. In addition, the golden triangle predicts the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control. It also predicts the maximal allowed size of the "foldable" protein domains, and the size of domains found in known protein structures is in a good agreement with this limit.

  5. A new tool for protein fold recognition: A Bayesian heuristic threading algorithm.

    Energy Technology Data Exchange (ETDEWEB)

    Crawford, O.

    1997-10-01

    This paper presents a new threading algorithm, designed to be used in protein fold recognition. Its purpose is to contribute toward the goal of predicting three-dimensional structures of proteins from knowledge of their amino-acid sequences alone. Sequences for new proteins are being discovered at a rapid rate, as a result of the Human Genome Project, and related genome research. Understanding of protein folding, and especially the ability to predict the 3D fold from the sequence, is crucial to the understanding of the function of these new proteins. This is considered by many to be the most important problem in contemporary molecular biology. Numerical tests of the speed and reliability of the algorithm are described, along with comparisons with two popular threading algorithms. For the systems examined, the new method constitutes a significant improvement.

  6. Criteria for folding in structure-based models of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Wołek, Karol; Cieplak, Marek, E-mail: mc@ifpan.edu.pl [Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668 Warsaw (Poland)

    2016-05-14

    In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show that the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching.

  7. 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.

  8. Cadmium impairs protein folding in the endoplasmic reticulum and induces the unfolded protein response.

    Science.gov (United States)

    Le, Quynh Giang; Ishiwata-Kimata, Yuki; Kohno, Kenji; Kimata, Yukio

    2016-08-01

    Cellular exposure to cadmium is known to strongly induce the unfolded protein response (UPR), which suggests that the endoplasmic reticulum (ER) is preferentially damaged by cadmium. According to recent reports, the UPR is induced both dependent on and independently of accumulation of unfolded proteins in the ER. In order to understand the toxic mechanism of cadmium, here we investigated how cadmium exposure leads to Ire1 activation, which triggers the UPR, using yeast Saccharomyces cerevisiae as a model organism. Cadmium poorly induced the UPR when Ire1 carried a mutation that impairs its ability to recognize unfolded proteins. Ire1 activation by cadmium was also attenuated by the chemical chaperone 4-phenylbutyrate. Cadmium caused sedimentation of BiP, the molecular chaperone in the ER, which suggests the ER accumulation of unfolded proteins. A green fluorescent protein-based reporter assay also indicated that cadmium damages the oxidative protein folding in the ER. We also found that an excess concentration of extracellular calcium attenuates the Ire1 activation by cadmium. Taken together, we propose that cadmium exposure leads to the UPR induction through impairment of protein folding in the ER. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. 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.

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

    Indian Academy of Sciences (India)

    3Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, Ballygunge Science. College, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700 019, India. [Ray S. K., Baruah V. J., Satapathy S. S. and Banerjee R. 2014 Cotranslational protein folding reveals the selective ...

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

    Indian Academy of Sciences (India)

    [Ray S. K., Baruah V. J., Satapathy S. S. and Banerjee R. 2014 Cotranslational protein folding reveals the selective use of synonymous codons along the coding sequence of a low expression gene. J. Genet. 93, 613–617]. Due to the degeneracy in the genetic code, many amino acids are encoded by more than one codon, ...

  12. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    Proteins are important biomolecules, which perform diverse structural and functional roles in living systems. Starting from a .... even be extended up to the level of protein secondary structural elements, as seen in protein topology cartoons [13]. Even though ... chemical interactions [8]. This distance map is a 2D symmetric, ...

  13. A 9-state hidden Markov model using protein secondary structure information for protein fold recognition.

    Science.gov (United States)

    Lee, Sun Young; Lee, Jong Yun; Jung, Kwang Su; Ryu, Keun Ho

    2009-06-01

    In protein fold recognition, the main disadvantage of hidden Markov models (HMMs) is the employment of large-scale model architectures which require large data sets and high computational resources for training. Also, HMMs must consider sequential information about secondary structures of proteins, to improve prediction performance and reduce model parameters. Therefore, we propose a novel method for protein fold recognition based on a hidden Markov model, called a 9-state HMM. The method can (i) reduce the number of states using secondary structure information about proteins for each fold and (ii) recognize protein folds more accurately than other HMMs.

  14. Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

    Directory of Open Access Journals (Sweden)

    Alistair G Irvine

    Full Text Available In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding. However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10(-5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding - differential affinity, rapid ligand exchange and conformational flexibility.

  15. Combining optimal control theory and molecular dynamics for protein folding.

    Science.gov (United States)

    Arkun, Yaman; Gur, Mert

    2012-01-01

    A new method to develop low-energy folding routes for proteins is presented. The novel aspect of the proposed approach is the synergistic use of optimal control theory with Molecular Dynamics (MD). In the first step of the method, optimal control theory is employed to compute the force field and the optimal folding trajectory for the Cα atoms of a Coarse-Grained (CG) protein model. The solution of this CG optimization provides an harmonic approximation of the true potential energy surface around the native state. In the next step CG optimization guides the MD simulation by specifying the optimal target positions for the Cα atoms. In turn, MD simulation provides an all-atom conformation whose Cα positions match closely the reference target positions determined by CG optimization. This is accomplished by Targeted Molecular Dynamics (TMD) which uses a bias potential or harmonic restraint in addition to the usual MD potential. Folding is a dynamical process and as such residues make different contacts during the course of folding. Therefore CG optimization has to be reinitialized and repeated over time to accomodate these important changes. At each sampled folding time, the active contacts among the residues are recalculated based on the all-atom conformation obtained from MD. Using the new set of contacts, the CG potential is updated and the CG optimal trajectory for the Cα atoms is recomputed. This is followed by MD. Implementation of this repetitive CG optimization-MD simulation cycle generates the folding trajectory. Simulations on a model protein Villin demonstrate the utility of the method. Since the method is founded on the general tools of optimal control theory and MD without any restrictions, it is widely applicable to other systems. It can be easily implemented with available MD software packages.

  16. Combining optimal control theory and molecular dynamics for protein folding.

    Directory of Open Access Journals (Sweden)

    Yaman Arkun

    Full Text Available A new method to develop low-energy folding routes for proteins is presented. The novel aspect of the proposed approach is the synergistic use of optimal control theory with Molecular Dynamics (MD. In the first step of the method, optimal control theory is employed to compute the force field and the optimal folding trajectory for the Cα atoms of a Coarse-Grained (CG protein model. The solution of this CG optimization provides an harmonic approximation of the true potential energy surface around the native state. In the next step CG optimization guides the MD simulation by specifying the optimal target positions for the Cα atoms. In turn, MD simulation provides an all-atom conformation whose Cα positions match closely the reference target positions determined by CG optimization. This is accomplished by Targeted Molecular Dynamics (TMD which uses a bias potential or harmonic restraint in addition to the usual MD potential. Folding is a dynamical process and as such residues make different contacts during the course of folding. Therefore CG optimization has to be reinitialized and repeated over time to accomodate these important changes. At each sampled folding time, the active contacts among the residues are recalculated based on the all-atom conformation obtained from MD. Using the new set of contacts, the CG potential is updated and the CG optimal trajectory for the Cα atoms is recomputed. This is followed by MD. Implementation of this repetitive CG optimization-MD simulation cycle generates the folding trajectory. Simulations on a model protein Villin demonstrate the utility of the method. Since the method is founded on the general tools of optimal control theory and MD without any restrictions, it is widely applicable to other systems. It can be easily implemented with available MD software packages.

  17. 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

  18. Twin-arginine-dependent translocation of folded proteins

    Science.gov (United States)

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-01-01

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF. PMID:22411976

  19. Hydrophobicity – Shake Flasks, Protein Folding and Drug Discovery

    Science.gov (United States)

    Sarkar, Aurijit; Kellogg, Glen E.

    2009-01-01

    Hydrophobic interactions are some of the most important interactions in nature. They are the primary driving force in a number of phenomena. This is mostly an entropic effect and can account for a number of biophysical events such as protein-protein or protein-ligand binding that are of immense importance in drug design. The earliest studies on this phenomenon can be dated back to the end of the 19th century when Meyer and Overton independently correlated the hydrophobic nature of gases to their anesthetic potency. Since then, significant progress has been made in this realm of science. This review briefly traces the history of hydrophobicity research along with the theoretical estimation of partition coefficients. Finally, the application of hydrophobicity estimation methods in the field of drug design and protein folding is discussed. PMID:19929828

  20. Multiple folding pathways for heterologously expressed human prion protein.

    Science.gov (United States)

    Jackson, G S; Hill, A F; Joseph, C; Hosszu, L; Power, A; Waltho, J P; Clarke, A R; Collinge, J

    1999-04-12

    Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

  1. A Folding Pathway Model of Mini-Protein BBA5

    Directory of Open Access Journals (Sweden)

    In-Ho Lee

    2015-01-01

    Full Text Available We present the folding pathway model of mini-protein BBA5, a bundle of secondary structures, α-helix and β-hairpin, by using action-derived molecular dynamics (ADMD simulations. From ten independent ADMD simulations, we extracted common features of the folding pathway of BBA5, from which we found that the early stage chain compaction was followed by the formation of C-terminal α-helix. The N-terminal β-hairpin was observed to form only after α-helix was stabilized. This result is in good agreement with the experimental observation that BBA5 mutants were moderately cooperative folders, and their C-terminal helical fragments were of higher secondary structure propensity while the N-terminal hairpin fragments were of a random coil spectrum. We found that the most flexible part of BBA5 is the N-terminal four residues. Although both are made of the identical ββα motif, the secondary structure formation sequence of BBA5 is found to be different from that of FSD-1. Finally, a description of the folding pathway in terms of principal component analysis is presented to characterize the folding dynamics in reduced dimensions. With only three principal components, we were able to describe 83.4% of the pathway.

  2. 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

  3. The effects of organic solvents on the folding pathway and associated thermodynamics of proteins: a microscopic view.

    Science.gov (United States)

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2016-01-18

    Protein folding is subject to the effects of solvation environment. A variety of organic solvents are used as additives for in vitro refolding of denatured proteins. Examination of the solvent effects on protein folding could be of fundamental importance to understand the molecular interactions in determining protein structure. This article investigated the folding of α-helix and β-hairpin structures in water and the solutions of two representative refolding additives (methanol (MeOH) and 1-Ethyl-3-methylimidazolium chloride (EMIM-Cl) ionic liquid) using REMD simulations. For both α-helix and β-hairpin in MeOH/water solution or α-helix in EMIM-Cl/water solution, the transient structures along the folding pathway are consistent with the counterparts in water but the relative statistical weights are changed, leading to the decrease in the overall folding free energy barrier. Accordingly, MeOH promotes the folding of both α-helix and β-hairpin but EMIM-Cl ionic liquid only promotes the folding of α-helix, consistent with experimental observations. The present study reveals for the first time the trivial effects on folding route but significant effects on folding thermodynamics from MeOH and EMIM-Cl, explaining the function of protein refolding additives and testifying the validity of the folding mechanism revealed by in vitro protein folding study using refolding additives.

  4. 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

  5. Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

    Science.gov (United States)

    Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

    2016-04-19

    The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein

  6. 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

  7. Causes and consequences of protein folding stress in aneuploid cells.

    Science.gov (United States)

    Donnelly, Neysan; Storchová, Zuzana

    2015-01-01

    Imbalanced chromosomal content, or aneuploidy, strongly affects the physiology of eukaryotic cells. The consequences of these effects are frequently detrimental, in particular in Metazoans. In humans, aneuploidy has been causatively linked to pathological conditions such as spontaneous abortions, trisomy syndromes and cancer. However, only in recent years have we witnessed an unraveling of the complex phenotypes that are caused by aneuploidy. Importantly, it has become apparent that aneuploidy evokes global and uniform changes that cannot be explained by the altered expression of the specific genes located on aneuploid chromosomes. Recent discoveries show that aneuploidy negatively affects protein folding; in particular, the functions of the molecular chaperone Heat Shock Protein 90 (HSP90) and the upstream regulator of heat shock-induced transcription, Heat Shock Factor 1 (HSF1), are impaired. Here we discuss the possible causes and consequences of this impairment and propose that the protein folding stress instigated by aneuploidy may be a common feature of conditions as variable as cancer and trisomy syndromes.

  8. Folding dynamics of a family of beta-sheet proteins

    Science.gov (United States)

    Rousseau, Denis

    2008-03-01

    Fatty acid binding proteins (FABP) consist of ten anti-parallel beta strands and two small alpha helices. The beta strands are arranged into two nearly orthogonal five-strand beta sheets that surround the interior cavity, which binds unsaturated long-chain fatty acids. In the brain isoform (BFABP), these are very important for the development of the central nervous system and neuron differentiation. Furthermore, BFABP is implicated in the pathogenesis of a variety of human diseases including cancer and neuronal degenerative disorders. In this work, site-directed spin labeling combined with EPR techniques have been used to study the folding mechanism of BFABP. In the first series of studies, we labeled the two Cys residues at position 5 and 80 in the wild type protein with an EPR spin marker; in addition, two singly labeled mutants at positions 5 and 80 in the C80A and C5A mutants, respectively, were also produced and used as controls. The changes in the distances between the two residues were examined by a pulsed EPR method, DEER (Double Electron Electron Resonance), as a function of guanidinium hydrochloride concentration. The results were compared with those from CW EPR, circular dichroism and fluorescence measurements, which provide the information regarding sidechain mobility, secondary structure and tertiary structure, respectively. The results will be discussed in the context of the folding mechanism of the family of fatty acid binding proteins.

  9. Work Done by Titin Protein Folding Assists Muscle Contraction

    Directory of Open Access Journals (Sweden)

    Jaime Andrés Rivas-Pardo

    2016-02-01

    Full Text Available Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6–8 pN. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle.

  10. 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.

  11. Extreme Folding

    Science.gov (United States)

    Demaine, Erik

    2012-02-01

    Our understanding of the mathematics and algorithms behind paper folding, and geometric folding in general, has increased dramatically over the past several years. These developments have found a surprisingly broad range of applications. In the art of origami, it has helped spur the technical origami revolution. In engineering and science, it has helped solve problems in areas such as manufacturing, robotics, graphics, and protein folding. On the recreational side, it has led to new kinds of folding puzzles and magic. I will give an overview of the mathematics and algorithms of folding, with a focus on new mathematics and sculpture.

  12. A weighted string kernel for protein fold recognition.

    Science.gov (United States)

    Nojoomi, Saghi; Koehl, Patrice

    2017-08-25

    Alignment-free methods for comparing protein sequences have proved to be viable alternatives to approaches that first rely on an alignment of the sequences to be compared. Much work however need to be done before those methods provide reliable fold recognition for proteins whose sequences share little similarity. We have recently proposed an alignment-free method based on the concept of string kernels, SeqKernel (Nojoomi and Koehl, BMC Bioinformatics, 2017, 18:137). In this previous study, we have shown that while Seqkernel performs better than standard alignment-based methods, its applications are potentially limited, because of biases due mostly to sequence length effects. In this study, we propose improvements to SeqKernel that follows two directions. First, we developed a weighted version of the kernel, WSeqKernel. Second, we expand the concept of string kernels into a novel framework for deriving information on amino acids from protein sequences. Using a dataset that only contains remote homologs, we have shown that WSeqKernel performs remarkably well in fold recognition experiments. We have shown that with the appropriate weighting scheme, we can remove the length effects on the kernel values. WSeqKernel, just like any alignment-based sequence comparison method, depends on a substitution matrix. We have shown that this matrix can be optimized so that sequence similarity scores correlate well with structure similarity scores. Starting from no information on amino acid similarity, we have shown that we can derive a scoring matrix that echoes the physico-chemical properties of amino acids. We have made progress in characterizing and parametrizing string kernels as alignment-based methods for comparing protein sequences, and we have shown that they provide a framework for extracting sequence information from structure.

  13. 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.

  14. Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins.

    Science.gov (United States)

    Delic, Marizela; Göngrich, Rebecca; Mattanovich, Diethard; Gasser, Brigitte

    2014-07-20

    Recombinant protein production has developed into a huge market with enormous positive implications for human health and for the future direction of a biobased economy. Limitations in the economic and technical feasibility of production processes are often related to bottlenecks of in vivo protein folding. Based on cell biological knowledge, some major bottlenecks have been overcome by the overexpression of molecular chaperones and other folding related proteins, or by the deletion of deleterious pathways that may lead to misfolding, mistargeting, or degradation. While important success could be achieved by this strategy, the list of reported unsuccessful cases is disappointingly long and obviously dependent on the recombinant protein to be produced. Singular engineering of protein folding steps may not lead to desired results if the pathway suffers from several limitations. In particular, the connection between folding quality control and proteolytic degradation needs further attention. Based on recent understanding that multiple steps in the folding and secretion pathways limit productivity, synergistic combinations of the cell engineering approaches mentioned earlier need to be explored. In addition, systems biology-based whole cell analysis that also takes energy and redox metabolism into consideration will broaden the knowledge base for future rational engineering strategies.

  15. 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.

  16. 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.

  17. 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.

  18. A Computational Approach to Studying Protein Folding Problems Considering the Crucial Role of the Intracellular Environment.

    Science.gov (United States)

    González-Pérez, Pedro P; Orta, Daniel J; Peña, Irving; Flores, Eduardo C; Ramírez, José U; Beltrán, Hiram I; Alas, Salomón J

    2017-10-01

    Intracellular protein folding (PF) is performed in a highly inhomogeneous, crowded, and correlated environment. Due to this inherent complexity, the study and understanding of PF phenomena is a fundamental issue in the field of computational systems biology. In particular, it is important to use a modeled medium that accurately reflects PF in natural systems. In the current study, we present a simulation wherein PF is carried out within an inhomogeneous modeled medium. Simulation resources included a two-dimensional hydrophobic-polar (HP) model, evolutionary algorithms, and the dual site-bond model. The dual site-bond model was used to develop an environment where HP beads could be folded. Our modeled medium included correlation lengths and fractal-like behavior, which were selected according to HP sequence lengths to induce folding in a crowded environment. Analysis of three benchmark HP sequences showed that the modeled inhomogeneous space played an important role in deeper energy folding and obtained better performance and convergence compared with homogeneous environments. Our computational approach also demonstrated that our correlated network provided a better space for PF. Thus, our approach represents a major advancement in PF simulations, not only for folding but also for understanding functional chemical structure and physicochemical properties of proteins in crowded molecular systems, which normally occur in nature.

  19. 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.

  20. Unexpected fold in the circumsporozoite protein target of malaria vaccines

    Energy Technology Data Exchange (ETDEWEB)

    Doud, Michael B.; Koksal, Adem C.; Mi, Li-Zhi; Song, Gaojie; Lu, Chafen; Springer, Timothy A. (Harvard-Med)

    2012-10-09

    Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an '{alpha}TSR' domain. The {alpha}TSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but {alpha}TSR does not. Interestingly, polymorphic T-cell epitopes map to specialized {alpha}TSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

  1. 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.

  2. Efficient conformational space exploration in ab initio protein folding simulation.

    Science.gov (United States)

    Ullah, Ahammed; Ahmed, Nasif; Pappu, Subrata Dey; Shatabda, Swakkhar; Ullah, A Z M Dayem; Rahman, M Sohel

    2015-08-01

    Ab initio protein folding simulation largely depends on knowledge-based energy functions that are derived from known protein structures using statistical methods. These knowledge-based energy functions provide us with a good approximation of real protein energetics. However, these energy functions are not very informative for search algorithms and fail to distinguish the types of amino acid interactions that contribute largely to the energy function from those that do not. As a result, search algorithms frequently get trapped into the local minima. On the other hand, the hydrophobic-polar (HP) model considers hydrophobic interactions only. The simplified nature of HP energy function makes it limited only to a low-resolution model. In this paper, we present a strategy to derive a non-uniform scaled version of the real 20×20 pairwise energy function. The non-uniform scaling helps tackle the difficulty faced by a real energy function, whereas the integration of 20×20 pairwise information overcomes the limitations faced by the HP energy function. Here, we have applied a derived energy function with a genetic algorithm on discrete lattices. On a standard set of benchmark protein sequences, our approach significantly outperforms the state-of-the-art methods for similar models. Our approach has been able to explore regions of the conformational space which all the previous methods have failed to explore. Effectiveness of the derived energy function is presented by showing qualitative differences and similarities of the sampled structures to the native structures. Number of objective function evaluation in a single run of the algorithm is used as a comparison metric to demonstrate efficiency.

  3. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions

    OpenAIRE

    Cieplak, Andrzej Stanis?aw

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of foldin...

  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. Folding energetics of ligand binding proteins. I. Theoretical model.

    Science.gov (United States)

    Rösgen, J; Hinz, H J

    2001-03-02

    Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx N + x D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data. Copyright 2001 Academic Press.

  6. 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.

  7. Universal distribution of protein evolution rates as a consequence of protein folding physics.

    Science.gov (United States)

    Lobkovsky, Alexander E; Wolf, Yuri I; Koonin, Eugene V

    2010-02-16

    The hypothesis that folding robustness is the primary determinant of the evolution rate of proteins is explored using a coarse-grained off-lattice model. The simplicity of the model allows rapid computation of the folding probability of a sequence to any folded conformation. For each robust folder, the network of sequences that share its native structure is identified. The fitness of a sequence is postulated to be a simple function of the number of misfolded molecules that have to be produced to reach a characteristic protein abundance. After fixation probabilities of mutants are computed under a simple population dynamics model, a Markov chain on the fold network is constructed, and the fold-averaged evolution rate is computed. The distribution of the logarithm of the evolution rates across distinct networks exhibits a peak with a long tail on the low rate side and resembles the universal empirical distribution of the evolutionary rates more closely than either distribution resembles the log-normal distribution. The results suggest that the universal distribution of the evolutionary rates of protein-coding genes is a direct consequence of the basic physics of protein folding.

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

    KAUST Repository

    Nath, Shuvra Kanti

    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.

  9. Modeling of folds and folding pathways for some protein families of (α + β)- and (α/β)-classes.

    Science.gov (United States)

    Gordeev, Alexey B; Efimov, Alexander V

    2013-01-01

    In this paper, updated structural trees for α/β-proteins containing five- and seven-segment (α/β)-motifs are represented. Novel structural motifs occurring in some families of (α + β)- and (α/β)-proteins are also characterized. Databases of these proteins have been compiled from the Protein Data Bank (PDB) and Structural Classification of Proteins (SCOP) and the corresponding structural trees have been constructed. The classification of these proteins has been developed and organized as an extension of the PCBOST database, which is available at http://strees.protres.ru . In total, the updated Protein Classification Based on Structural Trees database contains 11 structural trees, 106 levels, 635 folds, 4911 proteins and domains, and 14,202 PDB entries.

  10. 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.

  11. 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

  12. 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].

  13. Identifying common metalloprotease inhibitors by protein fold types using Fourier transform mass spectrometry.

    Science.gov (United States)

    Mitchell, Jennifer K; Pitcher, Desley; McArdle, Bernadette M; Alnefelt, Terese; Duffy, Sandra; Avery, Vicky; Quinn, Ronald J

    2007-12-01

    Fourteen natural products, known to inhibit other proteins of the Zincin-like fold class, were screened for inhibition of the Zincin-like fold metalloprotease thermolysin using mass spectrometry. Fourier Transform Mass Spectrometry was successful in identifying actinonin, a known inhibitor of astacin and stromelysin, to be an inhibitor of thermolysin. Molecular modelling studies have shown that specificity within the Zincin-like fold is determined by Protein Fold Topology.

  14. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  15. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Directory of Open Access Journals (Sweden)

    Axel Baumann

    Full Text Available Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando at molecular resolution.

  16. A New Heuristic Algorithm for Protein Folding in the HP Model.

    Science.gov (United States)

    Traykov, Metodi; Angelov, Slav; Yanev, Nicola

    2016-08-01

    This article presents an efficient heuristic for protein folding. The protein folding problem is to predict the compact three-dimensional structure of a protein based on its amino acid sequence. The focus is on an original integer programming model derived from a platform used for Contact Map Overlap problem.

  17. 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

    -control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully...

  18. Mechanism of acid-induced folding of proteins.

    Science.gov (United States)

    Goto, Y; Takahashi, N; Fink, A L

    1990-04-10

    We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

  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. 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...

  1. 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

  2. Low-dimensional, free-energy landscapes of protein-folding reactions by nonlinear dimensionality reduction

    Science.gov (United States)

    Das, Payel; Moll, Mark; Stamati, Hernán; Kavraki, Lydia E.; Clementi, Cecilia

    2006-01-01

    The definition of reaction coordinates for the characterization of a protein-folding reaction has long been a controversial issue, even for the “simple” case in which one single free-energy barrier separates the folded and unfolded ensemble. We propose a general approach to this problem to obtain a few collective coordinates by using nonlinear dimensionality reduction. We validate the usefulness of this method by characterizing the folding landscape associated with a coarse-grained protein model of src homology 3 as sampled by molecular dynamics simulations. The folding free-energy landscape projected on the few relevant coordinates emerging from the dimensionality reduction can correctly identify the transition-state ensemble of the reaction. The first embedding dimension efficiently captures the evolution of the folding process along the main folding route. These results clearly show that the proposed method can efficiently find a low-dimensional representation of a complex process such as protein folding. PMID:16785435

  3. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    Science.gov (United States)

    Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

    2013-12-13

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

  4. Probing folding free energy landscape of small proteins through ...

    Indian Academy of Sciences (India)

    Unknown

    simulations, where the inter amino acid interactions are given by a minimalistic model. (MM) we recently ... Analysis of Ntopo and RCO correlates the late stage folding with rearrange- ment of the side chain ... backbone atoms are numbered as i's, where i = 1, 2, 3…, etc. whereas the side residues are numbered as i′'s, ...

  5. 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.

  6. 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.

  7. Genetic Algorithms and Their Application to the Protein Folding Problem

    Science.gov (United States)

    1993-12-01

    Example of a Mutation ............................................................................. 16 Figure 4. A Three Amino Acid ProteinA ...given its primary structure. HH Figure 4. A Three Amino Acid ProteinA 28 3.1 Background Proteins are an integral part of everyday life. The function of

  8. Low-dimensional, free-energy landscapes of protein-folding reactions by nonlinear dimensionality reduction

    OpenAIRE

    Das, Payel; Moll, Mark; Stamati, Hernán; Kavraki, Lydia E.; Clementi, Cecilia

    2006-01-01

    The definition of reaction coordinates for the characterization of a protein-folding reaction has long been a controversial issue, even for the “simple” case in which one single free-energy barrier separates the folded and unfolded ensemble. We propose a general approach to this problem to obtain a few collective coordinates by using nonlinear dimensionality reduction. We validate the usefulness of this method by characterizing the folding landscape associated with a coarse-grained protein mo...

  9. 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...

  10. 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.

  11. 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.

  12. A molecular imaging biosensor detects in vivo protein folding and misfolding.

    Science.gov (United States)

    Sheahan, Anjali V; Sekar, Thillai V; Chen, Kai; Paulmurugan, Ramasamy; Massoud, Tarik F

    2016-07-01

    Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein. We comprehensively validated this biosensor in vitro, in cells, and by optically imaging protein folding and misfolding in living mice using several test proteins including enhanced green fluorescent protein, Renilla luciferase, Gaussia luciferase, and SIRT1. Applications of this novel biosensor are potentially far-reaching in both cell-based HTS approaches to discover new anti-misfolding drugs, and when using the same biosensor in validation studies of drug candidates in small animal models. Novel anti-misfolding drugs are needed as molecular therapeutics for many diseases. We developed first in vivo imaging protein folding biosensor to aid drug discovery. Biosensor created by flanking a test protein with rationally split Firefly luciferase. Biosensor validated by detecting folding of test proteins EGFP, Rluc, Gluc, and SIRT1. Generalizable molecular biosensor for translational applications in drug screening.

  13. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    TECS

    mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. ... state, it is necessary to observe parameters which are sensitive to the ... them. Thus, such measurements offer the possibility of monitoring various regions of a protein during the transition from the U to the N form. When coupled with the ...

  14. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  15. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding.

    Science.gov (United States)

    Wilson, C J; Zhan, H; Swint-Kruse, L; Matthews, K S

    2007-01-01

    In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

  16. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins

    Science.gov (United States)

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z.; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D.; Sheng, Yong; Crane, Denis I.; Florin, Timothy H.

    2013-01-01

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca2+ or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX’s suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER. PMID:23650437

  17. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, N; Bross, P; Jørgensen, M M

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts of such '......Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...... of such 'conformational disease' are illustrated by reference to cystic fibrosis, phenylketonuria and short-chain acyl-CoA dehydrogenase deficiency. Other cellular components such as chaperones and proteases, as well as environmental factors, may combine to modulate the phenotype of such disorders and this may open up...

  18. Heavy metal ions are potent inhibitors of protein folding.

    Science.gov (United States)

    Sharma, Sandeep K; Goloubinoff, Pierre; Christen, Philipp

    2008-07-25

    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. Cd2+, Hg2+ and Pb2+ 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.

  19. Effects of long-range electrostatic forces on simulated protein folding kinetics.

    Science.gov (United States)

    Robertson, Alex; Luttmann, Edgar; Pande, Vijay S

    2008-04-15

    Molecular dynamics simulations are a useful tool for characterizing protein folding pathways. There are several methods of treating electrostatic forces in these simulations with varying degrees of physical fidelity and computational efficiency. In this article, we compare the reaction field (RF) algorithm, particle-mesh Ewald (PME), and tapered cutoffs with increasing cutoff radii to address the impact of the electrostatics method employed on the folding kinetics. We quantitatively compare different methods by a correlation of quantitative measures of protein folding kinetics. The results of these comparisons show that for protein folding kinetics, the RF algorithm can quantitatively reproduce the kinetics of the more costly PME algorithm. These results not only assist the selection of appropriate algorithms for future simulations, but also give insight on the role that long-range electrostatic forces have in protein folding. (c) 2007 Wiley Periodicals, Inc.

  20. Direct observation of parallel folding pathways revealed using a symmetric repeat protein system.

    Science.gov (United States)

    Aksel, Tural; Barrick, Doug

    2014-07-01

    Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    -states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. This differentiation of a two-state from a multi-state structural ...

  2. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    TECS

    of monitoring various regions of a protein during the transition from the U to the N form. When coupled with the maximum entropy method (MEM) of analysis of intensity decay curves,. 1,2. TR-FRET measurements can resolve heterogeneity in terms of distributions of intramolecular distances. Much has been learnt about.

  3. 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.

  4. Quantifying the Sources of Kinetic Frustration in Folding Simulations of Small Proteins

    Science.gov (United States)

    2015-01-01

    Experiments and atomistic simulations of polypeptides have revealed structural intermediates that promote or inhibit conformational transitions to the native state during folding. We invoke a concept of “kinetic frustration” to quantify the prevalence and impact of these behaviors on folding rates within a large set of atomistic simulation data for 10 fast-folding proteins, where each protein’s conformational space is represented as a Markov state model of conformational transitions. Our graph theoretic approach addresses what conformational features correlate with folding inhibition and therefore permits comparison among features within a single protein network and also more generally between proteins. Nonnative contacts and nonnative secondary structure formation can thus be quantitatively implicated in inhibiting folding for several of the tested peptides. PMID:25136267

  5. Tracking of protein folding by chiral spectroscopic methods

    Czech Academy of Sciences Publication Activity Database

    Krupová, Monika; Andrushchenko, Valery; Bouř, Petr

    2016-01-01

    Roč. 23, č. 1 (2016), s. 36 ISSN 1211-5894. [Discussions in Structural Molecular Biology /14./. 17.03.2016-19.03.2016, Nové Hrady] R&D Projects: GA ČR(CZ) GA16-04902S; GA ČR GA15-09072S Institutional support: RVO:61388963 Keywords : proteins * fibrils * lanthanides * vibrational circular dichroism * circularly polarised luminescence Subject RIV: CF - Physical ; Theoretical Chemistry

  6. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Science.gov (United States)

    Cieplak, Andrzej Stanisław

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid

  7. Effective inter-residue contact definitions for accurate protein fold recognition

    Directory of Open Access Journals (Sweden)

    Yuan Chao

    2012-11-01

    Full Text Available Abstract Background Effective encoding of residue contact information is crucial for protein structure prediction since it has a unique role to capture long-range residue interactions compared to other commonly used scoring terms. The residue contact information can be incorporated in structure prediction in several different ways: It can be incorporated as statistical potentials or it can be also used as constraints in ab initio structure prediction. To seek the most effective definition of residue contacts for template-based protein structure prediction, we evaluated 45 different contact definitions, varying bases of contacts and distance cutoffs, in terms of their ability to identify proteins of the same fold. Results We found that overall the residue contact pattern can distinguish protein folds best when contacts are defined for residue pairs whose Cβ atoms are at 7.0 Å or closer to each other. Lower fold recognition accuracy was observed when inaccurate threading alignments were used to identify common residue contacts between protein pairs. In the case of threading, alignment accuracy strongly influences the fraction of common contacts identified among proteins of the same fold, which eventually affects the fold recognition accuracy. The largest deterioration of the fold recognition was observed for β-class proteins when the threading methods were used because the average alignment accuracy was worst for this fold class. When results of fold recognition were examined for individual proteins, we found that the effective contact definition depends on the fold of the proteins. A larger distance cutoff is often advantageous for capturing spatial arrangement of the secondary structures which are not physically in contact. For capturing contacts between neighboring β strands, considering the distance between Cα atoms is better than the Cβ−based distance because the side-chain of interacting residues on β strands sometimes point to

  8. Thermodynamics of model prions and its implications for the problem of prion protein folding.

    Science.gov (United States)

    Harrison, P M; Chan, H S; Prusiner, S B; Cohen, F E

    1999-02-19

    Prion disease is caused by the propagation of a particle containing PrPSc, a misfolded form of the normal cellular prion protein (PrPC). PrPC can re-fold to form PrPSc with loss of alpha-helical structure and formation of extensive beta-sheet structure. Here, we model this prion folding problem with a simple, low-resolution lattice model of protein folding. If model proteins are allowed to re-fold upon dimerization, a minor proportion of them (up to approximately 17%) encrypts an alternative native state as a homodimer. The structures in this homodimeric native state re-arrange so that they are very different in conformation from the monomeric native state. We find that model proteins that are relatively less stable as monomers are more susceptible to the formation of alternative native states as homodimers. These results suggest that less-stable proteins have a greater need for a well-designed energy landscape for protein folding to overcome an increased chance of encrypting substantially different native conformations stabilized by multimeric interactions. This conceptual framework for aberrant folding should be relevant in Alzheimer's disease and other disorders associated with protein aggregation. Copyright 1999 Academic Press.

  9. 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.

  10. 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

  11. 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.

  12. Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.

    Science.gov (United States)

    Karlsson, Amy J; Lim, Hyung-Kwon; Xu, Hansen; Rocco, Mark A; Bratkowski, Matthew A; Ke, Ailong; DeLisa, Matthew P

    2012-02-10

    A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Atomic interaction networks in the core of protein domains and their native folds.

    Science.gov (United States)

    Soundararajan, Venkataramanan; Raman, Rahul; Raguram, S; Sasisekharan, V; Sasisekharan, Ram

    2010-02-23

    Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be "signature" of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1-2 angstroms (mean 1.61A) C(alpha) RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the 'twilight' and 'midnight' zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence

  14. The energy landscape, folding pathways and the kinetics of a knotted protein.

    Directory of Open Access Journals (Sweden)

    Michael C Prentiss

    2010-07-01

    Full Text Available The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly.

  15. 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.

  16. 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)

  17. 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.

  18. Low-dimensional, free-energy landscapes of protein-folding reactions by nonlinear dimensionality reduction

    Science.gov (United States)

    Das, Payel; Moll, Mark; Stamati, Hernán; Kavraki, Lydia E.; Clementi, Cecilia

    2006-06-01

    The definition of reaction coordinates for the characterization of a protein-folding reaction has long been a controversial issue, even for the "simple" case in which one single free-energy barrier separates the folded and unfolded ensemble. We propose a general approach to this problem to obtain a few collective coordinates by using nonlinear dimensionality reduction. We validate the usefulness of this method by characterizing the folding landscape associated with a coarse-grained protein model of src homology 3 as sampled by molecular dynamics simulations. The folding free-energy landscape projected on the few relevant coordinates emerging from the dimensionality reduction can correctly identify the transition-state ensemble of the reaction. The first embedding dimension efficiently captures the evolution of the folding process along the main folding route. These results clearly show that the proposed method can efficiently find a low-dimensional representation of a complex process such as protein folding. reaction coordinate | transition state | manifold | embedding | ISOMAP

  19. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    Science.gov (United States)

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-28

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    Science.gov (United States)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  6. 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.

  7. 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.

  8. Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase.

    Science.gov (United States)

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2017-05-09

    Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. 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

  10. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    Directory of Open Access Journals (Sweden)

    Murat Cetinbaş

    Full Text Available Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics.

  11. 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.

  12. 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

    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......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...

  13. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Directory of Open Access Journals (Sweden)

    Andrzej Stanisław Cieplak

    Full Text Available Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and

  14. Protein structure prediction using a docking-based hierarchical folding scheme.

    Science.gov (United States)

    Kifer, Ilona; Nussinov, Ruth; Wolfson, Haim J

    2011-06-01

    The pathways by which proteins fold into their specific native structure are still an unsolved mystery. Currently, many methods for protein structure prediction are available, and most of them tackle the problem by relying on the vast amounts of data collected from known protein structures. These methods are often not concerned with the route the protein follows to reach its final fold. This work is based on the premise that proteins fold in a hierarchical manner. We present FOBIA, an automated method for predicting a protein structure. FOBIA consists of two main stages: the first finds matches between parts of the target sequence and independently folding structural units using profile-profile comparison. The second assembles these units into a 3D structure by searching and ranking their possible orientations toward each other using a docking-based approach. We have previously reported an application of an initial version of this strategy to homology based targets. Since then we have considerably enhanced our method's abilities to allow it to address the more difficult template-based target category. This allows us to now apply FOBIA to the template-based targets of CASP8 and to show that it is both very efficient and promising. Our method can provide an alternative for template-based structure prediction, and in particular, the docking-basedranking technique presented here can be incorporated into any profile-profile comparison based method. Copyright © 2011 Wiley-Liss, Inc.

  15. Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry.

    Science.gov (United States)

    Mortensen, Daniel N; Williams, Evan R

    2016-03-16

    The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.

  16. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    Science.gov (United States)

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

  17. 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.

  18. A rewired green fluorescent protein: folding and function in a nonsequential, noncircular GFP permutant.

    Science.gov (United States)

    Reeder, Philippa J; Huang, Yao-Ming; Dordick, Jonathan S; Bystroff, Christopher

    2010-12-28

    The sequential order of secondary structural elements in proteins affects the folding and activity to an unknown extent. To test the dependence on sequential connectivity, we reconnected secondary structural elements by their solvent-exposed ends, permuting their sequential order, called "rewiring". This new protein design strategy changes the topology of the backbone without changing the core side chain packing arrangement. While circular and noncircular permutations have been observed in protein structures that are not related by sequence homology, to date no one has attempted to rationally design and construct a protein with a sequence that is noncircularly permuted while conserving three-dimensional structure. Herein, we show that green fluorescent protein can be rewired, still functionally fold, and exhibit wild-type fluorescence excitation and emission spectra.

  19. Investigating protein folding and unfolding in electrospray nanodrops upon rapid mixing using theta-glass emitters.

    Science.gov (United States)

    Mortensen, Daniel N; Williams, Evan R

    2015-01-20

    Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 μs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 μs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 μs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously ( Fisher et al. Anal. Chem. 2014 , 86 , 4581 - 4588 ), a result that is attributed to the much smaller, ∼1.5 μm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 μs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.

  20. 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.)

  1. 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.

  2. Identification of novel restriction endonuclease-like fold families among hypothetical proteins.

    Science.gov (United States)

    Kinch, Lisa N; Ginalski, Krzysztof; Rychlewski, Leszek; Grishin, Nick V

    2005-01-01

    Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence similarity between protein families makes homology inference a challenging task and hinders new family identification with traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing PDB database.

  3. Exploring the correlation between the folding rates of proteins and the entanglement of their native states

    Science.gov (United States)

    Baiesi, Marco; Orlandini, Enzo; Seno, Flavio; Trovato, Antonio

    2017-12-01

    The folding of a protein towards its native state is a rather complicated process. However, there is empirical evidence that the folding time correlates with the contact order, a simple measure of the spatial organization of the native state of the protein. Contact order is related to the average length of the main chain loops formed by amino acids that are in contact. Here we argue that folding kinetics can also be influenced by the entanglement that loops may undergo within the overall three-dimensional protein structure. In order to explore such a possibility, we introduce a novel descriptor, which we call ‘maximum intrachain contact entanglement’. Specifically, we measure the maximum Gaussian entanglement between any looped portion of a protein and any other non-overlapping subchain of the same protein, which is easily computed by discretized line integrals on the coordinates of the Cα atoms. By analyzing experimental data sets of two-state and multi-state folders, we show that the new index is also a good predictor of the folding rate. Moreover, being only partially correlated with previous methods, it can be integrated with them to yield more accurate predictions.

  4. 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.

  5. 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

  6. Prediction of protein folds: extraction of new features, dimensionality reduction, and fusion of heterogeneous classifiers.

    Science.gov (United States)

    Ghanty, Pradip; Pal, Nikhil R

    2009-03-01

    Here, we consider a two-level (four classes in level 1 and 27 folds in level 2) protein fold determination problem. We propose several new features and use some existing features including frequencies of adjacent residues, frequencies of residues separated by one residue, and triplets (trio) of amino acid compositions (AACs). The dimensionality of the trio AAC features is drastically reduced using a neural network based novel online feature selection scheme. We also propose new sets of features called trio potential computed using the hydrophobicity values considering only the selected trio AACs. We demonstrate that the proposed features including the selected trio AACs and trio potential have good discriminating power for protein fold determination. As machine learning tools, we use multilayer perceptron network, radial basis function network, and support vector machine. To improve the recognition accuracies further, we use fusion of different classifiers using the same set of features as well as different sets of features. The effectiveness of our schemes is demonstrated with a benchmark structural classification of proteins (SCOP) dataset. Our system achieves 84.9% test accuracy for the SCOP structural class (four classes) determination and 68.6% test accuracy for the fold recognition with 27 folds. In order to demonstrate the consistency of feature sets and fusion schemes, we also perform the fivefold cross-validation experiments.

  7. Synthesis of PAF, an antifungal protein from P. chrysogenum, by native chemical ligation: native disulfide pattern and fold obtained upon oxidative refolding.

    Science.gov (United States)

    Váradi, Györgyi; Tóth, Gábor K; Kele, Zoltán; Galgóczy, László; Fizil, Ádám; Batta, Gyula

    2013-09-16

    The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.

  8. Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory

    Science.gov (United States)

    Satomura, Atsushi; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity. PMID:25970342

  9. Replica Exchange Wang—Landau Simulation of Lattice Protein Folding Funnels

    Science.gov (United States)

    Shi, Guangjie; Wüst, Thomas; Landau David, P.

    2017-10-01

    The resolution of Levinthal’s paradox concerning the ability of proteins to fold rapidly postulates the existence of a rough “folding funnel” in free energy space that guides the protein to its lowest free energy, native state. To study the folding of the protein ribonuclease A we have mapped it onto a 124 monomer, coarse-grained HP lattice model and onto an H0P model that also includes “neutral” 0-mers in addition to the hydrophobic H-mers and polar P-mers. Using Replica Exchange Wang-Landau sampling, we determined the density of states, g(E), which we then used to calculate the free energy of the protein vs end-to-end distance as a function of temperature. At low temperature the HP model shows a rather shallow and at free energy minimum, while the H0P model maintains a rough free energy funnel. Unlike the common, schematic figures, we find an asymmetric folding funnel that also changes shape substantially as the temperature decreases. Even the location of the free energy minimum shifts as the temperature decreases.

  10. Determination of Protein Folding Intermediate Structures Consistent with Data from Oxidative Footprinting Mass Spectrometry.

    Science.gov (United States)

    Heinkel, Florian; Gsponer, Jörg

    2016-01-29

    The mapping of folding landscapes remains an important challenge in protein chemistry. Pulsed oxidative labeling of exposed residues and their detection via mass spectrometry provide new means of taking time-resolved "snapshots" of the structural changes that occur during protein folding. However, such experiments have been so far only interpreted qualitatively. Here, we report the detailed structural interpretation of mass spectrometry data from fast photochemical oxidation of proteins (FPOP) experiments at atomic resolution in a biased molecular dynamics approach. We are able to calculate structures of the early folding intermediate of the model system barstar that are fully consistent with FPOP data and Φ values. Furthermore, structures calculated with both FPOP data and Φ values are significantly less compact and have fewer helical residues than intermediate structures calculated with Φ values only. This improves the agreement with the experimental β-Tanford value and CD measurements. The restraints that we introduce facilitate the structural interpretation of FPOP data and provide new means for refined structure calculations of transiently sampled states on protein folding landscapes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The Histone Database: an integrated resource for histones and histone fold-containing proteins

    Science.gov (United States)

    Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

  12. How Kinetics within the Unfolded State Affects Protein Folding: an Analysis Based on Markov State Models and an Ultra-Long MD Trajectory

    Science.gov (United States)

    Deng, Nan-jie; Dai, Wei

    2013-01-01

    Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultra-long MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤ 40 ns, compared with the folding time of ≈ 5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90 % of the unfolded states are sampled within the first 40 μs of the ultra-long MD trajectory, which on average explores ~27 % of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into “tubes”, and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with non-uniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently leading to non-exponential kinetics. A kinetic partition of

  13. All-Atom Four-Body Knowledge-Based Statistical Potentials to Distinguish Native Protein Structures from Nonnative Folds

    Directory of Open Access Journals (Sweden)

    Majid Masso

    2017-01-01

    Full Text Available Recent advances in understanding protein folding have benefitted from coarse-grained representations of protein structures. Empirical energy functions derived from these techniques occasionally succeed in distinguishing native structures from their corresponding ensembles of nonnative folds or decoys which display varying degrees of structural dissimilarity to the native proteins. Here we utilized atomic coordinates of single protein chains, comprising a large diverse training set, to develop and evaluate twelve all-atom four-body statistical potentials obtained by exploring alternative values for a pair of inherent parameters. Delaunay tessellation was performed on the atomic coordinates of each protein to objectively identify all quadruplets of interacting atoms, and atomic potentials were generated via statistical analysis of the data and implementation of the inverted Boltzmann principle. Our potentials were evaluated using benchmarking datasets from Decoys-‘R’-Us, and comparisons were made with twelve other physics- and knowledge-based potentials. Ranking 3rd, our best potential tied CHARMM19 and surpassed AMBER force field potentials. We illustrate how a generalized version of our potential can be used to empirically calculate binding energies for target-ligand complexes, using HIV-1 protease-inhibitor complexes for a practical application. The combined results suggest an accurate and efficient atomic four-body statistical potential for protein structure prediction and assessment.

  14. Structural Analysis of Protein Folding by the Long-Chain Archaeal Chaperone FKBP26

    Energy Technology Data Exchange (ETDEWEB)

    E Martinez-Hackert; W Hendrickson

    2011-12-31

    In the cell, protein folding is mediated by folding catalysts and chaperones. The two functions are often linked, especially when the catalytic module forms part of a multidomain protein, as in Methanococcus jannaschii peptidyl-prolyl cis/trans isomerase FKBP26. Here, we show that FKBP26 chaperone activity requires both a 50-residue insertion in the catalytic FKBP domain, also called 'Insert-in-Flap' or IF domain, and an 80-residue C-terminal domain. We determined FKBP26 structures from four crystal forms and analyzed chaperone domains in light of their ability to mediate protein-protein interactions. FKBP26 is a crescent-shaped homodimer. We reason that folding proteins are bound inside the large crescent cleft, thus enabling their access to inward-facing peptidyl-prolyl cis/trans isomerase catalytic sites and ipsilateral chaperone domain surfaces. As these chaperone surfaces participate extensively in crystal lattice contacts, we speculate that the observed lattice contacts reflect a proclivity for protein associations and represent substrate interactions by FKBP26 chaperone domains. Finally, we find that FKBP26 is an exceptionally flexible molecule, suggesting a mechanism for nonspecific substrate recognition.

  15. 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.

  16. 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.

  17. Remote protein homology detection and fold recognition using two-layer support vector machine classifiers.

    Science.gov (United States)

    Muda, Hilmi M; Saad, Puteh; Othman, Razib M

    2011-08-01

    Remote protein homology detection and fold recognition refer to detection of structural homology in proteins where there are small or no similarities in the sequence. To detect protein structural classes from protein primary sequence information, homology-based methods have been developed, which can be divided to three types: discriminative classifiers, generative models for protein families and pairwise sequence comparisons. Support Vector Machines (SVM) and Neural Networks (NN) are two popular discriminative methods. Recent studies have shown that SVM has fast speed during training, more accurate and efficient compared to NN. We present a comprehensive method based on two-layer classifiers. The 1st layer is used to detect up to superfamily and family in SCOP hierarchy using optimized binary SVM classification rules. It used the kernel function known as the Bio-kernel, which incorporates the biological information in the classification process. The 2nd layer uses discriminative SVM algorithm with string kernel that will detect up to protein fold level in SCOP hierarchy. The results obtained were evaluated using mean ROC and mean MRFP and the significance of the result produced with pairwise t-test was tested. Experimental results show that our approaches significantly improve the performance of remote protein homology detection and fold recognition for all three different version SCOP datasets (1.53, 1.67 and 1.73). We achieved 4.19% improvements in term of mean ROC in SCOP 1.53, 4.75% in SCOP 1.67 and 4.03% in SCOP 1.73 datasets when compared to the result produced by well-known methods. The combination of first layer and second layer of BioSVM-2L performs well in remote homology detection and fold recognition even in three different versions of datasets. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. 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.

  19. 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

  20. 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/.

  1. A hybrid approach to protein folding problem integrating constraint programming with local search.

    Science.gov (United States)

    Ullah, Abu Dayem; Steinhöfel, Kathleen

    2010-01-18

    The protein folding problem remains one of the most challenging open problems in computational biology. Simplified models in terms of lattice structure and energy function have been proposed to ease the computational hardness of this optimization problem. Heuristic search algorithms and constraint programming are two common techniques to approach this problem. The present study introduces a novel hybrid approach to simulate the protein folding problem using constraint programming technique integrated within local search. Using the face-centered-cubic lattice model and 20 amino acid pairwise interactions energy function for the protein folding problem, a constraint programming technique has been applied to generate the neighbourhood conformations that are to be used in generic local search procedure. Experiments have been conducted for a few small and medium sized proteins. Results have been compared with both pure constraint programming approach and local search using well-established local move set. Substantial improvements have been observed in terms of final energy values within acceptable runtime using the hybrid approach. Constraint programming approaches usually provide optimal results but become slow as the problem size grows. Local search approaches are usually faster but do not guarantee optimal solutions and tend to stuck in local minima. The encouraging results obtained on the small proteins show that these two approaches can be combined efficiently to obtain better quality solutions within acceptable time. It also encourages future researchers on adopting hybrid techniques to solve other hard optimization problems.

  2. Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies.

    Science.gov (United States)

    Khanal, Anil; Pan, Yan; Brown, Leonid S; Konermann, Lars

    2012-12-01

    Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR. Copyright © 2012 John Wiley & Sons, Ltd.

  3. 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.

  4. 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.

  5. Volume and energy folding landscape of prion protein revealed by pressure

    Directory of Open Access Journals (Sweden)

    Cordeiro Y.

    2005-01-01

    Full Text Available The main hypothesis for prion diseases proposes that the cellular protein (PrP C can be altered into a misfolded, ß-sheet-rich isoform, the PrP Sc (from scrapie. The formation of this abnormal isoform then triggers the transmissible spongiform encephalopathies. Here, we discuss the use of high pressure as a tool to investigate this structural transition and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The latest findings on the application of high pressure to the cellular prion protein and to the scrapie PrP forms will be summarized in this review, which focuses on the energetic and volumetric properties of prion folding and conversion.

  6. 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.

  7. 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.

  8. Folding of proteins with an all-atom Go-model.

    Science.gov (United States)

    Wu, L; Zhang, J; Qin, M; Liu, F; Wang, W

    2008-06-21

    The Go-like potential at a residual level has been successfully applied to the folding of proteins in many previous works. However, taking into consideration more detailed structural information in the atomic level, the definition of contacts used in these traditional Go-models may not be suitable for all-atom simulations. Here, in this work, we develop a rational definition of contacts considering the screening effect in the crowded intramolecular environment. In such a scheme, a large amount of screened atom pairs are excluded and the number of contacts is decreased compared to the case of the traditional definition. These contacts defined by such a new definition are compatible with the all-atom representation of protein structures. To verify the rationality of the new definition of contacts, the folding of proteins CI2 and SH3 is simulated by all-atom molecular dynamics simulations. A high folding cooperativity and good correlation of the simulated Phi-values with those obtained experimentally, especially for CI2, are found. This suggests that the all-atom Go-model is improved compared to the traditional Go-model. Based on the comparison of the Phi-values, the roles of side chains in the folding are discussed, and it is concluded that the side-chain structures are more important for local contacts in determining the transition state structures. Moreover, the relations between side chain and backbone orderings are also discussed.

  9. 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.

  10. 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.

  11. Protein-fold recognition using an improved single-source K diverse shortest paths algorithm.

    Science.gov (United States)

    Lhota, John; Xie, Lei

    2016-04-01

    Protein structure prediction, when construed as a fold recognition problem, is one of the most important applications of similarity search in bioinformatics. A new protein-fold recognition method is reported which combines a single-source K diverse shortest path (SSKDSP) algorithm with Enrichment of Network Topological Similarity (ENTS) algorithm to search a graphic feature space generated using sequence similarity and structural similarity metrics. A modified, more efficient SSKDSP algorithm is developed to improve the performance of graph searching. The new implementation of the SSKDSP algorithm empirically requires 82% less memory and 61% less time than the current implementation, allowing for the analysis of larger, denser graphs. Furthermore, the statistical significance of fold ranking generated from SSKDSP is assessed using ENTS. The reported ENTS-SSKDSP algorithm outperforms original ENTS that uses random walk with restart for the graph search as well as other state-of-the-art protein structure prediction algorithms HHSearch and Sparks-X, as evaluated by a benchmark of 600 query proteins. The reported methods may easily be extended to other similarity search problems in bioinformatics and chemoinformatics. The SSKDSP software is available at http://compsci.hunter.cuny.edu/~leixie/sskdsp.html. © 2016 Wiley Periodicals, Inc.

  12. 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.

  13. 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.

  14. Codon usage influences the local rate of translation elongation to regulate co-translational protein folding

    Science.gov (United States)

    Yu, Chien-Hung; Dang, Yunkun; Zhou, Zhipeng; Wu, Cheng; Zhao, Fangzhou; Sachs, Matthew S.; Liu, Yi

    2015-01-01

    Summary Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance rate of translation elongation, whereas non-optimal codons slow elognatioon. Codon usage also controls ribosome traffic on mRNA. These conclusions were further supported by ribosome profiling results in vitro and in vivo with template mRNAs designed to increase signal to noise. Finally, we demonstrate that codon usage regulates protein function by affecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding. PMID:26321254

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

    International Nuclear Information System (INIS)

    Rasia, Rodolfo M.; Lescop, Ewen; Palatnik, Javier F.; Boisbouvier, Jérôme; Brutscher, Bernhard

    2011-01-01

    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.

  16. IntFOLD: an integrated server for modelling protein structures and functions from amino acid sequences.

    Science.gov (United States)

    McGuffin, Liam J; Atkins, Jennifer D; Salehe, Bajuna R; Shuid, Ahmad N; Roche, Daniel B

    2015-07-01

    IntFOLD is an independent web server that integrates our leading methods for structure and function prediction. The server provides a simple unified interface that aims to make complex protein modelling data more accessible to life scientists. The server web interface is designed to be intuitive and integrates a complex set of quantitative data, so that 3D modelling results can be viewed on a single page and interpreted by non-expert modellers at a glance. The only required input to the server is an amino acid sequence for the target protein. Here we describe major performance and user interface updates to the server, which comprises an integrated pipeline of methods for: tertiary structure prediction, global and local 3D model quality assessment, disorder prediction, structural domain prediction, function prediction and modelling of protein-ligand interactions. The server has been independently validated during numerous CASP (Critical Assessment of Techniques for Protein Structure Prediction) experiments, as well as being continuously evaluated by the CAMEO (Continuous Automated Model Evaluation) project. The IntFOLD server is available at: http://www.reading.ac.uk/bioinf/IntFOLD/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Oxidative protein folding in the endoplasmic reticulum: tight links to the mitochondria-associated membrane (MAM).

    Science.gov (United States)

    Simmen, Thomas; Lynes, Emily M; Gesson, Kevin; Thomas, Gary

    2010-08-01

    The production of secretory proteins at the ER (endoplasmic reticulum) depends on a ready supply of energy and metabolites as well as the close monitoring of the chemical conditions that favor oxidative protein folding. ER oxidoreductases and chaperones fold nascent proteins into their export-competent three-dimensional structure. Interference with these protein folding enzymes leads to the accumulation of unfolded proteins within the ER lumen, causing an acute organellar stress that triggers the UPR (unfolded protein response). The UPR increases the transcription of ER chaperones commensurate with the load of newly synthesized proteins and can protect the cell from ER stress. Persistant stress, however, can force the UPR to commit cells to undergo apoptotic cell death, which requires the emptying of ER calcium stores. Conversely, a continuous ebb and flow of calcium occurs between the ER and mitochondria during resting conditions on a domain of the ER that forms close contacts with mitochondria, the MAM (mitochondria-associated membrane). On the MAM, ER folding chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57 and Ero1alpha regulate calcium flux from the ER through reversible, calcium and redox-dependent interactions with IP3Rs (inositol 1,4,5-trisphophate receptors) and with SERCAs (sarcoplasmic/endoplasmic reticulum calcium ATPases). During apoptosis progression and depending on the identity of the ER chaperone and oxidoreductase, these interactions increase or decrease, suggesting that the extent of MAM targeting of ER chaperones and oxidoreductases could shift the readout of ER-mitochondria calcium exchange from housekeeping to apoptotic. However, little is known about the cytosolic factors that mediate the on/off interactions between ER chaperones and oxidoreductases with ER calcium channels and pumps. One candidate regulator is the multi-functional molecule PACS-2 (phosphofurin acidic cluster sorting protein-2). Recent

  18. Why Do Protein Folding Rates Correlate with Metrics of Native Topology?

    Science.gov (United States)

    Faísca, Patrícia F. N.; Travasso, Rui D. M.; Parisi, Andrea; Rey, Antonio

    2012-01-01

    For almost 15 years, the experimental correlation between protein folding rates and the contact order parameter has been under scrutiny. Here, we use a simple simulation model combined with a native-centric interaction potential to investigate the physical roots of this empirical observation. We simulate a large set of circular permutants, thus eliminating dependencies of the folding rate on other protein properties (e.g. stability). We show that the rate-contact order correlation is a consequence of the fact that, in high contact order structures, the contact order of the transition state ensemble closely mirrors the contact order of the native state. This happens because, in these structures, the native topology is represented in the transition state through the formation of a network of tertiary interactions that are distinctively long-ranged. PMID:22558173

  19. 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/

  20. Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics.

    Directory of Open Access Journals (Sweden)

    M Luisa Romero-Romero

    Full Text Available Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.

  1. 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.

  2. Understanding protein flexibility through dimensionality reduction.

    Science.gov (United States)

    Teodoro, Miguel L; Phillips, George N; Kavraki, Lydia E

    2003-01-01

    This work shows how to decrease the complexity of modeling flexibility in proteins by reducing the number of dimensions necessary to model important macromolecular motions such as the induced-fit process. Induced fit occurs during the binding of a protein to other proteins, nucleic acids, or small molecules (ligands) and is a critical part of protein function. It is now widely accepted that conformational changes of proteins can affect their ability to bind other molecules and that any progress in modeling protein motion and flexibility will contribute to the understanding of key biological functions. However, modeling protein flexibility has proven a very difficult task. Experimental laboratory methods, such as x-ray crystallography, produce rather limited information, while computational methods such as molecular dynamics are too slow for routine use with large systems. In this work, we show how to use the principal component analysis method, a dimensionality reduction technique, to transform the original high-dimensional representation of protein motion into a lower dimensional representation that captures the dominant modes of motions of proteins. For a medium-sized protein, this corresponds to reducing a problem with a few thousand degrees of freedom to one with less than fifty. Although there is inevitably some loss in accuracy, we show that we can obtain conformations that have been observed in laboratory experiments, starting from different initial conformations and working in a drastically reduced search space.

  3. 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...

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis.

    Science.gov (United States)

    An, Philip; Winters, Dwight; Walker, Kenneth W

    2016-04-01

    Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. 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.

  13. Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein.

    Science.gov (United States)

    Alexander, Crispin G; Jürgens, Maike C; Shepherd, Dale A; Freund, Stefan M V; Ashcroft, Alison E; Ferguson, Neil

    2013-07-23

    HBc, the capsid-forming "core protein" of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry-mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.

  14. Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins

    Directory of Open Access Journals (Sweden)

    Jorge Cuellar

    2014-03-01

    Full Text Available Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg (habitat/body T = −1.9 to +2°C, and of the cow (body T = 37°C. We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.

  15. Determining Protein Folding Pathway and Associated Energetics through Partitioned Integrated-Tempering-Sampling Simulation.

    Science.gov (United States)

    Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2017-03-14

    Replica exchange molecular dynamics (REMD) and integrated-tempering-sampling (ITS) are two representative enhanced sampling methods which utilize parallel and integrated tempering approaches, respectively. In this work, a partitioned integrated-tempering-sampling (P-ITS) method is proposed which takes advantage of the benefits of both parallel and integrated tempering approaches. Using P-ITS, the folding pathways of a series of proteins with diverse native structures are explored on multidimensional free-energy landscapes, and the associated thermodynamics are evaluated. In comparison to the original form of ITS, P-ITS improves the sampling efficiency and measures the folding/unfolding thermodynamic quantities more consistently with experimental data. In comparison to REMD, P-ITS significantly reduces the requirement of computational resources and meanwhile achieves similar simulation results. The observed structural characterizations of transition and intermediate states of the proteins under study are in good agreement with previous experimental and simulation studies on the same proteins and homologues. Therefore, the P-ITS method has great potential in simulating the structural dynamics of complex biomolecular systems.

  16. 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.

  17. 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.

  18. Design of Surfactant Protein B Peptide Mimics Based on the Saposin Fold for Synthetic Lung Surfactants.

    Science.gov (United States)

    Walther, Frans J; Gordon, Larry M; Waring, Alan J

    2016-01-01

    Surfactant protein (SP)-B is a 79-residue polypeptide crucial for the biophysical and physiological function of endogenous lung surfactant. SP-B is a member of the Saposin or Saposin-like proteins (SAPLIP) family of proteins that share an overall three-dimensional folding pattern based on secondary structures and disulfide connectivity and exhibit a wide diversity of biological functions. Here we review the synthesis, molecular biophysics and activity of synthetic analogs of Saposin proteins designed to mimic those interactions of the parent proteins with lipids that enhance interfacial activity. Saposin proteins generally interact with target lipids as either monomers or multimers via well-defined amphipathic helices, flexible hinge domains, and insertion sequences. Based on the known 3D-structural motif for the Saposin family, we show how bioengineering techniques may be used to develop minimal peptide constructs that maintain desirable structural properties and activities in biomedical applications. One important application is the molecular design, synthesis and activity of Saposin mimics based on the SP-B structure. Synthetic lung surfactants containing active SP-B analogs may be potentially useful in treating diseases of surfactant deficiency or dysfunction including the neonatal respiratory distress syndrome and acute lung injury/acute respiratory distress syndrome.

  19. Conserved thioredoxin fold is present in Pisum sativum L. sieve element occlusion-1 protein

    Science.gov (United States)

    Umate, Pavan; Tuteja, Renu

    2010-01-01

    Homology-based three-dimensional model for Pisum sativum sieve element occlusion 1 (Ps.SEO1) (forisomes) protein was constructed. A stretch of amino acids (residues 320 to 456) which is well conserved in all known members of forisomes proteins was used to model the 3D structure of Ps.SEO1. The structural prediction was done using Protein Homology/analogY Recognition Engine (PHYRE) web server. Based on studies of local sequence alignment, the thioredoxin-fold containing protein [Structural Classification of Proteins (SCOP) code d1o73a_], a member of the glutathione peroxidase family was selected as a template for modeling the spatial structure of Ps.SEO1. Selection was based on comparison of primary sequence, higher match quality and alignment accuracy. Motif 1 (EVF) is conserved in Ps.SEO1, Vicia faba (Vf.For1) and Medicago truncatula (MT.SEO3); motif 2 (KKED) is well conserved across all forisomes proteins and motif 3 (IGYIGNP) is conserved in Ps.SEO1 and Vf.For1. PMID:20404566

  20. 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

  1. 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

  2. Transient laser spectroscopy of protein folding: detection and characterization of slow annealing processes

    Science.gov (United States)

    Subramaniam, Vinod; Bergenhem, Nils; Gafni, Ari; Steel, Duncan G.

    1995-09-01

    The unique structure of a protein is encoded in its characteristic sequence of amino acids; the processes by which this linear sequence collapses into a unique 3D structure remains an unsolved problem that represents one of the most challenging issues in fundamental biomolecular science. This so-called protein folding problem is the second half of the genetic code. Studies of this biological problem are complicated by the need to study dynamic behavior involving small populations of transient species in a solution environment. However, the use of advanced transient laser spectroscopy techniques based on intrinsic chromophores provides a powerful means to study this problem. Specifically, time-resolved phosphorescence of tryptophan (Trp) provides a means to study the dynamics associated with different regions of the protein surrounding the emitting Trp residue. Using these methodologies, we are able to study, in real time, the later stages of unfolding and refolding of the bacterial protein alkaline phosphatase, a nonspecific monoesterase. Results show the presence of several intermediate states, including states with significantly altered core structure that still exhibit complete biological activity. Moreover, the refolding of alkaline phosphatase following denaturation in either chaotropic denaturants or low pH reveals a relatively fast refolding leading to the biologically active state, while laser spectroscopy measurements show a soft core which is annealing to the native-like state on a time-scale long compared to the return of activity. The active refolded protein is also initially characterized by an increase in susceptibility to denaturant. The slow annealing of the core is consistent with the presence of high energy barriers that separate fully active, long-lived, kinetic intermediate states along the folding pathway, a description suggested in the rugged energy landscape model.

  3. 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.

  4. 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.

  5. Role of the osmolyte taurine on the folding of a model protein, hen egg white lysozyme, under a crowding condition.

    Science.gov (United States)

    Abe, Yoshito; Ohkuri, Takatoshi; Yoshitomi, Sachiko; Murakami, Shigeru; Ueda, Tadashi

    2015-05-01

    Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did β-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did β-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.

  6. Design of a rotamer library for coarse-grained models in protein-folding simulations.

    Science.gov (United States)

    Larriva, María; Rey, Antonio

    2014-01-27

    Rotamer libraries usually contain geometric information to trace an amino acid side chain, atom by atom, onto a protein backbone. These libraries have been widely used in protein design, structure refinement and prediction, homology modeling, and X-ray and NMR structure validation. However, they usually present too much information and are not always fully compatible with the coarse-grained models of the protein geometry that are frequently used to tackle the protein-folding problem through molecular simulation. In this work, we introduce a new backbone-dependent rotamer library for side chains compatible with low-resolution models in polypeptide chains. We have dispensed with an atomic description of proteins, representing each amino acid side chain by its geometric center (or centroid). The resulting rotamers have been estimated from a statistical analysis of a large structural database consisting of high-resolution X-ray protein structures. As additional information, each rotamer includes the frequency with which it has been found during the statistical analysis. More importantly, the library has been designed with a careful control to ensure that the vast majority of side chains in protein structures (at least 95% of residues) are properly represented. We have tested our library using an independent set of proteins, and our results support a good correlation between the reconstructed centroids from our rotamer library and those in the experimental structures. This new library can serve to improve the definition of side chain centroids in coarse-grained models, avoiding at the same time an excessive additional complexity in a geometric model for the polypeptide chain.

  7. 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.

  8. Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality.

    Science.gov (United States)

    de Marco, Ario

    2017-01-01

    Molecular and chemical chaperones /foldases can strongly contribute to improve the amounts and the structural quality of recombinant proteins. Several methodologies have been proposed to optimize their beneficial effects. This chapter presents a condensed summary of the biotechnological opportunities offered by this approach followed by a protocol describing the method we use for expressing disulfide bond-dependent recombinant antibodies in the cytoplasm of bacteria engineered to overexpress sulfhydryl oxidase and DsbC isomerase. The system is based on the possibility to trigger the foldase expression independently and before the induction of the target protein. As a consequence, the recombinant antibody synthesis starts only after enough foldases have accumulated to promote correct folding of the antibody.

  9. 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)

  10. Markov modeling of peptide folding in the presence of protein crowders

    Science.gov (United States)

    Nilsson, Daniel; Mohanty, Sandipan; Irbäck, Anders

    2018-02-01

    We use Markov state models (MSMs) to analyze the dynamics of a β-hairpin-forming peptide in Monte Carlo (MC) simulations with interacting protein crowders, for two different types of crowder proteins [bovine pancreatic trypsin inhibitor (BPTI) and GB1]. In these systems, at the temperature used, the peptide can be folded or unfolded and bound or unbound to crowder molecules. Four or five major free-energy minima can be identified. To estimate the dominant MC relaxation times of the peptide, we build MSMs using a range of different time resolutions or lag times. We show that stable relaxation-time estimates can be obtained from the MSM eigenfunctions through fits to autocorrelation data. The eigenfunctions remain sufficiently accurate to permit stable relaxation-time estimation down to small lag times, at which point simple estimates based on the corresponding eigenvalues have large systematic uncertainties. The presence of the crowders has a stabilizing effect on the peptide, especially with BPTI crowders, which can be attributed to a reduced unfolding rate ku, while the folding rate kf is left largely unchanged.

  11. 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

  12. Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

    Science.gov (United States)

    Bandyopadhyay, Boudhayan; Goldenzweig, Adi; Unger, Tamar; Adato, Orit; Fleishman, Sarel J; Unger, Ron; Horovitz, Amnon

    2017-12-15

    The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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.

  14. 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.

  15. Wavelet analysis of hemispheroid flow separation toward understanding human vocal fold pathologies

    Science.gov (United States)

    Plesniak, Daniel H.; Carr, Ian A.; Bulusu, Kartik V.; Plesniak, Michael W.

    2014-11-01

    Physiological flows observed in human vocal fold pathologies, such as polyps and nodules, can be modeled by flow over a wall-mounted protuberance. The experimental investigation of flow separation over a surface-mounted hemispheroid was performed using particle image velocimetry (PIV) and measurements of surface pressure in a low-speed wind tunnel. This study builds on the hypothesis that the signatures of vortical structures associated with flow separation are imprinted on the surface pressure distributions. Wavelet decomposition methods in one- and two-dimensions were utilized to elucidate the flow behavior. First, a complex Gaussian wavelet was used for the reconstruction of surface pressure time series from static pressure measurements acquired from ports upstream, downstream, and on the surface of the hemispheroid. This was followed by the application of a novel continuous wavelet transform algorithm (PIVlet 1.2) using a 2D-Ricker wavelet for coherent structure detection on instantaneous PIV-data. The goal of this study is to correlate phase shifts in surface pressure with Strouhal numbers associated with the vortex shedding. Ultimately, the wavelet-based analytical framework will be aimed at addressing pulsatile flows. This material is based in part upon work supported by the National Science Foundation under Grant Number CBET-1236351, and GW Center for Biomimetics and Bioinspired Engineering (COBRE).

  16. 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.

  17. 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.

  18. 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.

  19. Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar.

    Science.gov (United States)

    Baxa, Michael C; Yu, Wookyung; Adhikari, Aashish N; Ge, Liang; Xia, Zhen; Zhou, Ruhong; Freed, Karl F; Sosnick, Tobin R

    2015-07-07

    Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.

  20. The structure of a haemopexin-fold protein from cow pea (Vigna unguiculata) suggests functional diversity of haemopexins in plants

    International Nuclear Information System (INIS)

    Gaur, Vineet; Chanana, Veenu; Jain, Abha; Salunke, Dinakar M.

    2011-01-01

    The structure of CP4, a haemopexin-fold protein from cow pea (Vigna unguiculata), has been determined at 2.1 Å resolution. The haemopexin fold is present in almost all life forms and is utilized for carrying out diverse physiological functions. The structure of CP4, a haemopexin-fold protein from cow pea (Vigna unguiculata), was determined at 2.1 Å resolution. The protein exists as a monomer both in solution and in the crystal. The structure revealed a typical four-bladed β-propeller topology. The protein exhibits 42% sequence similarity to LS-24 from Lathyrus sativus, with substantial differences in the surface-charge distribution and in the oligomeric state. A structure-based sequence analysis of haemopexin-fold proteins of plant and mammalian origin established a sequence signature associated with the haemopexin motif. This signature sequence enabled the identification of other proteins with possible haemopexin-like topology of both plant and animal origin. Although CP4 shares a structural fold with LS-24 and other haemopexins, biochemical studies indicated possible functional differences between CP4 and LS-24. While both of these proteins exhibit spermine-binding potential, CP4 does not bind to haem, unlike LS-24

  1. Combining classifiers generated by multi-gene genetic programming for protein fold recognition using genetic algorithm.

    Science.gov (United States)

    Bardsiri, Mahshid Khatibi; Eftekhari, Mahdi; Mousavi, Reza

    2015-01-01

    In this study the problem of protein fold recognition, that is a classification task, is solved via a hybrid of evolutionary algorithms namely multi-gene Genetic Programming (GP) and Genetic Algorithm (GA). Our proposed method consists of two main stages and is performed on three datasets taken from the literature. Each dataset contains different feature groups and classes. In the first step, multi-gene GP is used for producing binary classifiers based on various feature groups for each class. Then, different classifiers obtained for each class are combined via weighted voting so that the weights are determined through GA. At the end of the first step, there is a separate binary classifier for each class. In the second stage, the obtained binary classifiers are combined via GA weighting in order to generate the overall classifier. The final obtained classifier is superior to the previous works found in the literature in terms of classification accuracy.

  2. 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.

  3. 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.

  4. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    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...

  5. A membrane cell for on-line hydrogen/deuterium exchange to study protein folding and protein-protein interactions by mass spectrometry.

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-09-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

  6. A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry*

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-01-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation. PMID:21610101

  7. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    Science.gov (United States)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α -helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation.

  8. Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes.

    Science.gov (United States)

    Jedrychowski, Mark P; Liu, Libin; Laflamme, Collette J; Karastergiou, Kalypso; Meshulam, Tova; Ding, Shi-Ying; Wu, Yuanyuan; Lee, Mi-Jeong; Gygi, Steven P; Fried, Susan K; Pilch, Paul F

    2015-11-01

    Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx). Adrx knockdown and overexpressing 3T3-L1 murine adipocyte cell lines and a knockout mouse model were used to assess the influence of Adrx on secreted proteins as well as the redox state of ER resident chaperones. The metabolic phenotypes of Adrx null mice were characterized and compared to WT mice. The correlation of Adrx levels BMI, adiponectin levels, and other inflammatory markers from adipose tissue of human subjects was also studied. Adiporedoxin functions via a CXXC active site, and is upstream of protein disulfide isomerase whose direct function is disulfide bond formation, and ultimately protein secretion. Over and under expression of Adrx in vitro enhances and reduces, respectively, the secretion of the disulfide-bonded proteins including adiponectin and collagen isoforms. On a chow diet, Adrx null mice have normal body weights, and glucose tolerance, are moderately hyperinsulinemic, have reduced levels of circulating adiponectin and are virtually free of adipocyte fibrosis resulting in a complex phenotype tending towards insulin resistance. Adrx protein levels in human adipose tissue correlate positively with adiponectin levels and negatively with the inflammatory marker phospho-Jun kinase. These data support the notion that Adrx plays a critical role in adipocyte biology and in the regulation of mouse and human metabolism via its modulation of adipocyte protein secretion.

  9. 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.

  10. The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold

    Directory of Open Access Journals (Sweden)

    Izaurralde Elisa

    2003-12-01

    Full Text Available Abstract Background Multimeric protein complexes have a role in many cellular pathways and are highly interconnected with various other proteins. The characterization of their domain composition and organization provides useful information on the specific role of each region of their sequence. Results We identified a new module, the PAM domain (PCI/PINT associated module, present in single subunits of well characterized multiprotein complexes, like the regulatory lid of the 26S proteasome, the COP-9 signalosome and the Sac3-Thp1 complex. This module is an around 200 residue long domain with a predicted TPR-like all-alpha-helical fold. Conclusions The occurrence of the PAM domain in specific subunits of multimeric protein complexes, together with the role of other all-alpha-helical folds in protein-protein interactions, suggest a function for this domain in mediating transient binding to diverse target proteins.

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

    Science.gov (United States)

    Pineda-Lucena, Antonio; Liao, Jack C C; Cort, John R; Yee, Adelinda; Kennedy, Michael A; Edwards, Aled M; Arrowsmith, Cheryl H

    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 ORF 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 beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta 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. A feature extraction technique using bi-gram probabilities of position specific scoring matrix for protein fold recognition.

    Science.gov (United States)

    Sharma, Alok; Lyons, James; Dehzangi, Abdollah; Paliwal, Kuldip K

    2013-03-07

    Discovering a three dimensional structure of a protein is a challenging task in biological science. Classifying a protein into one of its folds is an intermediate step for deciphering the three dimensional protein structure. The protein fold recognition can be done by developing feature extraction techniques to accurately extract all the relevant information from a protein sequence and then by employing a suitable classifier to label an unknown protein. Several feature extraction techniques have been developed in the past but with limited recognition accuracy only. In this work, we have developed a feature extraction technique which is based on bi-grams computed directly from Position Specific Scoring Matrices and demonstrated its effectiveness on a benchmark dataset. The proposed technique exhibits an absolute improvement of around 10% compared with existing feature extraction techniques. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. A tri-gram based feature extraction technique using linear probabilities of position specific scoring matrix for protein fold recognition.

    Science.gov (United States)

    Paliwal, Kuldip K; Sharma, Alok; Lyons, James; Dehzangi, Abdollah

    2014-03-01

    In biological sciences, the deciphering of a three dimensional structure of a protein sequence is considered to be an important and challenging task. The identification of protein folds from primary protein sequences is an intermediate step in discovering the three dimensional structure of a protein. This can be done by utilizing feature extraction technique to accurately extract all the relevant information followed by employing a suitable classifier to label an unknown protein. In the past, several feature extraction techniques have been developed but with limited recognition accuracy only. In this study, we have developed a feature extraction technique based on tri-grams computed directly from Position Specific Scoring Matrices. The effectiveness of the feature extraction technique has been shown on two benchmark datasets. The proposed technique exhibits up to 4.4% improvement in protein fold recognition accuracy compared to the state-of-the-art feature extraction techniques.

  14. 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

  15. Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding

    Science.gov (United States)

    Sherma, Nisha D.

    2014-01-01

    Abstract Significance: Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease. Recent Advances: Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures. Critical Issues: Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation. Future Directions: The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility. Antioxid. Redox Signal. 21, 511–531. PMID:24383618

  16. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    Science.gov (United States)

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

  17. Protein folding and unfolding pathways: The role of energy barriers, configurational entropy and internal energy. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

    Science.gov (United States)

    Olivares-Quiroz, L.

    2017-07-01

    In this Review, Finkelstein et al. [1] provide an accurate, descriptive and scientifically solid discussion on a topic that has attracted the attention of the biophysics community during the past decades. The so-called protein folding problem remains as one of the most challenging issues at the interface of Physics and Molecular Biology, not only due to its academic and scientific relevance by itself, but also due to the vast amount of potential practical applications in drug design, molecular engineering and nanosciences in general [2,3]. Unveiling the physical mechanisms underlying protein folding and unfolding has thus certainly become one of the most relevant challenges faced by modern interdisciplinary research [4].

  18. 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.

  19. 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.

  20. Green-lighting green fluorescent protein: faster and more efficient folding by eliminating a cis-trans peptide isomerization event.

    Science.gov (United States)

    Rosenman, David J; Huang, Yao-ming; Xia, Ke; Fraser, Keith; Jones, Victoria E; Lamberson, Colleen M; Van Roey, Patrick; Colón, Wilfredo; Bystroff, Christopher

    2014-04-01

    Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type. © 2014 The Protein Society.

  1. Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

    Science.gov (United States)

    Liu, Shu-su; Wei, Xuan; Dong, Xue; Xu, Liang; Liu, Jia; Jiang, Biao

    2015-07-25

    Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations. Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence. We identified twelve amino acid deletions that can retain the fluorescence of GFP(UV). Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence. Our results suggested that a "size-minimized" GFP may be developed by

  2. 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.

  3. 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.

  4. Multiphase Simulated Annealing Based on Boltzmann and Bose-Einstein Distribution Applied to Protein Folding Problem.

    Science.gov (United States)

    Frausto-Solis, Juan; Liñán-García, Ernesto; Sánchez-Hernández, Juan Paulo; González-Barbosa, J Javier; González-Flores, Carlos; Castilla-Valdez, Guadalupe

    2016-01-01

    A new hybrid Multiphase Simulated Annealing Algorithm using Boltzmann and Bose-Einstein distributions (MPSABBE) is proposed. MPSABBE was designed for solving the Protein Folding Problem (PFP) instances. This new approach has four phases: (i) Multiquenching Phase (MQP), (ii) Boltzmann Annealing Phase (BAP), (iii) Bose-Einstein Annealing Phase (BEAP), and (iv) Dynamical Equilibrium Phase (DEP). BAP and BEAP are simulated annealing searching procedures based on Boltzmann and Bose-Einstein distributions, respectively. DEP is also a simulated annealing search procedure, which is applied at the final temperature of the fourth phase, which can be seen as a second Bose-Einstein phase. MQP is a search process that ranges from extremely high to high temperatures, applying a very fast cooling process, and is not very restrictive to accept new solutions. However, BAP and BEAP range from high to low and from low to very low temperatures, respectively. They are more restrictive for accepting new solutions. DEP uses a particular heuristic to detect the stochastic equilibrium by applying a least squares method during its execution. MPSABBE parameters are tuned with an analytical method, which considers the maximal and minimal deterioration of problem instances. MPSABBE was tested with several instances of PFP, showing that the use of both distributions is better than using only the Boltzmann distribution on the classical SA.

  5. Multiphase Simulated Annealing Based on Boltzmann and Bose-Einstein Distribution Applied to Protein Folding Problem

    Directory of Open Access Journals (Sweden)

    Juan Frausto-Solis

    2016-01-01

    Full Text Available A new hybrid Multiphase Simulated Annealing Algorithm using Boltzmann and Bose-Einstein distributions (MPSABBE is proposed. MPSABBE was designed for solving the Protein Folding Problem (PFP instances. This new approach has four phases: (i Multiquenching Phase (MQP, (ii Boltzmann Annealing Phase (BAP, (iii Bose-Einstein Annealing Phase (BEAP, and (iv Dynamical Equilibrium Phase (DEP. BAP and BEAP are simulated annealing searching procedures based on Boltzmann and Bose-Einstein distributions, respectively. DEP is also a simulated annealing search procedure, which is applied at the final temperature of the fourth phase, which can be seen as a second Bose-Einstein phase. MQP is a search process that ranges from extremely high to high temperatures, applying a very fast cooling process, and is not very restrictive to accept new solutions. However, BAP and BEAP range from high to low and from low to very low temperatures, respectively. They are more restrictive for accepting new solutions. DEP uses a particular heuristic to detect the stochastic equilibrium by applying a least squares method during its execution. MPSABBE parameters are tuned with an analytical method, which considers the maximal and minimal deterioration of problem instances. MPSABBE was tested with several instances of PFP, showing that the use of both distributions is better than using only the Boltzmann distribution on the classical SA.

  6. 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.

  7. Engineering of functional replication protein a homologs based on insights into the evolution of oligonucleotide/oligosaccharide-binding folds.

    Science.gov (United States)

    Lin, Yuyen; Lin, Li-Jung; Sriratana, Palita; Coleman, Kelli; Ha, Taekjip; Spies, Maria; Cann, Isaac K O

    2008-09-01

    The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains Bacteria and Eukarya, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon Methanosarcina acetivorans harbors multiple functional RPAs named MacRPA1 (for M. acetivorans RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in Methanococcoides burtonii and Methanosaeta thermophila. We further created two RPAs that mimicked the RPAs in Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus through fusions of modules from MacRPA1 and M. thermautotrophicus RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.

  8. Identification and analysis of key residues involved in folding and binding of protein-carbohydrate complexes.

    Science.gov (United States)

    Gromiha, Michael; Shanmugam, N R Siva; Selvin, J Fermin Angelo; Veluraja, K

    2018-02-21

    Protein-carbohydrate interactions play vital roles in several biological processes in living organisms. The comparative analysis of binding site residues along with stabilizing residues in protein-carbohydrate complexes provides ample insights to understand the structure, function and recognition mechanism. In this work, we have identified 2.45% binding site residues in a non-redundant dataset of 1130 complexes using distance-based criteria and 7.07% stabilizing residues using the concepts of hydrophobicity, long-range interactions and conservation of residues. Further, 5.9% of binding and 2.04% of stabilizing residues are common to each other, which are termed as key residues. The key residues have been analyzed based on protein classes, carbohydrate types, gene ontology functional classifications, amino acid preference and structure-based parameters. We found that all-β, α+β and α/β have more key residues than other protein classes and most of the KRs are present in β-strands, which shows their importance in stability and binding of complexes. On the ligand side, L-saccharide has the highest number of key residues and it has a high percentage of KRs in SRs and BRs than other carbohydrate types. Further, polar and charged residues have a high tendency to serve as key residues. Classifications based on gene ontology terms revealed that Lys is preferred in all the three groups: molecular functions, biological processes and cellular components. Key residues have 6 to 9 contacts within the protein and make only one contact with the carbohydrate ligand. These contacts are dominant to form polar-nonpolar contacts followed by the contacts between charged atoms. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. This analysis helps in understanding the interplay between stability and binding in protein

  9. 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.

  10. 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.

  11. 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.

  12. A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant

    OpenAIRE

    Reeder, Philippa J.; Huang, Yao-Ming; Dordick, Jonathan S.; Bystroff, Christopher

    2010-01-01

    The sequential order of secondary structural elements in proteins affects the folding and activity to an unknown extent. To test the dependence on sequential connectivity, secondary structural elements were reconnected by their solvent-exposed ends, permuting their sequential order, called “re-wiring.” This new protein design strategy changes the topology of the backbone without changing the core sidechain packing arrangement. While circular and non-circular permutations have been observed in...

  13. Loss of dispersion energy changes the stability and folding/unfolding equilibrium of the Trp-Cage protein

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Vondrášek, Jiří; Hobza, Pavel

    2009-01-01

    Roč. 113, č. 16 (2009), s. 5657-5660 ISSN 1520-6106 R&D Projects: GA MŠk LC512; GA ČR GA203/06/1727 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : Trp-Cage protein * dispersion energy * protein folding Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.471, year: 2009

  14. Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold.

    Science.gov (United States)

    Tan, Kemin; Cao, Nan; Cheng, Bokun; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

    2016-01-16

    The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52Å resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel β-sheet stabilized by a crossing-over α-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin; Cao, Nan; Cheng, Bokun; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

    2016-01-16

    The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 angstrom resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel beta-sheet stabilized by a crossing-over alpha-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.

  16. FoldIndex((c)) : a simple tool to predict whether a given protein sequence is intrinsically unfolded

    NARCIS (Netherlands)

    Prilusky, J; Felder, CE; Zeev-Ben-Mordehai, T; Rydberg, EH; Man, O; Beckmann, J.S.; Silman, I.; Sussman, J.L.

    2005-01-01

    An easy-to-use, versatile and freely available graphic web server, FoldIndex© is described: it predicts if a given protein sequence is intrinsically unfolded implementing the algorithm of Uversky and co-workers, which is based on the average residue hydrophobicity and net charge of the sequence.

  17. Folding equilibrium constants of telomere G-quadruplexes in free state or associated with proteins determined by isothermal differential hybridization.

    Science.gov (United States)

    Wang, Quan; Ma, Li; Hao, Yu-Hua; Tan, Zheng

    2010-11-15

    Guanine rich (G-rich) nucleic acids form G-quadruplex structures that are implicated in many biological processes, pharmaceutical applications, and molecular machinery. The folding equilibrium constant (K(F)) of the G-quadruplex not only determines its stability and competition against duplex formation in genomic DNA but also defines its recognition by proteins and drugs and technical specifications. The K(F) is most conveniently derived from thermal melting analysis that has so far yielded extremely diversified results for the human telomere G-quadruplex. Melting analysis cannot be used for nucleic acids associated with proteins, thus has difficulty to study how protein association affects the folding equilibrium of G-quadruplex structure. In this work, we established an isothermal differential hybridization (IDH) method that is able to determine the K(F) of G-quadruplex, either alone or associated with proteins. Using this method, we studied the folding equilibrium of the core sequence G(3)(T(2)AG(3))(3) from vertebrate telomere in K(+) and Na(+) solutions and how it is affected by proteins associated at its adjacent regions. Our results show that the K(F) obtained for the free G-quadruplex is within 1 order of magnitude of most of those obtained by melting analysis and protein binding beside a G-quadruplex can dramatically destabilize the G-quadruplex.

  18. Understanding, improving and using green fluorescent proteins.

    Science.gov (United States)

    Cubitt, A B; Heim, R; Adams, S R; Boyd, A E; Gross, L A; Tsien, R Y

    1995-11-01

    Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.

  19. Statistical dictionaries for hypothetical in silico model of the early-stage intermediate in protein folding

    Science.gov (United States)

    Kalinowska, Barbara; Fabian, Piotr; Stąpor, Katarzyna; Roterman, Irena

    2015-07-01

    The polypeptide chain folding process appears to be a multi-stage phenomenon. The scientific community has recently devoted much attention to early stages of this process, with numerous attempts at simulating them—either experimentally or in silico. This paper presents a comparative analysis of the predicted and observed results of folding simulations. The proposed technique, based on statistical dictionaries, yields a global accuracy of 57 %—a marked improvement over older approaches (with an accuracy of approximately 46 %).

  20. 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

    . 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...... active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important...... factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1...

  1. A discriminative method for protein remote homology detection and fold recognition combining Top-n-grams and latent semantic analysis.

    Science.gov (United States)

    Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

    2008-12-01

    Protein remote homology detection and fold recognition are central problems in bioinformatics. Currently, discriminative methods based on support vector machine (SVM) are the most effective and accurate methods for solving these problems. A key step to improve the performance of the SVM-based methods is to find a suitable representation of protein sequences. In this paper, a novel building block of proteins called Top-n-grams is presented, which contains the evolutionary information extracted from the protein sequence frequency profiles. The protein sequence frequency profiles are calculated from the multiple sequence alignments outputted by PSI-BLAST and converted into Top-n-grams. The protein sequences are transformed into fixed-dimension feature vectors by the occurrence times of each Top-n-gram. The training vectors are evaluated by SVM to train classifiers which are then used to classify the test protein sequences. We demonstrate that the prediction performance of remote homology detection and fold recognition can be improved by combining Top-n-grams and latent semantic analysis (LSA), which is an efficient feature extraction technique from natural language processing. When tested on superfamily and fold benchmarks, the method combining Top-n-grams and LSA gives significantly better results compared to related methods. The method based on Top-n-grams significantly outperforms the methods based on many other building blocks including N-grams, patterns, motifs and binary profiles. Therefore, Top-n-gram is a good building block of the protein sequences and can be widely used in many tasks of the computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the prediction of protein binding sites.

  2. A DsbA-Deficient Periplasm Enables Functional Display of a Protein with Redox-Sensitive Folding on M13 Phage.

    Science.gov (United States)

    Chen, Minyong; Samuelson, James C

    2016-06-14

    The requirements for target protein folding in M13 phage display are largely underappreciated. Here we chose Fbs1, a carbohydrate binding protein, as a model to address this issue. Importantly, folding of Fbs1 is impaired in an oxidative environment. Fbs1 can be displayed on M13 phage using the SRP or Sec pathway. However, the displayed Fbs1 protein is properly folded only when Fbs1 is translocated via the SRP pathway and displayed using Escherichia coli cells with a DsbA-negative periplasm. This study indicates M13 phage display may be improved using a system specifically designed according to the folding requirements of each target protein.

  3. A critical comparison of coarse-grained structure-based approaches and atomic models of protein folding.

    Science.gov (United States)

    Hu, Jie; Chen, Tao; Wang, Moye; Chan, Hue Sun; Zhang, Zhuqing

    2017-05-31

    Structure-based coarse-grained Gō-like models have been used extensively in deciphering protein folding mechanisms because of their simplicity and tractability. Meanwhile, explicit-solvent molecular dynamics (MD) simulations with physics-based all-atom force fields have been applied successfully to simulate folding/unfolding transitions for several small, fast-folding proteins. To explore the degree to which coarse-grained Gō-like models and their extensions to incorporate nonnative interactions are capable of producing folding processes similar to those in all-atom MD simulations, here we systematically compare the computed unfolded states, transition states, and transition paths obtained using coarse-grained models and all-atom explicit-solvent MD simulations. The conformations in the unfolded state in common Gō models are more extended, and are thus more in line with experiment, than those from all-atom MD simulations. Nevertheless, the structural features of transition states obtained by the two types of models are largely similar. In contrast, the folding transition paths are significantly more sensitive to modeling details. In particular, when common Gō-like models are augmented with nonnative interactions, the predicted dimensions of the unfolded conformations become similar to those computed using all-atom MD. With this connection, the large deviations of all-atom MD from simple diffusion theory are likely caused in part by the presence of significant nonnative effects in folding processes modelled by current atomic force fields. The ramifications of our findings to the application of coarse-grained modeling to more complex biomolecular systems are discussed.

  4. Comparison of two ESI-MS based H/D exchange methods for extracting protein folding energies

    Science.gov (United States)

    Liyanage, Rohana; Devarapalli, Nagarjuna; Puckett, Latisha M.; Phan, N. H.; Gidden, Jennifer; Stites, Wesley E.; Lay, Jackson O., Jr.

    2009-10-01

    In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate and mGdHCl values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained and mGdHCl values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear

  5. Solution structures of proteins from NMR data and modeling: Alternative folds for neutrophil peptide 5

    International Nuclear Information System (INIS)

    Levy, R.M.; Bassolino, D.A.; Kitchen, D.B.; Pardi, A.

    1989-01-01

    The structure of neutrophil peptide 5 in solution has recently reported. The structure determination was accomplished by using a distance geometry algorithm and 107 interproton distances constrains obtained from 2D NMR data. In each of the eight independent solutions to the distance geometry equations, the overall fold of the polypeptide backbone was identical and the root mean square (rms) deviation between backbone atoms of the superimposed structures was small. In this paper the authors report additional NP-5 structures obtained by using a new structure generation algorithm: a Monte Carlo search in torsion angle space. These structures have a large rms backbone deviation from the distance geometry structures. The backbone topologies differ in significant respects from the distance geometry structures and from each other. Structures are found that are pseudo mirror images of part or all of the fold corresponding to that first obtained with the distance geometry procedure. The results demonstrate that the previously accepted criteria for defining the accuracy and precision of a peptide structure generated from NMR data are inadequate. An energetic analysis of structures corresponding to the different folding topologies has been carried out. The molecular mechanics energies obtained by minimization and molecular dynamics refinement provide sufficient information to eliminate certain alternative structures. On the basis of a careful comparison of the different trial structures with the experimental data, it is concluded that the NP-5 peptide fold which was originally reported is most consistent with the data

  6. The alpha/beta-Hydrolase Fold 3DM Database (ABHDB) as a Tool for Protein Engineering

    NARCIS (Netherlands)

    Kourist, R.; Jochens, H.; Bartsch, S.; Kuipers, R.K.P.; Padhi, S.K.; Gall, M.; Bottcher, D.; Joosten, H.J.; Bornscheuer, U.T.

    2010-01-01

    Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/ß-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition

  7. 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.

  8. Understanding protein evolution: from protein physics to Darwinian selection.

    Science.gov (United States)

    Zeldovich, Konstantin B; Shakhnovich, Eugene I

    2008-01-01

    Efforts in whole-genome sequencing and structural proteomics start to provide a global view of the protein universe, the set of existing protein structures and sequences. However, approaches based on the selection of individual sequences have not been entirely successful at the quantitative description of the distribution of structures and sequences in the protein universe because evolutionary pressure acts on the entire organism, rather than on a particular molecule. In parallel to this line of study, studies in population genetics and phenomenological molecular evolution established a mathematical framework to describe the changes in genome sequences in populations of organisms over time. Here, we review both microscopic (physics-based) and macroscopic (organism-level) models of protein-sequence evolution and demonstrate that bridging the two scales provides the most complete description of the protein universe starting from clearly defined, testable, and physiologically relevant assumptions.

  9. 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

    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......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...

  10. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  11. Use of multiple picosecond high-mass molecular dynamics simulations to predict crystallographic B-factors of folded globular proteins

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    2016-09-01

    Full Text Available Predicting crystallographic B-factors of a protein from a conventional molecular dynamics simulation is challenging, in part because the B-factors calculated through sampling the atomic positional fluctuations in a picosecond molecular dynamics simulation are unreliable, and the sampling of a longer simulation yields overly large root mean square deviations between calculated and experimental B-factors. This article reports improved B-factor prediction achieved by sampling the atomic positional fluctuations in multiple picosecond molecular dynamics simulations that use uniformly increased atomic masses by 100-fold to increase time resolution. Using the third immunoglobulin-binding domain of protein G, bovine pancreatic trypsin inhibitor, ubiquitin, and lysozyme as model systems, the B-factor root mean square deviations (mean ± standard error of these proteins were 3.1 ± 0.2–9 ± 1 Å2 for Cα and 7.3 ± 0.9–9.6 ± 0.2 Å2 for Cγ, when the sampling was done for each of these proteins over 20 distinct, independent, and 50-picosecond high-mass molecular dynamics simulations with AMBER forcefield FF12MC or FF14SB. These results suggest that sampling the atomic positional fluctuations in multiple picosecond high-mass molecular dynamics simulations may be conducive to a priori prediction of crystallographic B-factors of a folded globular protein.

  12. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  13. Protein folding and quality control in the endoplasmic reticulum: Recent lessons from yeast and mammalian cell systems.

    Science.gov (United States)

    Brodsky, Jeffrey L; Skach, William R

    2011-08-01

    The evolution of eukaryotes was accompanied by an increased need for intracellular communication and cellular specialization. Thus, a more complex collection of secreted and membrane proteins had to be synthesized, modified, and folded. The endoplasmic reticulum (ER) thereby became equipped with devoted enzymes and associated factors that both catalyze the production of secreted proteins and remove damaged proteins. A means to modify ER function to accommodate and destroy misfolded proteins also evolved. Not surprisingly, a growing number of human diseases are linked to various facets of ER function. Each of these topics will be discussed in this article, with an emphasis on recent reports in the literature that employed diverse models. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Understanding the neurospecificity of Prion protein signaling.

    Science.gov (United States)

    Schneider, Benoit; Pietri, Mathea; Pradines, Elodie; Loubet, Damien; Launay, Jean-Marie; Kellermann, Odile; Mouillet-Richard, Sophie

    2011-01-01

    The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.

  15. Aspergillus niger protein estA defines a new class of fungal esterases within the alfa/beta hydrolase fold superfamily of proteins

    NARCIS (Netherlands)

    Bourne, Y.; Hasper, A.A.; Chahinian, H.; Juin, M.; Graaff, de L.H.

    2004-01-01

    From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipaseacetylcholinesterase

  16. ReFOLD: a server for the refinement of 3D protein models guided by accurate quality estimates.

    Science.gov (United States)

    Shuid, Ahmad N; Kempster, Robert; McGuffin, Liam J

    2017-07-03

    ReFOLD is a novel hybrid refinement server with integrated high performance global and local Accuracy Self Estimates (ASEs). The server attempts to identify and to fix likely errors in user supplied 3D models of proteins via successive rounds of refinement. The server is unique in providing output for multiple alternative refined models in a way that allows users to quickly visualize the key residue locations, which are likely to have been improved. This is important, as global refinement of a full chain model may not always be possible, whereas local regions, or individual domains, can often be much improved. Thus, users may easily compare the specific regions of the alternative refined models in which they are most interested e.g. key interaction sites or domains. ReFOLD was used to generate hundreds of alternative refined models for the CASP12 experiment, boosting our group's performance in the main tertiary structure prediction category. Our successful refinement of initial server models combined with our built-in ASEs were instrumental to our second place ranking on Template Based Modeling (TBM) and Free Modeling (FM)/TBM targets. The ReFOLD server is freely available at: http://www.reading.ac.uk/bioinf/ReFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. 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

  18. 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.

  19. A comparative method for finding and folding RNA secondary structures within protein-coding regions

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Meyer, Irmtraud Margret; Forsberg, Roald

    2004-01-01

    that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known...... secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD....

  20. Numerical impact simulation of gradually increased kinetic energy transfer has the potential to break up folded protein structures resulting in cytotoxic brain tissue edema.

    Science.gov (United States)

    von Holst, Hans; Li, Xiaogai

    2013-07-01

    Although the consequences of traumatic brain injury (TBI) and its treatment have been improved, there is still a substantial lack of understanding the mechanisms. Numerical simulation of the impact can throw further lights on site and mechanism of action. A finite element model of the human head and brain tissue was used to simulate TBI. The consequences of gradually increased kinetic energy transfer was analyzed by evaluating the impact intracranial pressure (ICP), strain level, and their potential influences on binding forces in folded protein structures. The gradually increased kinetic energy was found to have the potential to break apart bonds of Van der Waals in all impacts and hydrogen bonds at simulated impacts from 6 m/s and higher, thereby superseding the energy in folded protein structures. Further, impacts below 6 m/s showed none or very slight increase in impact ICP and strain levels, whereas impacts of 6 m/s or higher showed a gradual increase of the impact ICP and strain levels reaching over 1000 KPa and over 30%, respectively. The present simulation study shows that the free kinetic energy transfer, impact ICP, and strain levels all have the potential to initiate cytotoxic brain tissue edema by unfolding protein structures. The definition of mild, moderate, and severe TBI should thus be looked upon as the same condition and separated only by a gradual severity of impact.

  1. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

    2013-01-01

    of this classification suggests that the balance between favoring and disfavoring structural features determines if a pair of proteins interacts or not. Our results are in agreement with previous works and support the funnel-like intermolecular energy landscape theory that explains PPIs. We have used these features...

  2. TDP-43 Proteinopathies: A New Player in Neurodegenerative Diseases with Defective Protein Folding

    Directory of Open Access Journals (Sweden)

    Suna Lahut

    2012-03-01

    Full Text Available The proteome is the sum of all proteins inside a cell, and proteostasis (protein homeostasis is the stable condition of the proteome. Proteostasis is essential for the cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins challenge the proteostasis machinery and the vitality of the cell. There is increasing evidence that the accumulation of damaged proteins not only has direct consequences on the efficiency and fidelity of cellular processes but, when not corrected, that they initiate a cascade of dysfunction, which in humans is associated with a plethora of diseases of protein conformation, referred to as proteinopathies. Alzheimer’s Disease (AD, Parkinson’s Disease (PD, Huntington’s Disease (HD, Amyotrophic Lateral Sclerosis (ALS, cancer and diabetes, whose frequencies have drastically increased in countries with aging populations, are all consequences of misfolded proteins. This paper focuses on TDP-43, which excelled as a key protein in neurodegenerative processes because of its association with different diseases, especially with ALS and Frontotemporal Lobar Dementia (FTLD, the two best studied examples of TDP-43 proteinopathies.

  3. Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

    Energy Technology Data Exchange (ETDEWEB)

    Kozlov, G.; Määttänen, P; Schrag, J; Hura, G; Gabrielli, L; Cygler, M; Thomas, D; Gehring, K

    2009-01-01

    Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

  4. TDP-43 Proteinopathies: A New Player in Neurodegenerative Diseases with Defective Protein Folding

    Directory of Open Access Journals (Sweden)

    Suna Lahut

    2012-03-01

    Full Text Available The proteome is the sum of all proteins inside a cell, and proteostasis (protein homeostasis is the stable condition of the proteome. Proteostasis is essential for the cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins challenge the proteostasis machinery and the vitality of the cell. There is increasing evidence that the accumulation of damaged proteins not only has direct consequences on the efficiency and fidelity of cellular processes but, when not corrected, that they initiate a cascade of dysfunction, which in humans is associated with a plethora of diseases of protein conformation, referred to as proteinopathies. Alzheimer’s Disease (AD, Parkinson’s Disease (PD, Huntington’s Disease (HD, Amyotrophic Lateral Sclerosis (ALS, cancer and diabetes, whose frequencies have drastically increased in countries with aging populations, are all consequences of misfolded proteins. This paper focuses on TDP-43, which excelled as a key protein in neurodegenerative processes because of its association with different diseases, especially with ALS and Frontotemporal Lobar Dementia (FTLD, the two best studied examples of TDP-43 proteinopathies

  5. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  6. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

    Science.gov (United States)

    Wang, Tao; Darwin, K. Heran; Li, Huilin

    2010-01-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analogue of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein bearing little sequence or structural resemblance to the highly structured ubiquitin. Thus it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled-coils that recognize Pup. Mpa binds unstructured Pup via hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work revealed a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This critical difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment of tuberculosis. PMID:20953180

  7. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    Energy Technology Data Exchange (ETDEWEB)

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  8. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

    Science.gov (United States)

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

    2016-06-20

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/.

  9. GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Marzec, Michal; Eletto, Davide; Argon, Yair

    2012-01-01

    Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94...

  10. The Mitochondrial Disulfide Relay System: Roles in Oxidative Protein Folding and Beyond

    Directory of Open Access Journals (Sweden)

    Manuel Fischer

    2013-01-01

    Full Text Available Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS. The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes in vitro and in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.

  11. Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion

    OpenAIRE

    O’Brien, Darragh P.; Hernandez, Belen; Durand, Dominique; Hourdel, Véronique; Sotomayor-Pérez, Ana-Cristina; Vachette, Patrice; Ghomi, Mahmoud; Chamot-Rooke, Julia; Ladant, Daniel; Brier, Sébastien; Chenal, Alexandre

    2016-01-01

    International audience; Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a f...

  12. %MinMax: A versatile tool for calculating and comparing synonymous codon usage and its impact on protein folding.

    Science.gov (United States)

    Rodriguez, Anabel; Wright, Gabriel; Emrich, Scott; Clark, Patricia L

    2018-01-01

    Most amino acids can be encoded by more than one synonymous codon, but these are rarely used with equal frequency. In many coding sequences the usage patterns of rare versus common synonymous codons is nonrandom and under selection. Moreover, synonymous substitutions that alter these patterns can have a substantial impact on the folding efficiency of the encoded protein. This has ignited broad interest in exploring synonymous codon usage patterns. For many protein chemists, biophysicists and structural biologists, the primary motivation for codon analysis is identifying and preserving usage patterns most likely to impact high-yield production of functional proteins. Here we describe the core functions and new features of %MinMax, a codon usage calculator freely available as a web-based portal and downloadable script (http://www.codons.org). %MinMax evaluates the relative usage frequencies of the synonymous codons used to encode a protein sequence of interest and compares these results to a rigorous null model. Crucially, for analyzing codon usage in common host organisms %MinMax requires only the coding sequence as input; with a user-input codon frequency table, %MinMax can be used to evaluate synonymous codon usage patterns for any coding sequence from any fully sequenced genome. %MinMax makes no assumptions regarding the impact of transfer ribonucleic acid concentrations or other molecular-level interactions on translation rates, yet its output is sufficient to predict the effects of synonymous codon substitutions on cotranslational folding mechanisms. A simple calculation included within %MinMax can be used to harmonize codon usage frequencies for heterologous gene expression. © 2017 The Protein Society.

  13. The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

    Science.gov (United States)

    A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

  14. Identification of Capsid/Coat Related Protein Folds and Their Utility for Virus Classification

    OpenAIRE

    Nasir, Arshan; Caetano-Anoll?s, Gustavo

    2017-01-01

    The viral supergroup includes the entire collection of known and unknown viruses that roam our planet and infect life forms. The supergroup is remarkably diverse both in its genetics and morphology and has historically remained difficult to study and classify. The accumulation of protein structure data in the past few years now provides an excellent opportunity to re-examine the classification and evolution of viruses. Here we scan completely sequenced viral proteomes from all genome types an...

  15. Probing the Ca2+-assisted pi-pi interaction during Ca2+-dependent protein folding

    Czech Academy of Sciences Publication Activity Database

    Matyska Lišková, Petra; Fišer, Radovan; Macek, Pavel; Chmelík, Josef; Sýkora, Jan; Bednárová, Lucie; Konopásek, I.; Bumba, Ladislav

    2016-01-01

    Roč. 12, č. 2 (2016), s. 531-541 ISSN 1744-683X R&D Projects: GA ČR(CZ) GAP207/11/0717; GA ČR(CZ) GBP208/12/G016; GA MŠk LO1509 Institutional support: RVO:61388971 ; RVO:61388963 ; RVO:61388955 Keywords : METAL-ION-BINDING * NEISSERIA-MENINGITIDIS * RTX PROTEINS Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.889, year: 2016

  16. Recurrent oligomers in proteins: an optimal scheme reconciling accurate and concise backbone representations in automated folding and design studies.

    Science.gov (United States)

    Micheletti, C; Seno, F; Maritan, A

    2000-09-01

    A novel scheme is introduced to capture the spatial correlations of consecutive amino acids in naturally occurring proteins. This knowledge-based strategy is able to carry out optimally automated subdivisions of protein fragments into classes of similarity. The goal is to provide the minimal set of protein oligomers (termed "oligons" for brevity) that is able to represent any other fragment. At variance with previous studies in which recurrent local motifs were classified, our concern is to provide simplified protein representations that have been optimised for use in automated folding and/or design attempts. In such contexts, it is paramount to limit the number of degrees of freedom per amino acid without incurring loss of accuracy of structural representations. The suggested method finds, by construction, the optimal compromise between these needs. Several possible oligon lengths are considered. It is shown that meaningful classifications cannot be done for lengths greater than six or smaller than four. Different contexts are considered for which oligons of length five or six are recommendable. With only a few dozen oligons of such length, virtually any protein can be reproduced within typical experimental uncertainties. Structural data for the oligons are made publicly available.

  17. 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.

  18. 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.

  19. Redesigning the type II' β-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

    Science.gov (United States)

    Madan, Bharat; Sokalingam, Sriram; Raghunathan, Govindan; Lee, Sun-Gu

    2014-10-01

    Both Type I' and Type II' β-turns have the same sense of the β-turn twist that is compatible with the β-sheet twist. They occur predominantly in two residue β-hairpins, but the occurrence of Type I' β-turns is two times higher than Type II' β-turns. This suggests that Type I' β-turns may be more stable than Type II' β-turns, and Type I' β-turn sequence and structure can be more favorable for protein folding than Type II' β-turns. Here, we redesigned the native Type II' β-turn in GFP to Type I' β-turn, and investigated its effect on protein folding and stability. The Type I' β-turns were designed based on the statistical analysis of residues in natural Type I' β-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β-turns in natural β-hairpins can be further optimized by converting the sequence to Type I'. © 2014 Wiley Periodicals, Inc.

  20. Role of Gln 85 of human CYP27A1 in 25-hydroxyvitamin D(3)-binding and protein folding.

    Science.gov (United States)

    Sawada, Natsumi; Yamamoto, Keiko; Yamada, Sachiko; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Inouye, Kuniyo; Sakaki, Toshiyuki

    2007-03-30

    CYP27A1 catalyzes vitamin D(3) 25-hydroxylation and further hydroxylation at C-1alpha, C-24 or C-26(27). Molecular modeling of human CYP27A1 and docking with 25-hydroxyvitamin D(3) predicted that Gln 85 might be important for 1alpha-hydroxylation activity of CYP27A1 by forming a hydrogen bond with the 25-OH group of 25-hydroxyvitamin D(3). Expectedly, the mutant Q85H expressed in Escherichia coli showed no detectable 1alpha-hydroxylation activity toward 25-hydroxyvitamin D(3). In addition, Q85H prefers 24-hydroxylation toward 25-hydroxyvitamin D(3) whereas the wild-type prefers 26(27)-hydroxylation. A molecular modeling study also suggests that Gln 85 of CYP27A1 simultaneously interacts with Asn 107 and the hydroxyl group of the substrate. The fact that Q85L did not contain a heme molecule suggests that the hydrogen bond between Gln 85 and Asn 107 is important for protein folding of CYP27A1. Based on these results, it is possible that Gln 85 plays essential roles in both substrate-binding and protein folding.

  1. BIOSYNTHESIS, CHARACTERIZATION AND APPLICATION OF TIO2 NANOPARTICLES IN BIOCATALYSIS AND PROTEIN FOLDING

    Directory of Open Access Journals (Sweden)

    Razi Ahmad,

    2013-08-01

    Full Text Available The nano-TiO2 was synthesized using Lactobacillus sp. and characterized by XRD and TEM. The X-ray diffraction showed that TiO2 nanoparticles were crystalline in nature. TEM images revealed that these particles are irregular in shape with an average particle size of 50–100 nm. The biosynthesized nanoparticles were used for the immobilization and refolding of thermally inactivated alpha amylase enzyme. The enzyme after adsorption on TiO2 nanoparticles retained 71% of enzyme activity. The immobilized enzyme was found to be thermally more stable as compared to the free enzyme. When the enzyme was heated to 60°C for 60 min the free enzyme loses all of its activity whereas the adsorbed enzyme retained 82% of its activity.The adsorbed/immobilized protein could be reused five times without any loss in enzyme activity. The operational stability data also shows that after immobilization the stability of alpha amylase increases. To study the nanoparticles-protein interaction, alpha amylase enzyme was inactivated by heating at 60°C for 1 hour. The thermally inactivated alpha amylase when incubated with the biosynthesized TiO2 nanoparticles regains nearly 65% activity after 2.0 hour. Thus TiO2 nanoparticles assist in refolding of the enzyme.

  2. The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli*

    Science.gov (United States)

    Mehner, Denise; Osadnik, Hendrik; Lünsdorf, Heinrich; Brüser, Thomas

    2012-01-01

    Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons. PMID:22689583

  3. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    Daisuke Ishibashi

    Full Text Available Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK, derived from a patient with Gerstmann-Sträussler-Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.

  4. A Novel, Highly Stable Fold of the Immunoglobulin Binding Domain of Streptococcal Protein G

    Science.gov (United States)

    Gronenborn, Angela M.; Filpula, David R.; Essig, Nina Z.; Achari, Aniruddha; Whitlow, Marc; Wingfield, Paul T.; Marius Clore, G.

    1991-08-01

    The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH_2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (overset{circ}{mathrm A}) for the backbone atoms, 0.65 overset{circ}{mathrm A} for all atoms, and 0.39 overset{circ}{mathrm A} for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded β sheet, on top of which lies a long helix. The central two strands (β 1 and β 4), comprising the NH_2- and COOH-termini, are parallel, and the outer two strands (β 2 and β 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87^circC).

  5. 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.

  6. Single-Molecule Protein Folding: A Study of the Surface-Mediated Conformational Dynamics of a Model Amphipathic Peptide

    Science.gov (United States)

    Cunningham, Joy; English, Douglas

    2004-03-01

    Most surface-active polypeptides, composed of 10-50 amino acids, are devoid of well-defined tertiary structure. The conformation of these proteins is greatly dependent upon their environment and may assume totally different characteristics in an aqueous environment, in a detergent micelle, or in an organic solvent. Most antimicrobial peptides are helix-forming and are activated upon interaction with a membrane-mimicking environment. We are seeking to physically characterize the mechanism of membrane-peptide interaction through studying a simple model peptide, MT-1. MT-1 was designed as a nonhomologous analogue of melittin, the principle component in bee venom. We are using single molecule spectroscopy to examine the induction of secondary structure upon interaction of MT-1 with various membrane-mimicking interfaces. Specifically, we monitor coil-to-helix transition through single molecule fluorescence resonance energy transfer (sm-FRET) to determine conformational distributions of folded and unfolded peptides at an interface. Studies with MT-1 will focus upon the biologically relevant issues of orientation, aggregation, and folding at surfaces using both ensemble and single molecule experiments.

  7. Methionine sulfoxides on prion protein Helix-3 switch on the alpha-fold destabilization required for conversion.

    Directory of Open Access Journals (Sweden)

    Giorgio Colombo

    Full Text Available BACKGROUND: The conversion of the cellular prion protein (PrP(C into the infectious form (PrP(Sc is the key event in prion induced neurodegenerations. This process is believed to involve a multi-step conformational transition from an alpha-helical (PrP(C form to a beta-sheet-rich (PrP(Sc state. In addition to the conformational difference, PrP(Sc exhibits as covalent signature the sulfoxidation of M213. To investigate whether such modification may play a role in the misfolding process we have studied the impact of methionine oxidation on the dynamics and energetics of the HuPrP(125-229 alpha-fold. METHODOLOGY/PRINCIPAL FINDINGS: Using molecular dynamics simulation, essential dynamics, correlated motions and signal propagation analysis, we have found that substitution of the sulfur atom of M213 by a sulfoxide group impacts on the stability of the native state increasing the flexibility of regions preceding the site of the modification and perturbing the network of stabilizing interactions. Together, these changes favor the population of alternative states which maybe essential in the productive pathway of the pathogenic conversion. These changes are also observed when the sulfoxidation is placed at M206 and at both, M206 and M213. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the sulfoxidation of Helix-3 methionines might be the switch for triggering the initial alpha-fold destabilization required for the productive pathogenic conversion.

  8. Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities

    Czech Academy of Sciences Publication Activity Database

    Durech, M.; Trčka, F.; Man, Petr; Blackburn, E.A.; Hernychová, L.; Dvořáková, P.; Coufalová, D.; Kavan, Daniel; Vojtěšek, B.; Muller, P.

    2016-01-01

    Roč. 15, č. 5 (2016), s. 1710-1727 ISSN 1535-9476 R&D Projects: GA MŠk(CZ) LO1509 EU Projects: Wellcome Trust(CZ) 01527/Z/13/Z Institutional support: RVO:61388971 Keywords : HEAT-SHOCK-PROTEIN * MOLECULAR CHAPERONE DNAK * SUBSTRATE-BINDING DOMAIN * INVASIVE BREAST-CANCER Subject RIV: CE - Biochemistry Impact factor: 6.540, year: 2016

  9. Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

    Science.gov (United States)

    Ren, Weitong; Li, Wenfei; Wang, Jun; Zhang, Jian; Wang, Wei

    2017-10-26

    Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

  10. 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

  11. Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

    Science.gov (United States)

    Shih, Ying-Chun; Chen, Chao-Ling; Zhang, Yan; Mellor, Rebecca L; Kanter, Evelyn M; Fang, Yun; Wang, Hua-Chi; Hung, Chen-Ting; Nong, Jing-Yi; Chen, Hui-Ju; Lee, Tzu-Han; Tseng, Yi-Shuan; Chen, Chiung-Nien; Wu, Chau-Chung; Lin, Shuei-Liong; Yamada, Kathryn A; Nerbonne, Jeanne M; Yang, Kai-Chien

    2018-03-13

    Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure (HF). Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting co-expression gene network analysis on RNA sequencing data from failing human heart, we identified thioredoxin domain containing 5 (TXNDC5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum (ER) protein, as a potential novel mediator of cardiac fibrosis and we completed experiments to test this hypothesis directly. Objective: To determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. Methods and Results: RNASeq and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles (LV) and in hypertrophied/failing mouse LV. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 (TGFβ1) and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under TGFβ1 stimulation in vitro. Knockdown of TXNDC5 attenuated TGFβ1-induced hCF activation and ECM protein upregulation independent of SMAD3, whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing JNK activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. TGFβ1-induced TXNDC5 upregulation in hCF was dependent on ER stress and

  12. Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35.

    Science.gov (United States)

    Pocanschi, Cosmin L; Popot, Jean-Luc; Kleinschmidt, Jörg H

    2013-03-01

    Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55-64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1-2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5-9 kJ/mol for the fast process and ~29-37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.

  13. A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria.

    Science.gov (United States)

    Fleming, Karen G

    2015-10-05

    In vitro folding studies of outer membrane beta-barrels have been invaluable in revealing the lipid effects on folding rates and efficiencies as well as folding free energies. Here, the biophysical results are summarized, and these kinetic and thermodynamic findings are considered in terms of the requirements for folding in the context of the cellular environment. Because the periplasm lacks an external energy source the only driving forces for sorting and folding available within this compartment are binding or folding free energies and their associated rates. These values define functions for periplasmic chaperones and suggest a biophysical mechanism for the BAM complex. © 2015 The Author(s).

  14. Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

    Science.gov (United States)

    Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

    2017-04-01

    Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Holden, Paul; Bächinger, Hans Peter

    2017-10-20

    Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/ SERPINH1 ) and 65-kDa FK506-binding protein (FKBP65/ FKBP10 ), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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.

  17. 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.

  18. Crystal structure of ATVORF273, a new fold for a thermo- and acido-stable protein from the Acidianus two-tailed virus

    DEFF Research Database (Denmark)

    Felisberto-Rodrigues, Catarina; Blangy, Stéphanie; Goulet, Adeline

    2012-01-01

    in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV[Formula: see text]. ATV[Formula: see text] forms tetramers in solution and a molecular envelope is provided...... for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV[Formula: see text......] displays a new [Formula: see text] protein fold, consistent with the absence of homologues of this protein in public sequence databases....

  19. Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation.

    Science.gov (United States)

    Tehver, Riina; Thirumalai, D

    2008-04-04

    The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (TR-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the

  20. 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.

  1. Mechanistic understanding of protein-silicone oil interactions.

    Science.gov (United States)

    Li, Jinjiang; Pinnamaneni, Swathi; Quan, Yong; Jaiswal, Archana; Andersson, Fredrik I; Zhang, Xiaochun

    2012-06-01

    To investigate interactions between protein and silicone oil so that we can provide some mechanistic understanding of protein aggregation in silicone oil lubricated syringes and its prevention by formulation additives such as Polysorbate 80 and Poloxamer 188. Interfacial tension values of silicone oil/water interface of abatacept solutions with and without formulation additives were obtained under equilibrium conditions using Attension Theta optical tensiometer. Their adsorption and desorption profiles were measured using Quartz Crystal Microbalancing with Dissipation monitoring (QCM-D). The degree of aggregation of abatacept was assessed based on size exclusion measurement. Adsorption of abatacept at the oil/water interface was shown. Polysorbat 80 was more effective than Poloxamer 188 in preventing abatacept adsorption. Moreover, it was noted that some of the adsorbed abatacept molecules were not desorbed readily upon buffer rinse. Finally, no homogeneous aggregation was observed at room temperature and a slight increase of aggregation was only observed for samples measured at 40°C which can be prevented using Polysorbate 80. Interfacial adsorption of proteins is the key step and maybe responsible for the phenomenon of soluble-protein loss when contacting silicone oil and the irreversible adsorption of protein may be associated with protein denaturation/aggregation.

  2. The structure of mouse cytomegalovirus m04 protein obtained from sparse NMR data reveals a conserved fold of the m02-m06 viral immune modulator family.

    Science.gov (United States)

    Sgourakis, Nikolaos G; Natarajan, Kannan; Ying, Jinfa; Vogeli, Beat; Boyd, Lisa F; Margulies, David H; Bax, Ad

    2014-09-02

    Immunoevasins are key proteins used by viruses to subvert host immune responses. Determining their high-resolution structures is key to understanding virus-host interactions toward the design of vaccines and other antiviral therapies. Mouse cytomegalovirus encodes a unique set of immunoevasins, the m02-m06 family, that modulates major histocompatibility complex class I (MHC-I) antigen presentation to CD8+ T cells and natural killer cells. Notwithstanding the large number of genetic and functional studies, the structural biology of immunoevasins remains incompletely understood, largely because of crystallization bottlenecks. Here we implement a technology using sparse nuclear magnetic resonance data and integrative Rosetta modeling to determine the structure of the m04/gp34 immunoevasin extracellular domain. The structure reveals a β fold that is representative of the m02-m06 family of viral proteins, several of which are known to bind MHC-I molecules and interfere with antigen presentation, suggesting its role as a diversified immune regulation module. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. 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/.

  4. The UlaG protein family defines novel structural and functional motifs grafted on an ancient RNase fold

    Directory of Open Access Journals (Sweden)

    Coll Miquel

    2011-09-01

    Full Text Available Abstract Background Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT. A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-β-lactamase (MBL enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gram-negative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk

  5. Recovering kinetics from a simplified protein folding model using replica exchange simulations: a kinetic network and effective stochastic dynamics.

    Science.gov (United States)

    Zheng, Weihua; Andrec, Michael; Gallicchio, Emilio; Levy, Ronald M

    2009-08-27

    We present an approach to recover kinetics from a simplified protein folding model at different temperatures using the combined power of replica exchange (RE), a kinetic network, and effective stochastic dynamics. While RE simulations generate a large set of discrete states with the correct thermodynamics, kinetic information is lost due to the random exchange of temperatures. We show how we can recover the kinetics of a 2D continuous potential with an entropic barrier by using RE-generated discrete states as nodes of a kinetic network. By choosing the neighbors and the microscopic rates between the neighbors appropriately, the correct kinetics of the system can be recovered by running a kinetic simulation on the network. We fine-tune the parameters of the network by comparison with the effective drift velocities and diffusion coefficients of the system determined from short-time stochastic trajectories. One of the advantages of the kinetic network model is that the network can be built on a high-dimensional discretized state space, which can consist of multiple paths not consistent with a single reaction coordinate.

  6. Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

    Science.gov (United States)

    Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

    2015-01-30

    Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Folding of small proteins by Monte Carlo simulations with chemical shift restraints without the use of molecular fragment replacement or structural homology.

    Science.gov (United States)

    Robustelli, Paul; Cavalli, Andrea; Dobson, Christopher M; Vendruscolo, Michele; Salvatella, Xavier

    2009-06-04

    It has recently been shown that protein structures can be determined from nuclear magnetic resonance (NMR) chemical shifts using a molecular fragment replacement strategy. In these approaches, structural motifs are selected from existing protein structures on the basis of chemical shift and sequence homology and assembled to generate new structures. Here, we demonstrate that it is also possible to determine structures of proteins by directly incorporating experimental NMR chemical shifts as structural restraints in conformational searches, without the use of structural homology and molecular fragment replacement. In this approach, a protein is folded from an extended conformation to its native state using a simulated annealing procedure that minimizes an energy function that combines a standard force field with a term that penalizes the differences between experimental and calculated chemical shifts. We provide an initial demonstration of this procedure by determining the structure of two small proteins, with alpha and beta folds, respectively.

  8. Mechanistic understanding of fouling of protein A chromatography resin.

    Science.gov (United States)

    Pathak, Mili; Rathore, Anurag S

    2016-08-12

    This paper aims to provide a thorough understanding of how fouling of Protein A resin takes place. Binding and mass transport properties of widely used agarose-based Protein A resin, MabSelect SuRe™, have been examined to understand the mechanism of resin fouling. There could be various factors that impact resin fouling. These include product/impurity build-up due to components in the feed material and ligand degradation due to the use of harsh buffers. To unravel their contributions, cycling studies were performed with and without product loading. The results presented in this paper provide a lucid understanding of the causative factors that limit Protein A chromatographic resin lifetime. The capacity fall for protein A resin at the end of 100th cycle due to use of feed material was found to be five times greater than that without using feed material. Compared to the fresh resin, the cycled resin samples shows 24% reduction in particle porosity and 51% reduction in pore mass transfer coefficient. Transmission electron microscopy (TEM) was used to qualitatively monitor accumulation of foulants on the cycled resin. Fouled resin sample contained a dense residue in the interior and exterior of resin particle both as a film at the bead surface and as granules. The surface activation energy increased five times in the case of fouled resin sample. The major event in fouling was identified as the non-specific adsorption of the feed material components on resin, signaling that pore diffusion is the rate limiting step. It is anticipated that these findings will assist in development of a more robust and economical downstream manufacturing process for monoclonal antibody purification. Copyright © 2016 Elsevier B.V. All rights reserved.

  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…

  10. 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.

  11. Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.

    Science.gov (United States)

    Zybailov, Boris; Friso, Giulia; Kim, Jitae; Rudella, Andrea; Rodríguez, Verenice Ramírez; Asakura, Yukari; Sun, Qi; van Wijk, Klaas J

    2009-08-01

    The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate

  12. Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart.

    Science.gov (United States)

    Melkani, Girish C; Trujillo, Adriana S; Ramos, Raul; Bodmer, Rolf; Bernstein, Sanford I; Ocorr, Karen

    2013-01-01

    Amyloid-like inclusions have been associated with Huntington's disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of

  13. Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects.

    Science.gov (United States)

    Taskent, Humeyra; Cho, Jae-Hyun; Raleigh, Daniel P

    2008-05-02

    Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed alpha-beta protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 degrees C. Plots of the log of the observed first-order rate constant versus denaturant concentration, "chevron plots," displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford beta parameter, beta(T), shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of Delta G versus Delta G degrees, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.

  14. Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.

    Science.gov (United States)

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J; Robinson, Colin

    2012-03-02

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.

  15. 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

  16. 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.

  17. The Protein Disulfide Isomerase of Botrytis cinerea: An ER Protein Involved in Protein Folding and Redox Homeostasis Influences NADPH Oxidase Signaling Processes

    Directory of Open Access Journals (Sweden)

    Robert Marschall

    2017-05-01

    Full Text Available Botrytis cinerea is a filamentous plant pathogen, which infects hundreds of plant species; within its lifestyle, the production of reactive oxygen species (ROS and a balanced redox homeostasis are essential parameters. The pathogen is capable of coping with the plant’s oxidative burst and even produces its own ROS to enhance the plant’s oxidative burst. Highly conserved NADPH oxidase (Nox complexes produce the reactive molecules. The membrane-associated complexes regulate a large variety of vegetative and pathogenic processes. Besides their commonly accepted function at the plasma membrane, recent studies reveal that Nox complexes are also active at the membrane of the endoplasmic reticulum. In this study, we identified the essential ER protein BcPdi1 as new interaction partner of the NoxA complex in B. cinerea. Mutants that lack this ER chaperone display overlapping phenotypes to mutants of the NoxA signaling pathway. The protein appears to be involved in all major developmental processes, such as the formation of sclerotia, conidial anastomosis tubes and infection cushions (IC’s and is needed for full virulence. Moreover, expression analyses and reporter gene studies indicate that BcPdi1 affects the redox homeostasis and unfolded protein response (UPR-related genes. Besides the close association between BcPdi1 and BcNoxA, interaction studies provide evidence that the ER protein might likewise be involved in Ca2+ regulated processes. Finally, we were able to show that the potential key functions of the protein BcPdi1 might be affected by its phosphorylation state.

  18. Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling

    NARCIS (Netherlands)

    Tark-Dame, M.; Jerabek, H.; Manders, E.M.M.; Heermann, D.W.; van Driel, R.

    2014-01-01

    Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type-specific transcription. We know remarkably little about the principles that control

  19. Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

    OpenAIRE

    Simkovic, Felix; Thomas, Jens M. H.; Keegan, Ronan M.; Winn, Martyn D.; Mayans, Olga; Rigden, Daniel J.

    2016-01-01

    For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based) structure prediction. Such models can be used in structure solution by molecular replacement (MR) where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles sea...

  20. Interrogating and predicting tolerated sequence diversity in protein folds: application to E. elaterium trypsin inhibitor-II cystine-knot miniprotein.

    Directory of Open Access Journals (Sweden)

    Jennifer L Lahti

    2009-09-01

    Full Text Available Cystine-knot miniproteins (knottins are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.

  1. Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability using Mass Spectrometry

    OpenAIRE

    Tran, Duc T.; Banerjee, Sambuddha; Alayash, Abdu I.; Crumbliss, Alvin L.; Fitzgerald, Michael C.

    2012-01-01

    Described here is a mass spectrometry based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol, which involves evaluating the denaturant dependence of this slow H/D exchange reaction in proteins, allows the global and/or subglobal unfolding/refolding properties of proteins and protein-ligand complexes to be probed. The protocol is developed using several m...

  2. Kinetics of Peptide Folding in Lipid Membranes

    Science.gov (United States)

    Oh, Kwang-Im; Smith-Dupont, Kathryn B.; Markiewicz, Beatrice N.; Gai, Feng

    2015-01-01

    Despite our extensive understanding of water-soluble protein folding kinetics, much less is known about the folding dynamics and mechanisms of membrane proteins. However, recent studies have shown that for relatively simple systems, such as peptides that form a transmembrane α-helix, helical dimer, or helix-turn-helix, it is possible to assess the kinetics of several important steps, including peptide binding to the membrane from aqueous solution, peptide folding on the membrane surface, helix insertion into the membrane, and helix-helix association inside the membrane. Herein, we provide a brief review of these studies and also suggest new initiation and probing methods that could lead to improved temporal and structural resolution in future experiments. PMID:25808575

  3. 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

  4. Understanding protein–protein interactions by genetic suppression

    Indian Academy of Sciences (India)

    Unknown

    Protein–protein interactions influence many cellular processes and it is increasingly being felt that even a weak and remote interplay between two subunits of a protein or between two proteins in a complex may govern the fate of a par- ticular biochemical pathway. In a bacterial system where the complete genome ...

  5. Understanding protein–protein interactions by genetic suppression

    Indian Academy of Sciences (India)

    Protein–protein interactions influence many cellular processes and it is increasingly being felt that even a weak and remote interplay between two subunits of a protein or between two proteins in a complex may govern the fate of a particular biochemical pathway. In a bacterial system where the complete genome sequence ...

  6. Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design.

    Science.gov (United States)

    AhYoung, Andrew P; Koehl, Antoine; Cascio, Duilio; Egea, Pascal F

    2015-09-01

    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. © 2015 The Protein Society.

  7. Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole.

    Science.gov (United States)

    Martinez, E; Jimenez, M A; Seguí-Real, B; Vandekerckhove, J; Sandoval, I V

    1997-04-18

    To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the

  8. The canonical twin-arginine translocase components are not required for secretion of folded green fluorescent protein from the ancestral strain of Bacillus subtilis.

    Science.gov (United States)

    Snyder, Anthony J; Mukherjee, Sampriti; Glass, J Kyle; Kearns, Daniel B; Mukhopadhyay, Suchetana

    2014-05-01

    Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

  9. Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae.

    Science.gov (United States)

    Bergdahl, Basti; Gorwa-Grauslund, Marie F; van Niel, Ed W J

    2014-04-23

    Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of

  10. Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

    KAUST Repository

    Yagi, Hiromasa

    2013-06-01

    Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution. © 2013 Elsevier Ltd. All rights reserved.

  11. Understanding of Protein Synthesis in a Living Cell

    Science.gov (United States)

    Mustapha, Y.; Muhammad, S.

    2006-01-01

    The assembly of proteins takes place in the cytoplasm of a cell. There are three main steps. In initiation, far left, all the necessary parts of the process are brought together by a small molecule called a ribosome. During elongation, amino acids, the building blocks of proteins, are joined to one another in a long chain. The sequence in which…

  12. A strategy for finding classes of minima on a hypersurface: implications for approaches to the protein folding problem.

    Science.gov (United States)

    Head-Gordon, T; Stillinger, F H; Arrecis, J

    1991-12-15

    Locating the native structure of a given protein is a task made difficult by the complexity of the potential energy hypersurface and by the huge number of local minima it contains. We have explored a strategy (the "antlion" method) for hypersurface modification that suppresses all minima but that of the native structure. Transferrable penalty functions with general applicability for modifying a hypersurface to retain the desired minimum are identified, and two blocked oligopeptides (alanine dipeptide and tetrapeptide) are used for specific numerical illustration of the dramatic simplification that ensues. In addition, an intermediary role for neural networks to manage some aspects of the antlion strategy applied to large polypeptides and proteins is introduced.

  13. Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells

    DEFF Research Database (Denmark)

    Schelhaas, Mario; Malmström, Johan; Pelkmans, Lucas

    2007-01-01

    Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated...... viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about...

  14. 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.

  15. 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.

  16. 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.

  17. Protein self-assembly and lipid binding in the folding of the potassium channel KcsA

    NARCIS (Netherlands)

    Barrera, F.N.; Renard, M.L.; Poveda, J.A.; de Kruijff, B.; Killian, J.A.; González-Ros, J.M.

    2008-01-01

    Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl â-D-maltoside (DDM) micelles

  18. 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

    . 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...

  19. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.

    2006-01-01

    The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6.......8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum...... protein 29 (ERp29); 78-kDa glucose-related protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non-GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN...

  20. Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae.

    Directory of Open Access Journals (Sweden)

    Preethi Ragunathan

    Full Text Available Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.

  1. Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation ▿

    OpenAIRE

    Whitman, Shannon D.; Smith, Everett Clinton; Dutch, Rebecca Ellis

    2009-01-01

    Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, H...

  2. A disorder-induced domino-like destabilization mechanism governs the folding and functional dynamics of the repeat protein IκBα.

    Directory of Open Access Journals (Sweden)

    Srinivasan Sivanandan

    Full Text Available The stability of the repeat protein IκBα, a transcriptional inhibitor in mammalian cells, is critical in the functioning of the NF-κB signaling module implicated in an array of cellular processes, including cell growth, disease, immunity and apoptosis. Structurally, IκBα is complex, with both ordered and disordered regions, thus posing a challenge to the available computational protocols to model its conformational behavior. Here, we introduce a simple procedure to model disorder in systems that undergo binding-induced folding that involves modulation of the contact map guided by equilibrium experimental observables in combination with an Ising-like Wako-Saitô-Muñoz-Eaton model. This one-step procedure alone is able to reproduce a variety of experimental observables, including ensemble thermodynamics (scanning calorimetry, pre-transitions, m-values and kinetics (roll-over in chevron plot, intermediates and their identity, and is consistent with hydrogen-deuterium exchange measurements. We further capture the intricate distance-dynamics between the domains as measured by single-molecule FRET by combining the model predictions with simple polymer physics arguments. Our results reveal a unique mechanism at work in IκBα folding, wherein disorder in one domain initiates a domino-like effect partially destabilizing neighboring domains, thus highlighting the effect of symmetry-breaking at the level of primary sequences. The offshoot is a multi-state and a dynamic conformational landscape that is populated by increasingly partially folded ensembles upon destabilization. Our results provide, in a straightforward fashion, a rationale to the promiscuous binding and short intracellular half-life of IκBα evolutionarily engineered into it through repeats with variable stabilities and expand the functional repertoire of disordered regions in proteins.

  3. Folding kinetics of the S100A11 protein dimer studied by time-resolved electrospray mass spectrometry and pulsed hydrogen-deuterium exchange.

    Science.gov (United States)

    Pan, Jingxi; Rintala-Dempsey, Anne C; Li, Yu; Shaw, Gary S; Konermann, Lars

    2006-03-07

    This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

  4. Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold.

    Science.gov (United States)

    Henriques de Jesus, Maria Perestrello Ramos; Zygadlo Nielsen, Agnieszka; Busck Mellor, Silas; Matthes, Annemarie; Burow, Meike; Robinson, Colin; Erik Jensen, Poul

    2017-11-01

    Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

  5. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G. (LNGB-Mexico)

    2012-04-18

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

  6. Stability Mechanisms of Laccase Isoforms using a Modified FoldX Protocol Applicable to Widely Different Proteins

    DEFF Research Database (Denmark)

    Christensen, Niels J.; Kepp, Kasper P.

    2013-01-01

    ) and thermostability (Topt ∼ 45–80 °C) and with 67–77% sequence identity. The extended protocol uses (i) statistical averaging, (ii) a molecular-dynamics-validated “compromise” homology model to minimize bias that causes proteins close in sequence to a structural template to be too stable due to having the benefits...... with experimental distributions of stability effects from mutation. The residues causing the differential stability of the four isoforms are consistent with a range of compiled laccase wild type data, suggesting that we may have identified general drivers of laccase stability. Several sites near Cu, notably 79, 241...

  7. Dietary Guidelines should reflect new understandings about adult protein needs

    Directory of Open Access Journals (Sweden)

    Layman Donald K

    2009-03-01

    Full Text Available Abstract Dietary Guidelines for Americans provide nutrition advice aimed at promoting healthy dietary choices for life-long health and reducing risk of chronic diseases. With the advancing age of the population, the 2010 Dietary Guidelines confront increasing risks for age-related problems of obesity, osteoporosis, type 2 diabetes, Metabolic Syndrome, heart disease, and sarcopenia. New research demonstrates that the meal distribution and amount of protein are important in maintaining body composition, bone health and glucose homeostasis. This editorial reviews the benefits of dietary protein for adult health, addresses omissions in current nutrition guidelines, and offers concepts for improving the Dietary Guidelines.

  8. The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus.

    Science.gov (United States)

    Koudounas, Konstantinos; Thomopoulou, Margarita; Michaelidis, Christos; Zevgiti, Efstathia; Papakostas, Georgios; Tserou, Paraskevi; Daras, Gerasimos; Hatzopoulos, Polydefkis

    2017-07-01

    Oleuropein, a terpene-derived glycosylated secoiridoid biosynthesized exclusively by members of the Oleaceae family, is involved in a two-component defense system comprising a β-glucosidase that activates oleuropein into a toxic glutaraldehyde-like structure. Oleuropein and its deglycosylated derivatives have high pharmaceutical interest. In this study we determined that the in planta heterologous expressed OeGLU, an oleuropein-specific β-glucosidase from olive ( Olea europaea ), had enzymatic kinetics similar to the olive native enzyme. The C terminus encompassing the nuclear localization signal sequesters the enzyme in the nucleus, and predetermines the protein-protein recognition and homodimerization. Biochemical analysis revealed that OeGLU is a homomultimer with high M r In silico prediction modeling of the complex structure and bimolecular fluorescence complementation analyses revealed that the C terminus of OeGLU is essential for the proper assembly of an octameric form, a key conformational feature that determines the activity of the enzyme. Our results demonstrate that intrinsic characteristics of the OeGLU ensure separation from oleuropein and keep the dual-partner defensive system conditionally inactive. Upon cell destruction, the dual-partner defense system is activated and olive massively releases the arsenal of defense. © 2017 American Society of Plant Biologists. All Rights Reserved.

  9. Understanding the structural parameters of biocompatible nanoparticles dictating protein fouling

    Czech Academy of Sciences Publication Activity Database

    de Castro, C. E.; Mattei, B.; Riske, K. A.; Jäger, Eliezer; Jäger, Alessandro; Štěpánek, Petr; Giacomelli, F. C.

    2014-01-01

    Roč. 30, č. 32 (2014), s. 9770-9779 ISSN 0743-7463 R&D Projects: GA ČR GAP208/10/1600 Institutional support: RVO:61389013 Keywords : nanoparticles * light scattering * protein adsorption Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.457, year: 2014

  10. Model systems for understanding absorption tuning by opsin proteins

    DEFF Research Database (Denmark)

    Nielsen, Mogens Brøndsted

    2009-01-01

    This tutorial review reports on model systems that have been synthesised and investigated for elucidating how opsin proteins tune the absorption of the protonated retinal Schiff base chromophore. In particular, the importance of the counteranion is highlighted. In addition, the review advocates...

  11. Using Antifreeze Proteins to understand ice microstructure evolution

    Science.gov (United States)

    Bayer-Giraldi, Maddalena; Azuma, Nobuhiko; Takata, Morimasa; Weikusat, Christian; Kondo, Hidemasa; Kipfstuhl, Sepp

    2017-04-01

    Polar ice sheets are considered a unique climate archive. The chemical analysis of its impurities and the development of its microstructure with depth give insight in past climate conditions as well as in the development of the ice sheet with time and deformation. Microstructural patterns like small grain size observed in specific depths are thought to be linked to the retarding effect of impurities on ice grain growth. Clear evidence of size or chemical composition of the impurities causing this effect is missing, but in this context a major role of nanoparticles has been suggested. In order to shed light on different mechanisms by which nanoparticles can control microstructure development we used antifreeze proteins (AFPs) as proxies for particles in ice. These proteins are small nanoparticles, approx. 5 nm in size, with the special characteristics of firmly binding to ice through several hydrogen bonds. We used AFPs from the sea-ice microalgae Fragilariopsis cylindrus (fcAFPs) in bubble-free, small-grained polycrystalline ice obtained by the phase-transition size refinement method. We explain how fcAFP bind to ice by presenting the 3-D-protein structure model inferred by X-ray structure analysis, and show the importance of the chemical interaction between particles and ice in controlling normal grain growth, comparing fcAFPs to other protein nanoparticles. We used modifications of fcAFPs for particle localization through fluorescence spectroscopy. Furthermore, the effect of fcAFPs on the driving factors for ice deformation during creep, i.e. on internal dislocations due to incorporation within the lattice and on the mobility of grain boundaries due to pinning, makes these proteins particularly interesting in studying the process of ice deformation.

  12. Understanding, predicting and controlling the physicochemical functionality of rice protein ingredients

    OpenAIRE

    Amagliani, Luca

    2016-01-01

    The aim of this research was to characterise the nutrient composition and protein profile of a range of intact and hydrolysed rice protein ingredients, to benchmark their physicochemical properties against those of selected commercial dairy protein ingredients, and to develop tailored solutions for understanding, predicting, modifying and controlling their functionality in food systems. The rice protein ingredients studied had protein contents in the range 32-78%, and lower levels of calcium ...

  13. Vapor treatment of electrospray droplets: evidence for the folding of initially denatured proteins on the sub-millisecond time-scale.

    Science.gov (United States)

    Kharlamova, Anastasia; DeMuth, J Corinne; McLuckey, Scott A

    2012-01-01

    The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK(a) or pK(b) of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry. © American Society for Mass Spectrometry, 2011

  14. Differential rates of protein folding and cellular trafficking for the Hendra virus F and G proteins: implications for F-G complex formation.

    Science.gov (United States)

    Whitman, Shannon D; Smith, Everett Clinton; Dutch, Rebecca Ellis

    2009-09-01

    Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.

  15. 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.

  16. Choosing the optimal spectroscopic toolkit to understand protein function.

    Science.gov (United States)

    Hough, Michael A

    2017-06-30

    Spectroscopy was one of the earliest methods used to study the properties and reactions of proteins, and remains one of the most powerful and widely used approaches to this day. A sometimes bewildering range of spectroscopies is now available, applicable to different sample states, timescales and indeed biological questions. This editorial describes some of the most relevant spectroscopic methods together with a selection of illustrative examples. © 2017 The Author(s).

  17. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric (Van Andel); (NU Singapore)

    2012-02-21

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  18. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Science.gov (United States)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  19. Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR*

    Science.gov (United States)

    Corazza, Alessandra; Rennella, Enrico; Schanda, Paul; Mimmi, Maria Chiara; Cutuil, Thomas; Raimondi, Sara; Giorgetti, Sofia; Fogolari, Federico; Viglino, Paolo; Frydman, Lucio; Gal, Maayan; Bellotti, Vittorio; Brutscher, Bernhard; Esposito, Gennaro

    2010-01-01

    β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant. PMID:20028983

  20. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Shiva Kumar

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  1. Optimized set of two-dimensional experiments for fast sequential assignment, secondary structure determination, and backbone fold validation of 13C/15N-labelled proteins

    International Nuclear Information System (INIS)

    Bersch, Beate; Rossy, Emmanuel; Coves, Jacques; Brutscher, Bernhard

    2003-01-01

    NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional 1 H- 15 N HSQC-like spectra. The 1 H- 15 N correlation peaks are frequency shifted by an amount ± ω X along the 15 N dimension, where ω X is the C α , C β , or H α frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces

  2. 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...

  3. The parallel universe of RNA folding.

    Science.gov (United States)

    Batey, R T; Doudna, J A

    1998-05-01

    How do large RNA molecules find their active conformations among a universe of possible structures? Two recent studies reveal that RNA folding is a rapid and ordered process, with surprising similarities to protein folding mechanisms.

  4. Computational study of pH-dependent oligomerization and ligand binding in Alt a 1, a highly allergenic protein with a unique fold.

    Science.gov (United States)

    Garrido-Arandia, María; Bretones, Jorge; Gómez-Casado, Cristina; Cubells, Nuria; Díaz-Perales, Araceli; Pacios, Luis F

    2016-05-01

    Alt a 1 is a highly allergenic protein from Alternaria fungi responsible for several respiratory diseases. Its crystal structure revealed a unique β-barrel fold that defines a new family exclusive to fungi and forms a symmetrical dimer in a butterfly-like shape as well as tetramers. Its biological function is as yet unknown but its localization in cell wall of Alternaria spores and its interactions in the onset of allergy reactions point to a function to transport ligands. However, at odds with binding features in β-barrel proteins, monomeric Alt a 1 seems unable to harbor ligands because the barrel is too narrow. Tetrameric Alt a 1 is able to bind the flavonoid quercetin, yet the stability of the aggregate and the own ligand binding are pH-dependent. At pH 6.5, which Alt a 1 would meet when secreted by spores in bronchial epithelium, tetramer-quercetin complex is stable. At pH 5.5, which Alt a 1 would meet in apoplast when infecting plants, the complex breaks down. By means of a combined computational study that includes docking calculations, empirical pKa estimates, Poisson-Boltzmann electrostatic potentials, and Molecular Dynamics simulations, we identified a putative binding site at the dimeric interface between subunits in tetramer. We propose an explanation on the pH-dependence of both oligomerization states and protein-ligand affinity of Alt a 1 in terms of electrostatic variations associated to distinct protonation states at different pHs. The uniqueness of this singular protein can thus be tracked in the combination of all these features.

  5. Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds.

    Science.gov (United States)

    Simkovic, Felix; Thomas, Jens M H; Keegan, Ronan M; Winn, Martyn D; Mayans, Olga; Rigden, Daniel J

    2016-07-01

    For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based) structure prediction. Such models can be used in structure solution by molecular replacement (MR) where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions ('decoys'), is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue-residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing.

  6. Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

    Directory of Open Access Journals (Sweden)

    Felix Simkovic

    2016-07-01

    Full Text Available For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based structure prediction. Such models can be used in structure solution by molecular replacement (MR where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions (`decoys', is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue–residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing.

  7. 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.

  8. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  9. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  10. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yujia Zhao

    2016-12-01

    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  11. The evolutionary history of protein fold families and proteomes confirms that the archaeal ancestor is more ancient than the ancestors of other superkingdoms

    Directory of Open Access Journals (Sweden)

    Kim Kyung Mo

    2012-01-01

    Full Text Available Abstract Background The entire evolutionary history of life can be studied using myriad sequences generated by genomic research. This includes the appearance of the first cells and of superkingdoms Archaea, Bacteria, and Eukarya. However, the use of molecular sequence information for deep phylogenetic analyses is limited by mutational saturation, differential evolutionary rates, lack of sequence site independence, and other biological and technical constraints. In contrast, protein structures are evolutionary modules that are highly conserved and diverse enough to enable deep historical exploration. Results Here we build phylogenies that describe the evolution of proteins and proteomes. These phylogenetic trees are derived from a genomic census of protein domains defined at the fold family (FF level of structural classification. Phylogenomic trees of FF structures were reconstructed from genomic abundance levels of 2,397 FFs in 420 proteomes of free-living organisms. These trees defined timelines of domain appearance, with time spanning from the origin of proteins to the present. Timelines are divided into five different evolutionary phases according to patterns of sharing of FFs among superkingdoms: (1 a primordial protein world, (2 reductive evolution and the rise of Archaea, (3 the rise of Bacteria from the common ancestor of Bacteria and Eukarya and early development of the three superkingdoms, (4 the rise of Eukarya and widespread organismal diversification, and (5 eukaryal diversification. The relative ancestry of the FFs shows that reductive evolution by domain loss is dominant in the first three phases and is responsible for both the diversification of life from a universal cellular ancestor and the appearance of superkingdoms. On the other hand, domain gains are predominant in the last two phases and are responsible for organismal diversification, especially in Bacteria and Eukarya. Conclusions The evolution of functions that are

  12. MiAMP1, a novel protein from macadamia integrifolia adopts a greek key β-barrel fold unique amongst plant antimicrobial proteins

    International Nuclear Information System (INIS)

    McManus, A.M.; Nielsen, K.J.; Craik, D.J.; Marcus, J.P.; Harrison, S.J.; Green, J.L.; Manners, J.M.

    1999-01-01

    Full text: MiAMP1 is a recently discovered 76 amino acid, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein. A study of its antimicrobial activity revealed that it inhibited the growth of several microbial plant pathogens in vitro but had no effect on mammalian or plant cells. For these reasons, MiAMP1 is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ( 15 N) 2D NMR spectroscopy and subsequent simulated annealing calculations. MiAMP1 is made up of eight β-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key β-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the β-Barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action

  13. Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein.

    Science.gov (United States)

    Chen, Zhiqiang; Huang, Chao; Chennamsetty, Naresh; Xu, Xuankuo; Li, Zheng Jian

    2016-08-19

    Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. An orthogonal technique, differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4-5 range. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120h, and links the aggregate stability to such conditions as resin

  14. Stretching to understand proteins - a survey of the protein data bank.

    Science.gov (United States)

    Sułkowska, Joanna I; Cieplak, Marek

    2008-01-01

    We make a survey of resistance of 7510 proteins to mechanical stretching at constant speed as studied within a coarse-grained molecular dynamics model. We correlate the maximum force of resistance with the native structure, predict proteins which should be especially strong, and identify the nature of their force clamps.

  15. Mycobacterium tuberculosis copper-regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer.

    Science.gov (United States)

    Nowicka, Urszula; Hoffman, Morgan; Randles, Leah; Shi, Xiaoshan; Khavrutskii, Lyuba; Stefanisko, Karen; Tarasova, Nadya I; Darwin, K Heran; Walters, Kylie J

    2016-02-01

    Multiple genes in Mycobacterium tuberculosis (Mtb) are regulated by copper including socAB (small orf induced by copper A and B), which is induced by copper and repressed by RicR (regulated in copper repressor). socA and socB encode hypothetical proteins of 61 and 54 amino acids, respectively. Here, we use biophysical and computational methods to evaluate the SocB structure. We find that SocB lacks evidence for secondary structure, with no thermal cooperative unfolding event, according to circular dichroism measurements. 2D NMR spectra similarly exhibit hallmarks of a disordered structural state, which is also supported by analyzing SocB diffusion. Altogether, these findings suggest that by itself SocB is intrinsically disordered. Interestingly, SocB interacts with a synthetic phospholipid bilayer and becomes helical, which suggests that it may be membrane-associated. © 2015 Wiley Periodicals, Inc.

  16. Understanding complex structures in fold-and-thrust belts. Integration of geometric and growth strata analyses, paleomagnetism, AMS and analogue models in the Western termination of the Southern Pyrenees

    Science.gov (United States)

    Pueyo, Emilio L.; Sánchez, Elisa; Oliva-Urcia, Belén; José Ramón, Ma

    2014-05-01

    Classic 2D approaches have helped the understanding of the geometry and kinematics of fold-and-thrust belts belts (FAT belts) but are insufficient to unravel many natural cases. This is because deformation is 3D from the geometric point of view and, thus, cylindrical features may be considered as a simplification. On the other hand, deformation kinematics is usually complex, diachronic and poliphasic in real cases. Therefore, FAT belts have to be always considered in 4D. In this sense, the Southern Pyrenees is a perfect location to study the evolution of FAT belts because of the exceptional outcropping conditions of growth strata, the proven diachronic kinematics and the non-coaxial interference of deformation events. Within the vast catalogue of complex structures that includes superposed folding, conical and plunging folds, oblique thrust ramps, etc here, we have selected the westernmost termination of the South Pyrenean sole thrust to illustrate how the integration of geometric and kinematic analysis can help unraveling complex structures in FAT belts. The San Marzal pericline (4 km2 surface extension) is the lateral termination of the Sto. Domingo deca-kilometric fold. San Marzal looks like a large 70° plunging cylindrical structure. However the large magnitude (≡ 60-70°) of vertical axis rotations accommodated between its flanks cannot be explained without a conical geometry. In this work we will show how the structural analysis performed on this structure has disentangled its complex geometry. This analyses comprises several hundreds of bedding data, joints and veins and more than 150 standard paleomagnetic and AMS sites. Besides, we will show how the kinematic information derived from magnetostratigraphic sections (more than 8 km of sampled profiles) has helped to constraint the folding and rotation ages and velocities. Finally, all these complex geometric and kinematic features have inspired us to build an analogue model where we can explore the 3D

  17. Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

    DEFF Research Database (Denmark)

    O'Reilly, Linda P; Andresen, Brage S; Engel, Paul C

    2005-01-01

    was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though......Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When...... the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed...

  18. A network biology approach to understanding the importance of chameleon proteins in human physiology and pathology.

    Science.gov (United States)

    Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Marashi, Sayed-Amir

    2017-02-01

    Chameleon proteins are proteins which include sequences that can adopt α-helix-β-strand (HE-chameleon) or α-helix-coil (HC-chameleon) or β-strand-coil (CE-chameleon) structures to operate their crucial biological functions. In this study, using a network-based approach, we examined the chameleon proteins to give a better knowledge on these proteins. We focused on proteins with identical chameleon sequences with more than or equal to seven residues long in different PDB entries, which adopt HE-chameleon, HC-chameleon, and CE-chameleon structures in the same protein. One hundred and ninety-one human chameleon proteins were identified via our in-house program. Then, protein-protein interaction (PPI) networks, Gene ontology (GO) enrichment, disease network, and pathway enrichment analyses were performed for our derived data set. We discovered that there are chameleon sequences which reside in protein-protein interaction regions between two proteins critical for their dual function. Analysis of the PPI networks for chameleon proteins introduced five hub proteins, namely TP53, EGFR, HSP90AA1, PPARA, and HIF1A, which were presented in four PPI clusters. The outcomes demonstrate that the chameleon regions are in critical domains of these proteins and are important in the development and treatment of human cancers. The present report is the first network-based functional study of chameleon proteins using computational approaches and might provide a new perspective for understanding the mechanisms of diseases helping us in developing new medical therapies along with discovering new proteins with chameleon properties which are highly important in cancer.

  19. 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

  20. 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.

  1. 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

  2. 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

    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...

  3. Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint

    NARCIS (Netherlands)

    Wierenga, Rik K.; Terpstra, Peter; Hol, Wim G.J.

    1986-01-01

    An amino acid sequence "fingerprint” has been derived that can be used to test if a particular sequence will fold into a βαβ-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding βαβ-folds. This fingerprint is in fact a set

  4. Towards a better understanding of the specificity of protein-protein interaction

    Czech Academy of Sciences Publication Activity Database

    Kysilka, Jiří; Vondrášek, Jiří

    2012-01-01

    Roč. 25, č. 11 (2012), s. 604-615 ISSN 0952-3499 R&D Projects: GA ČR GAP208/10/0725; GA ČR GAP302/10/0427; GA MŠk(CZ) LH11020 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : protein-protein interaction * molecular recognition * x-ray structure analysis * empirical potentials * side chain-side chain interaction * interaction energy * bioinformatics Subject RIV: CE - Biochemistry Impact factor: 3.006, year: 2012

  5. Chromophore-protein coupling beyond nonpolarizable models: understanding absorption in green fluorescent protein

    NARCIS (Netherlands)

    Daday, C.; Curutchet, C.; Sinicropi, A.; Mennucci, B.; Filippi, Claudia

    2015-01-01

    The nature of the coupling of the photoexcited chromophore with the environment in a prototypical system like green fluorescent protein (GFP) is to date not understood, and its description still defies state-of-the-art multiscale approaches. To identify which theoretical framework of the

  6. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  7. C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.

    Science.gov (United States)

    Chalat, Madhavan; Moleschi, Kody; Molday, Robert S

    2017-02-01

    ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2. © 2017 Chalat et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication 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).

  8. Functional implications of the beta-helical protein fold: differences in chemical and thermal stabilities of Erwinia chrysanthemi EC16 pectate lyases B, C, and E.

    Science.gov (United States)

    Hurlbert, J C; Preston, J F

    2000-09-15

    Colonization of plant tissue by the phytopathogen Erwinia chrysanthemi EC16 is aided by the activities of the pectate lyase isozymes (PLs), which depolymerize the polygalacturonic acid component (PGA) of plant cell walls. The bacterium secretes four pectate lyases (PLa, PLb, PLc, and PLe), two of which, PLc and PLe, have been shown to fold into a similar domain motif, the beta-helix. To understand the rationale behind the evolution and retention of these isoforms, the susceptibilities of pectate lyases B, C, and E to chemical and thermal denaturation and the resulting enzymatic inactivation were examined. With guanidine hydrochloride used as a denaturant, all three pectate lyases denatured with transition midpoint guanidine hydrochloride concentrations (Cm) of 1.3, 1.1, and 1.8 M for PLb, PLc, and PLe, respectively. Lyase activity decreased in direct response to loss of secondary structure in all enzymes. Pectate lyases B and C demonstrated increased enzymatic activity at temperatures above 30 degrees C, with maximal activity observed at 40 degrees C for PLb and 35 degrees C for PLc. Transition midpoints (Tm) as measured by circular dichroism were at 46.9 degrees C for PLb and 44.3 degrees C for PLc, indicating detectable conformational changes accompanying thermal inactivation. Decreased enzymatic activity of PLe was observed at all temperatures above 30 degrees C, and the enzyme was found to possess a Tm at 38.9 degrees C. The data demonstrate structural differences among these enzymes that may be the basis for different enzymatic efficiencies under the potential array of environmental conditions experienced by the bacterium. These differences, in turn, may play a part in the retention of these isozymes as virulence factors, allowing the successful colonization of susceptible plant hosts.

  9. 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.

  10. The interactions between CdTe quantum dots and proteins: understanding nano-bio interface

    Directory of Open Access Journals (Sweden)

    Shreeram S. Joglekar

    2017-01-01

    Full Text Available Despite remarkable developments in the nanoscience, relatively little is known about the physical (electrostatic interactions of nanoparticles with bio macromolecules. These interactions can influence the properties of both nanoparticles and the bio-macromolecules. Understanding this bio-interface is a prerequisite to utilize both nanoparticles and biomolecules for bioengineering. In this study, luminescent, water soluble CdTe quantum dots (QDs capped with mercaptopropionic acid (MPA were synthesized by organometallic method and then interaction between nanoparticles (QDs and three different types of proteins (BSA, Lysozyme and Hemoglobin were investigated by fluorescence spectroscopy at pH= 7.4. Based on fluorescence quenching results, Stern-Volmer quenching constant (Ksv, binding constant (Kq and binding sites (n for proteins were calculated. The results show that protein structure (e.g.,globular, metalloprotein, etc. has a significant role in Protein-Quantum dots interactions and each type of protein influence physicochemical properties of Quantum dots differently.

  11. 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.

  12. Integrating Model-Based Learning and Animations for Enhancing Students' Understanding of Proteins Structure and Function

    Science.gov (United States)

    Barak, Miri; Hussein-Farraj, Rania

    2013-01-01

    This paper describes a study conducted in the context of chemistry education reforms in Israel. The study examined a new biochemistry learning unit that was developed to promote in-depth understanding of 3D structures and functions of proteins and nucleic acids. Our goal was to examine whether, and to what extent teaching and learning via…

  13. Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus.

    Science.gov (United States)

    Gu, Zhenyu; Zitzewitz, Jill A; Matthews, C Robert

    2007-04-27

    To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good

  14. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta......The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro...

  15. Are protein hubs faster folders? Exploration based on Escherichia coli proteome.

    Science.gov (United States)

    Xu, Hong-Rui; Cheng, Jun-Feng; Hu, Xiao-Pan; Chu, Ying-Ying; Ma, Bin-Guang

    2016-12-01

    Protein hubs in protein-protein interaction network are especially important due to their central roles in the entire network. Despite of their importance, the folding kinetics of hub proteins in comparison with non-hubs is still unknown. In this work, the folding rates for protein hubs and non-hubs were predicted and compared for the interactome of Escherichia coli K12, and the results showed that hub proteins fold faster than non-hub proteins. A possible explanation might be that protein hubs have more and fast-folding structural conformations than non-hubs, which leads to the notion of "hub of hubs" in the protein conformation space. It was found that the sequence and structure features relevant to protein folding rates are also different between hub and non-hub proteins. Moreover, the interacting proteins tend to have similar folding rates. These results gave insightful implications for understanding the interplay between the mechanisms of protein folding and interaction.

  16. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Nielsen, Morten Schallburg; Brejning, Jeanette; García, R.

    1997-01-01

    /LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids...

  17. An attempt to understand kidney's protein handling function by comparing plasma and urine proteomes.

    Directory of Open Access Journals (Sweden)

    Lulu Jia

    Full Text Available BACKGROUND: With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form "reference profiles" of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. METHODOLOGY/PRINCIPAL FINDINGS: After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched. CONCLUSION/SIGNIFICANCE: The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma and output (urine, it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It

  18. Statistical theory of neutral protein evolution by random site mutations

    Indian Academy of Sciences (India)

    Administrator

    Abstract. Understanding the features of the protein conformational space represents a key component to characterize protein structural evolution at the molecular level. This problem is approached in a two- fold manner; simple lattice models are used to represent protein structures with the ability of a protein sequence to fold ...

  19. Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

    Science.gov (United States)

    Massiah, Michael A; Wright, Katharine M; Du, Haijuan

    2016-04-01

    This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. Copyright © 2016 John Wiley & Sons, Inc.

  20. Understanding gene essentiality by finely characterizing hubs in the yeast protein interaction network.

    Science.gov (United States)

    Pang, Kaifang; Sheng, Huanye; Ma, Xiaotu

    2010-10-08

    The centrality-lethality rule, i.e., high-degree proteins or hubs tend to be more essential than low-degree proteins in the yeast protein interaction network, reveals that a protein's central position indicates its important function, but whether and why hubs tend to be more essential have been heavily debated. Here, we integrated gene expression and functional module data to classify hubs into four types: non-co-expressed non-co-cluster hubs, non-co-expressed co-cluster hubs, co-expressed non-co-cluster hubs and co-expressed co-cluster hubs. We found that all the four hub types are more essential than non-hubs, but they also show different enrichments in essential proteins. Non-co-expressed non-co-cluster hubs play key role in organizing different modules formed by the other three hub types, but they are less important to the survival of the yeast cell. Among the four hub types, co-expressed co-cluster hubs, which likely correspond to the core components of stable protein complexes, are the most essential. These results demonstrated that our classification of hubs into four types could better improve the understanding of gene essentiality. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Toward understanding life under subzero conditions: the significance of exploring psychrophilic "cold-shock" proteins.

    Science.gov (United States)

    Kuhn, Emanuele

    2012-11-01

    Understanding the behavior of proteins under freezing conditions is vital for detecting and locating extraterrestrial life in cold environments, such as those found on Mars and the icy moons of Jupiter and Saturn. This review highlights the importance of studying psychrophilic "cold-shock" proteins, a topic that has yet to be explored. A strategy for analyzing the psychrophilic RNA helicase protein CsdA (Psyc_1082) from Psychrobacter arcticus 273-4 as a key protein for life under freezing temperatures is proposed. The experimental model presented here was developed based on previous data from investigations of Escherichia coli, P. arcticus 273-4, and RNA helicases. P. arcticus 273-4 is considered a model for life in freezing environments. It is capable of growing in temperatures as cold as -10°C by using physiological strategies to survive not only in freezing temperatures but also under low-water-activity and limited-nutrient-availability conditions. The analyses of its genome, transcriptome, and proteome revealed specific adaptations that allow it to inhabit freezing environments by adopting a slow metabolic strategy rather than a cellular dormancy state. During growth at subzero temperatures, P. arcticus 273-4 genes related to energy metabolism and carbon substrate incorporation are downregulated, and genes for maintenance of membranes, cell walls, and nucleic acid motion are upregulated. At -6°C, P. arcticus 273-4 does not upregulate the expression of either RNA or protein chaperones; however, it upregulates the expression of its cold-shock induced DEAD-box RNA helicase protein A (CsdA - Psyc_1082). CsdA - Psyc_1082 was investigated as a key helper protein for sustaining life in subzero conditions. Proving CsdA - Psyc_1082 to be functional as a key protein for life under freezing temperatures may extend the known minimum growth temperature of a mesophilic cell and provide key information about the mechanisms that underlie cold-induced biological systems in

  2. Domain decomposition-based structural condensation of large protein structures for understanding their conformational dynamics.

    Science.gov (United States)

    Kim, Jae In; Na, Sungsoo; Eom, Kilho

    2011-01-15

    Normal mode analysis (NMA) with coarse-grained model, such as elastic network model (ENM), has allowed the quantitative understanding of protein dynamics. As the protein size is increased, there emerges the expensive computational process to find the dynamically important low-frequency normal modes due to diagonalization of massive Hessian matrix. In this study, we have provided the domain decomposition-based structural condensation method that enables the efficient computations on low-frequency motions. Specifically, our coarse-graining method is established by coupling between model condensation (MC; Eom et al., J Comput Chem 2007, 28, 1400) and component mode synthesis (Kim et al., J Chem Theor Comput 2009, 5, 1931). A protein structure is first decomposed into substructural units, and then each substructural unit is coarse-grained by MC. Once the NMA is implemented to coarse-grained substructural units, normal modes and natural frequencies for each coarse-grained substructural unit are assembled by using geometric constraints to provide the normal modes and natural frequencies for whole protein structure. It is shown that our coarse-graining method enhances the computational efficiency for analysis of large protein complexes. It is clearly suggested that our coarse-graining method provides the B-factors of 100 large proteins, quantitatively comparable with those obtained from original NMA, with computational efficiency. Moreover, the collective behaviors and/or the correlated motions for model proteins are well delineated by our suggested coarse-grained models, quantitatively comparable with those computed from original NMA. It is implied that our coarse-grained method enables the computationally efficient studies on conformational dynamics of large protein complex.

  3. THE ALPHA/BETA-HYDROLASE FOLD

    NARCIS (Netherlands)

    OLLIS, DL; CHEAH, E; CYGLER, M; FROLOW, F; FRANKEN, SM; HAREL, M; REMINGTON, SJ; SILMAN, [No Value; SCHRAG, J; SUSSMAN, JL; VERSCHUEREN, KHG; GOLDMAN, A

    We have identified a new protein fold-the alpha/beta-hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta-sheet, not barrel, of eight beta-sheets connected by alpha-helices. These

  4. Role of His101 in the Protein Folding/Unfolding of a Goose-Type Lysozyme from Ostrich (Struthio camelus) Egg White.

    Science.gov (United States)

    Somboonpatarakun, Chalermchai; Fukamizo, Tamo; Araki, Tomohiro; Klaynongsruang, Sompong

    2016-12-01

    To understand the role of His101 in protein structure stabilization of goose-type (G-type) lysozyme, we conducted thermal unfolding/refolding experiments using native G-type lysozyme from ostrich egg white (nOEL), the recombinant G-type lysozyme (rOEL), and the mutant lysozyme, in which His101 is mutated to alanine (H101A-OEL). Thermal stability on lytic activity and in-gel refolding experiments provided similar profiles for all three OELs. Circular dichroism (CD) spectroscopy was used to determine the secondary structure of three OELs as a function of temperature. Unfolding/refolding experiments (30-90 °C) monitored by CD spectroscopy revealed an unfolding transition at 65-67 °C and a complete refolding at almost the same temperature. Notably, a slightly lower thermal stability was observed for H101A-OEL, corresponding to the calculated difference in transition free energy of thermal unfolding (∆∆G m ) between rOEL and H101A-OEL of -0.63 kcal/mol. To assess the effects of H101A mutation on the electrostatic behavior, we examined the pH-activity profile of the three OELs. nOEL and rOEL exhibit bimodal relationship between pH and lytic activity showing optima at pH 3.0 and 7.0, while optima for H101A-OEL activity were pH 4.0 and 6.0. Electrostatic environment surrounding His101 was affected by the H101A mutation resulting in the slightly lower thermal stability.

  5. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, E.; Blicher, T.

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation......)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule....

  6. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation......(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule....

  7. Integrating Model-Based Learning and Animations for Enhancing Students' Understanding of Proteins Structure and Function

    Science.gov (United States)

    Barak, Miri; Hussein-Farraj, Rania

    2013-04-01

    This paper describes a study conducted in the context of chemistry education reforms in Israel. The study examined a new biochemistry learning unit that was developed to promote in-depth understanding of 3D structures and functions of proteins and nucleic acids. Our goal was to examine whether, and to what extent teaching and learning via model-based learning and animations of biomolecules affect students' chemical understanding. Applying the mixed methods research paradigm, pre- and post-questionnaires as well as class-observations were employed in the collection, analysis, and interpretation of data. The research population included 175 grade twelve students, divided into three research groups: (a) hands-on exploration of animations, (b) teacher's demonstrations of animations, (c) traditional learning using textbooks. Findings indicated that the integration of model-based learning and 3D animations enhanced students' understanding of proteins' structure and function and their ability to transfer across different levels of chemistry understanding. Findings also indicated that teachers' demonstrations of animations may enhance students' `knowledge'—a low order thinking skill; however, in order to enhance higher levels of thinking, students should be able to explore 3D animations on their own. Applying constructivist and interpretative analysis of the data, three themes were raised, corresponding to cognitive, affective, and social aspects of learning while exploring web-based models and animations.

  8. The Structure of the Neurotoxin- Associated Protein HA33/A from Clostridium botulinum Suggests a Reoccurring Beta-Trefoil Fold in the Progenitor Toxin Complex

    National Research Council Canada - National Science Library

    Arndt, Joseph W; Gu, Jenny; Jaroszewski, Lukasz; Schwarzenbacher, Robert; Hanson, Michael A; Lebeda, Frank L; Stevens, Raymond C

    2004-01-01

    The hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinum neurotoxin secreted complexes and is critical in toxin protection, internalization, and possibly activation...

  9. Physical models have gender‐specific effects on student understanding of protein structure–function relationships

    Science.gov (United States)

    Harris, Michelle A.; Chang, Wesley S.; Dent, Erik W.; Nordheim, Erik V.; Franzen, Margaret A.

    2016-01-01

    Abstract Understanding how basic structural units influence function is identified as a foundational/core concept for undergraduate biological and biochemical literacy. It is essential for students to understand this concept at all size scales, but it is often more difficult for students to understand structure–function relationships at the molecular level, which they cannot as effectively visualize. Students need to develop accurate, 3‐dimensional mental models of biomolecules to understand how biomolecular structure affects cellular functions at the molecular level, yet most traditional curricular tools such as textbooks include only 2‐dimensional representations. We used a controlled, backward design approach to investigate how hand‐held physical molecular model use affected students' ability to logically predict structure–function relationships. Brief (one class period) physical model use increased quiz score for females, whereas there was no significant increase in score for males using physical models. Females also self‐reported higher learning gains in their understanding of context‐specific protein function. Gender differences in spatial visualization may explain the gender‐specific benefits of physical model use observed. © 2016 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 44(4):326–335, 2016. PMID:26923186

  10. Physical models have gender-specific effects on student understanding of protein structure-function relationships.

    Science.gov (United States)

    Forbes-Lorman, Robin M; Harris, Michelle A; Chang, Wesley S; Dent, Erik W; Nordheim, Erik V; Franzen, Margaret A

    2016-07-08

    Understanding how basic structural units influence function is identified as a foundational/core concept for undergraduate biological and biochemical literacy. It is essential for students to understand this concept at all size scales, but it is often more difficult for students to understand structure-function relationships at the molecular level, which they cannot as effectively visualize. Students need to develop accurate, 3-dimensional mental models of biomolecules to understand how biomolecular structure affects cellular functions at the molecular level, yet most traditional curricular tools such as textbooks include only 2-dimensional representations. We used a controlled, backward design approach to investigate how hand-held physical molecular model use affected students' ability to logically predict structure-function relationships. Brief (one class period) physical model use increased quiz score for females, whereas there was no significant increase in score for males using physical models. Females also self-reported higher learning gains in their understanding of context-specific protein function. Gender differences in spatial visualization may explain the gender-specific benefits of physical model use observed. © 2016 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 44(4):326-335, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

  11. 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.

  12. High-level expression and improved folding of proteins by using the vp39 late promoter enhanced with homologous DNA regions.

    Science.gov (United States)

    Ishiyama, Seiji; Ikeda, Masahiro

    2010-11-01

    Some recombinant proteins expressed by baculovirus expression vector systems (BEVS) aggregate because the BEVS can produce large amounts of protein late during infection, when post-translational modification and protein quality control mechanisms are inactive. For expression during earlier stages than that driven by the polyhedrin (polh) very late promoter, transfer vectors were generated in which this promoter was replaced with a green fluorescent protein (GFP) gene controlled by a vp39 late promoter modified to contain HR3, one of the homologous DNA regions (HRs) of Bombyx mori nuclear polyhedrosis virus (BmNPV). The rise times of the fluorescence of GFP expressed by using recombinant viruses carrying the modified vp39 promoter were earlier than those associated with either the polh promoter or the native vp39 promoter lacking HR3. In transient expression assays, the vp39 late promoter in transfer vectors behaved like a delayed-early promoter, and was enhanced by HR3, and required IE-1 protein and various viral gene products encoded on both sides of BmNPV polh. When the vp39 promoter with HR3 was used, the aggregation of several foreign proteins expressed by the BEVS was markedly decreased. This study provides a new option for the expression of sufficiently quality-controlled proteins by using the vp39 promoter and HR3 in BEVS early in baculovirus infection, when the infection has caused little damage in the host cells.

  13. Downhill versus barrier-limited folding of BBL 2: mechanistic insights from kinetics of folding monitored by independent tryptophan probes.

    Science.gov (United States)

    Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Teufel, Daniel P; Ferguson, Neil; Fersht, Alan R

    2009-04-10

    Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of an alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics.

  14. Understanding the GPCR biased signaling through G protein and arrestin complex structures.

    Science.gov (United States)

    Zhou, X Edward; Melcher, Karsten; Xu, H Eric

    2017-08-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are important drug targets for many human diseases. The determination of the 3-D structure of GPCRs and their signaling complexes has promoted our understanding of GPCR biology and provided templates for structure-based drug discovery. In this review, we focus on the recent structure work on GPCR signaling complexes, the β2-adrenoreceptor-Gs and the rhodopsin-arrestin complexes in particular, and highlight the structural features of GPCR complexes involved in G protein- and arrestin-mediated signal transduction. The crystal structures reveal distinct structural mechanisms by which GPCRs recruit a G protein and an arrestin. A comparison of the two complex structures provides insight into the molecular mechanism of functionally selective GPCR signaling, and a structural basis for the discovery of G protein- and arrestin-biased treatments of human diseases related to GPCR signal transduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

    Science.gov (United States)

    Serfiotis-Mitsa, Dimitra; Herbert, Andrew P.; Roberts, Gareth A.; Soares, Dinesh C.; White, John H.; Blakely, Garry W.; Uhrín, Dušan; Dryden, David T. F.

    2010-01-01

    Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic. PMID:20007596

  16. Vocal Fold Paralysis

    Science.gov (United States)

    ... decades-long project to develop an electrical stimulation technology to help people avoid having a tracheotomy when both vocal folds are paralyzed. The device, which currently is being tested in animals and people, uses an implanted pacemaker to stimulate ...

  17. Crystal structures of MW1337R and lin2004: Representatives of a novel protein family that adopt a four-helical bundle fold

    Energy Technology Data Exchange (ETDEWEB)

    Kozbial, Piotr; Xu, Qingping; Chiu, Hsiu-Ju; McMullan, Daniel; Krishna, S. Sri; Miller, Mitchell D.; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Clayton, Thomas; Deller, Marc; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K.; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Koesema, Eric; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Murphy, Kevin D.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L.; Spraggon, Glen; Trout, Christina V.; ban den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Wolf, Guenter; Zubieta, Chloe; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A. (Scripps); (SSRL); (JCSG); (UCSD); (Burnham)

    2009-08-28

    To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs, which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown