Sample records for protein folding mechanisms
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When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.
Muñoz, Victor; Cerminara, Michele
2016-09-01
Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).
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Glycoprotein folding and quality-control mechanisms in protein-folding diseases
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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.
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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
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Pappu, Rohit V.; Nussinov, Ruth
2009-03-01
multi-scale dynamical problem when one considers the synergies between protein expression, spontaneous folding, chaperonin-assisted folding, protein targeting, the kinetics of post-translational modifications, protein degradation, and of course the drive to avoid aggregation. Further, there is growing recognition that cells not only tolerate but select for proteins that are intrinsically disordered. These proteins are essential for many crucial activities, and yet their inability to fold in isolation makes them prone to proteolytic processing and aggregation. In the series of papers that make up this special focus on protein folding in physical biology, leading researchers provide insights into diverse cross-sections of problems in protein folding. Barrick provides a concise review of what we have learned from the study of two-state folders and draws attention to how several unanswered questions are being approached using studies on large repeat proteins. Dissecting the contribution of hydration-mediated interactions to driving forces for protein folding and assembly has been extremely challenging. There is renewed interest in using hydrostatic pressure as a tool to access folding intermediates and decipher the role of partially hydrated states in folding, misfolding, and aggregation. Silva and Foguel review many of the nuances that have been uncovered by perturbing hydrostatic pressure as a thermodynamic parameter. As noted above, protein folding in vivo is expected to be considerably more complex than the folding of two-state proteins in dilute solutions. Lucent et al review the state-of-the-art in the development of quantitative theories to explain chaperonin-assisted folding in vivo. Additionally, they highlight unanswered questions pertaining to the processing of unfolded/misfolded proteins by the chaperone machinery. Zhuang et al present results that focus on the effects of surface tethering on transition state ensembles and folding mechanisms of a model two
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Mechanical Modeling and Computer Simulation of Protein Folding
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…
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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.
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Protein folding and the organization of the protein topology universe
DEFF Research Database (Denmark)
Lindorff-Larsen,, Kresten; Røgen, Peter; Paci, Emanuele
2005-01-01
residues and, in addition, that the topology of the transition state is closer to that of the native state than to that of any other fold in the protein universe. Here, we review the evidence for these conclusions and suggest a molecular mechanism that rationalizes these findings by presenting a view...... of protein folds that is based on the topological features of the polypeptide backbone, rather than the conventional view that depends on the arrangement of different types of secondary-structure elements. By linking the folding process to the organization of the protein structure universe, we propose...
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Intermediates and the folding of proteins L and G
Energy Technology Data Exchange (ETDEWEB)
Brown, Scott; Head-Gordon, Teresa
2003-07-01
We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G that are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted {beta}-1 and {beta}-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third {beta}-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.
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Roles of beta-turns in protein folding: from peptide models to protein engineering.
Marcelino, Anna Marie C; Gierasch, Lila M
2008-05-01
Reverse turns are a major class of protein secondary structure; they represent sites of chain reversal and thus sites where the globular character of a protein is created. It has been speculated for many years that turns may nucleate the formation of structure in protein folding, as their propensity to occur will favor the approximation of their flanking regions and their general tendency to be hydrophilic will favor their disposition at the solvent-accessible surface. Reverse turns are local features, and it is therefore not surprising that their structural properties have been extensively studied using peptide models. In this article, we review research on peptide models of turns to test the hypothesis that the propensities of turns to form in short peptides will relate to the roles of corresponding sequences in protein folding. Turns with significant stability as isolated entities should actively promote the folding of a protein, and by contrast, turn sequences that merely allow the chain to adopt conformations required for chain reversal are predicted to be passive in the folding mechanism. We discuss results of protein engineering studies of the roles of turn residues in folding mechanisms. Factors that correlate with the importance of turns in folding indeed include their intrinsic stability, as well as their topological context and their participation in hydrophobic networks within the protein's structure.
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Protein solubility and folding enhancement by interaction with RNA.
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Seong Il Choi
Full Text Available While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.
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Transiently disordered tails accelerate folding of globular proteins.
Mallik, Saurav; Ray, Tanaya; Kundu, Sudip
2017-07-01
Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. © 2017 Federation of European Biochemical Societies.
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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.
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Bergasa-Caceres, Fernando; Rabitz, Herschel A.
2014-01-01
A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.
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Experimental investigation of protein folding and misfolding.
Dobson, Christopher M
2004-09-01
Newly synthesised proteins need to fold, often to intricate and close-packed structures, in order to function. The underlying mechanism by which this complex process takes place both in vitro and in vivo is now becoming understood, at least in general terms, as a result of the application of a wide range of biophysical and computational methods used in combination with the techniques of biochemistry and protein engineering. It is increasingly apparent, however, that folding is not only crucial for generating biological activity, but that it is also coupled to a wide range of processes within the cell, ranging from the trafficking of proteins to specific organelles to the regulation of cell growth and differentiation. Not surprisingly, therefore, the failure of proteins to fold appropriately, or to remain correctly folded, is associated with a large number of cellular malfunctions that give rise to disease. Misfolding, and its consequences such as aggregation, can be investigated by extending the types of techniques used to study the normal folding process. Application of these techniques is enabling the development of a unified description of the interconversion and regulation of the different conformational states available to proteins in living systems. Such a description proves a generic basis for understanding the fundamental links between protein misfolding and its associated clinical disorders, such as Alzheimer's disease and Type II diabetes, and for exploring novel therapeutic strategies directed at their prevention and treatment on a rational basis.
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Finkelstein, A. V.; Galzitskaya, O. V.
2004-04-01
Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.
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Synthetic oligorotaxanes exert high forces when folding under mechanical load
Sluysmans, Damien; Hubert, Sandrine; Bruns, Carson J.; Zhu, Zhixue; Stoddart, J. Fraser; Duwez, Anne-Sophie
2018-01-01
Folding is a ubiquitous process that nature uses to control the conformations of its molecular machines, allowing them to perform chemical and mechanical tasks. Over the years, chemists have synthesized foldamers that adopt well-defined and stable folded architectures, mimicking the control expressed by natural systems1,2. Mechanically interlocked molecules, such as rotaxanes and catenanes, are prototypical molecular machines that enable the controlled movement and positioning of their component parts3-5. Recently, combining the exquisite complexity of these two classes of molecules, donor-acceptor oligorotaxane foldamers have been synthesized, in which interactions between the mechanically interlocked component parts dictate the single-molecule assembly into a folded secondary structure6-8. Here we report on the mechanochemical properties of these molecules. We use atomic force microscopy-based single-molecule force spectroscopy to mechanically unfold oligorotaxanes, made of oligomeric dumbbells incorporating 1,5-dioxynaphthalene units encircled by cyclobis(paraquat-p-phenylene) rings. Real-time capture of fluctuations between unfolded and folded states reveals that the molecules exert forces of up to 50 pN against a mechanical load of up to 150 pN, and displays transition times of less than 10 μs. While the folding is at least as fast as that observed in proteins, it is remarkably more robust, thanks to the mechanically interlocked structure. Our results show that synthetic oligorotaxanes have the potential to exceed the performance of natural folding proteins.
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Maity, Hiranmay; Reddy, Govardhan
2018-04-01
Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.
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BiP clustering facilitates protein folding in the endoplasmic reticulum.
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Marc Griesemer
2014-07-01
Full Text Available The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER: translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'. Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.
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Understanding ensemble protein folding at atomic detail
International Nuclear Information System (INIS)
Wallin, Stefan; Shakhnovich, Eugene I
2008-01-01
Although far from routine, simulating the folding of specific short protein chains on the computer, at a detailed atomic level, is starting to become a reality. This remarkable progress, which has been made over the last decade or so, allows a fundamental aspect of the protein folding process to be addressed, namely its statistical nature. In order to make quantitative comparisons with experimental kinetic data a complete ensemble view of folding must be achieved, with key observables averaged over the large number of microscopically different folding trajectories available to a protein chain. Here we review recent advances in atomic-level protein folding simulations and the new insight provided by them into the protein folding process. An important element in understanding ensemble folding kinetics are methods for analyzing many separate folding trajectories, and we discuss techniques developed to condense the large amount of information contained in an ensemble of trajectories into a manageable picture of the folding process. (topical review)
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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.
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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...
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Protein folding simulations: from coarse-grained model to all-atom model.
Zhang, Jian; Li, Wenfei; Wang, Jun; Qin, Meng; Wu, Lei; Yan, Zhiqiang; Xu, Weixin; Zuo, Guanghong; Wang, Wei
2009-06-01
Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL
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Improving Protein Fold Recognition by Deep Learning Networks
Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin
2015-12-01
For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.
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Improving Protein Fold Recognition by Deep Learning Networks.
Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin
2015-12-04
For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.
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Makabe, Koki; Nakamura, Takashi; Dhar, Debanjan; Ikura, Teikichi; Koide, Shohei; Kuwajima, Kunihiro
2018-04-27
Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. Copyright © 2018. Published by Elsevier Ltd.
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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.
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How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?
Bonetti, Daniela; Troilo, Francesca; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano
2018-04-24
The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Solvent Effects on Protein Folding/Unfolding
García, A. E.; Hillson, N.; Onuchic, J. N.
Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.
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Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo
2017-12-01
Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent
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Frustration in Condensed Matter and Protein Folding
Li, Z.; Tanner, S.; Conroy, B.; Owens, F.; Tran, M. M.; Boekema, C.
2014-03-01
By means of computer modeling, we are studying frustration in condensed matter and protein folding, including the influence of temperature and Thomson-figure formation. Frustration is due to competing interactions in a disordered state. The key issue is how the particles interact to reach the lowest frustration. The relaxation for frustration is mostly a power function (randomly assigned pattern) or an exponential function (regular patterns like Thomson figures). For the atomic Thomson model, frustration is predicted to decrease with the formation of Thomson figures at zero kelvin. We attempt to apply our frustration modeling to protein folding and dynamics. We investigate the homogeneous protein frustration that would cause the speed of the protein folding to increase. Increase of protein frustration (where frustration and hydrophobicity interplay with protein folding) may lead to a protein mutation. Research is supported by WiSE@SJSU and AFC San Jose.
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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.
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Self-organized critical model for protein folding
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.
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In vitro folding of inclusion body proteins.
Rudolph, R; Lilie, H
1996-01-01
Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.
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Effects of knot type in the folding of topologically complex lattice proteins
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.
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Heterochiral Knottin Protein: Folding and Solution Structure.
Mong, Surin K; Cochran, Frank V; Yu, Hongtao; Graziano, Zachary; Lin, Yu-Shan; Cochran, Jennifer R; Pentelute, Bradley L
2017-10-31
Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.
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Evolution of a protein folding nucleus.
Xia, Xue; Longo, Liam M; Sutherland, Mason A; Blaber, Michael
2016-07-01
The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric β-trefoil protein by ϕ-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the β-trefoil. The results show structural acquisition of the FN during gene fusion events, incorporating novel turn structure created by gene fusion. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. FN plasticity by way of circular permutation is made possible by the intrinsic C3 cyclic symmetry of the β-trefoil architecture, identifying a possible selective advantage that helps explain the prevalence of cyclic structural symmetry in the proteome. © 2015 The Protein Society.
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High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.
Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A
2016-11-23
Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.
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SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition.
Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina
2007-05-22
Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at http://svm-fold.c2b2.columbia.edu. Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach
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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.
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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.
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Solitons and protein folding: An In Silico experiment
International Nuclear Information System (INIS)
Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.
2015-01-01
Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics
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Protein folding simulations by generalized-ensemble algorithms.
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.
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Impact of hydrodynamic interactions on protein folding rates depends on temperature
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.
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Visualization of protein folding funnels in lattice models.
Directory of Open Access Journals (Sweden)
Antonio B Oliveira
Full Text Available Protein folding occurs in a very high dimensional phase space with an exponentially large number of states, and according to the energy landscape theory it exhibits a topology resembling a funnel. In this statistical approach, the folding mechanism is unveiled by describing the local minima in an effective one-dimensional representation. Other approaches based on potential energy landscapes address the hierarchical structure of local energy minima through disconnectivity graphs. In this paper, we introduce a metric to describe the distance between any two conformations, which also allows us to go beyond the one-dimensional representation and visualize the folding funnel in 2D and 3D. In this way it is possible to assess the folding process in detail, e.g., by identifying the connectivity between conformations and establishing the paths to reach the native state, in addition to regions where trapping may occur. Unlike the disconnectivity maps method, which is based on the kinetic connections between states, our methodology is based on structural similarities inferred from the new metric. The method was developed in a 27-mer protein lattice model, folded into a 3×3×3 cube. Five sequences were studied and distinct funnels were generated in an analysis restricted to conformations from the transition-state to the native configuration. Consistent with the expected results from the energy landscape theory, folding routes can be visualized to probe different regions of the phase space, as well as determine the difficulty in folding of the distinct sequences. Changes in the landscape due to mutations were visualized, with the comparison between wild and mutated local minima in a single map, which serves to identify different trapping regions. The extension of this approach to more realistic models and its use in combination with other approaches are discussed.
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Microwave-enhanced folding and denaturation of globular proteins
DEFF Research Database (Denmark)
Bohr, Henrik; Bohr, Jakob
2000-01-01
It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from...... its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves...
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Ligand-promoted protein folding by biased kinetic partitioning.
Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M
2017-04-01
Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.
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International Nuclear Information System (INIS)
Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N.; Schug, Alexander
2015-01-01
The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism
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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)
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Prediction of the optimal set of contacts to fold the smallest knotted protein
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.
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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.
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Targeting the OB-Folds of Replication Protein A with Small Molecules
Directory of Open Access Journals (Sweden)
Victor J. Anciano Granadillo
2010-01-01
Full Text Available Replication protein A (RPA is the main eukaryotic single-strand (ss DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.
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Effects of a mutation on the folding mechanism of a β-hairpin
Juraszek, J.; Bolhuis, P.G.
2009-01-01
The folding mechanism of a protein is determined by its primary sequence. Yet, how the mechanism is changed by a mutation is still poorly understood, even for basic secondary structures such as β-hairpins. We perform an extensive simulation study of the effects of mutating the GB1 β-hairpin into
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Why and how does native topology dictate the folding speed of a protein?
Rustad, Mark; Ghosh, Kingshuk
2012-11-01
, we find our new topology based metric (combining ACO, COC1, and COC2) scales as N0.54, N being the number of amino acids in a protein. This is in remarkable agreement with a previous argument based on random systems that predict protein folding speed depends on exp (- N0.5). The first principle calculation presented here provides deeper insights to the role of topology in protein folding and unifies many parallel arguments, seemingly disconnected, demonstrating the existence of universal mechanism in protein folding kinetics that can be understood from simple polymer physics based principles.
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Light-activated control of protein channel assembly mediated by membrane mechanics
Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.
2016-12-01
Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.
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Examining a Thermodynamic Order Parameter of Protein Folding.
Chong, Song-Ho; Ham, Sihyun
2018-05-08
Dimensionality reduction with a suitable choice of order parameters or reaction coordinates is commonly used for analyzing high-dimensional time-series data generated by atomistic biomolecular simulations. So far, geometric order parameters, such as the root mean square deviation, fraction of native amino acid contacts, and collective coordinates that best characterize rare or large conformational transitions, have been prevailing in protein folding studies. Here, we show that the solvent-averaged effective energy, which is a thermodynamic quantity but unambiguously defined for individual protein conformations, serves as a good order parameter of protein folding. This is illustrated through the application to the folding-unfolding simulation trajectory of villin headpiece subdomain. We rationalize the suitability of the effective energy as an order parameter by the funneledness of the underlying protein free energy landscape. We also demonstrate that an improved conformational space discretization is achieved by incorporating the effective energy. The most distinctive feature of this thermodynamic order parameter is that it works in pointing to near-native folded structures even when the knowledge of the native structure is lacking, and the use of the effective energy will also find applications in combination with methods of protein structure prediction.
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Mechanisms of protein misfolding in conformational lung diseases.
LENUS (Irish Health Repository)
McElvaney, N G
2012-08-01
Genetic or environmentally-induced alterations in protein structure interfere with the correct folding, assembly and trafficking of proteins. In the lung the expression of misfolded proteins can induce a variety of pathogenetic effects. Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two major clinically relevant pulmonary disorders associated with protein misfolding. Both are genetic diseases the primary causes of which are expression of mutant alleles of the cystic fibrosis transmembrane conductance regulator (CFTR) and SERPINA1, respectively. The most common and best studied mutant forms of CFTR and AAT are ΔF508 CFTR and the Glu342Lys mutant of AAT called ZAAT, respectively. Non-genetic mechanisms can also damage protein structure and induce protein misfolding in the lung. Cigarette-smoke contains oxidants and other factors that can modify a protein\\'s structure, and is one of the most significant environmental causes of protein damage within the lung. Herein we describe the mechanisms controlling the folding of wild type and mutant versions of CFTR and AAT proteins, and explore the consequences of cigarette-smoke-induced effects on the protein folding machinery in the lung.
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Cotranslational protein folding reveals the selective use of ...
Indian Academy of Sciences (India)
to fold properly by decelerating the translation rate at these sites. Thus the cotranslational protein folding is believed to be true for many proteins and is an important selection factor for the selective codon usage to optimize proper gene expres- sion and function (Komar 2009). A web server CS and S has been created by ...
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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.
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Nanoscale Dewetting Transition in Protein Complex Folding
Hua, Lan; Huang, Xuhui; Liu, Pu; Zhou, Ruhong; Berne, Bruce J.
2011-01-01
In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding. PMID:17608515
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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.
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There and back again: Two views on the protein folding puzzle.
Finkelstein, Alexei V; Badretdin, Azat J; Galzitskaya, Oxana V; Ivankov, Dmitry N; Bogatyreva, Natalya S; Garbuzynskiy, Sergiy O
2017-07-01
The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the
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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.
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Aggregation of natively folded proteins: a theoretical approach
International Nuclear Information System (INIS)
Trovato, Antonio; Maritan, Amos; Seno, Flavio
2007-01-01
The reliable identification of β-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. While the list of aggregation related diseases is growing, it has also been shown that many proteins that are normally well behaved can be induced to aggregate in vitro. This fact suggests the existence of a unified framework that could explain both folding and aggregation. By assuming this universal behaviour, we have recently introduced an algorithm (PASTA: prediction of amyloid structure aggregation), which is based on a sequence-specific energy function derived from the propensity of two residue types to be found paired in neighbouring strands within β-sheets in globular proteins. The algorithm is able to predict the most aggregation-prone portions of several proteins initially unfolded, in excellent agreement with experimental results. Here, we apply the method to a set of proteins which are known to aggregate, but which are natively folded. The quality of the prediction is again very high, corroborating the hypothesis that the amyloid structure is stabilized by the same physico-chemical determinants as those operating in folded proteins
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Coherent topological phenomena in protein folding
DEFF Research Database (Denmark)
Bohr, Henrik; Brunak, Søren; Bohr, Jakob
1997-01-01
A theory is presented for coherent topological phenomena in protein dynamics with implications for protein folding and stability. We discuss the relationship to the writhing number used in knot diagrams of DNA. The winding state defines a long-range order along the backbone of a protein with long......-range excitations, `wring' modes, that play an important role in protein denaturation and stability. Energy can be pumped into these excitations, either thermally or by an external force....
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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.
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Quantification of Drive-Response Relationships Between Residues During Protein Folding.
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.
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Energy Technology Data Exchange (ETDEWEB)
NONE
2000-03-01
Efforts were exerted to achieve efficient expression of proteins of hyperthermophilic bacteria, hyperthermophilic archaeabacteria in particular, using a heterogene expression system in which Escherichia coli was the host. In an effort to search for genes related to protein folding and to elucidate the mechanism of folding, chaperonin and prefoldin subunit genes, out of various factors participating in protein folding in hyperthermophilic archaeabacteria, were cloned, and expressed in Escherichia coli. As a system for analyzing protein folding reaction, an experimental system was established on a substrate comprising isopropyl malate dehydrogenase, citrate synthase, glucose dehydrogenase, and a green fluorescent protein. Studies were further conducted to elucidate the mechanism of expression of enzyme genes in Escherichia coli for the establishment of a mass production method for useful enzymes. Also carried out was the research and development of an element technology evaluation system involving protein expression. (NEDO)
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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
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Protein fold recognition using geometric kernel data fusion.
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.
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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...
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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].
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Recent developments in the theory of protein folding: searching for the global energy minimum.
Scheraga, H A
1996-04-16
Statistical mechanical theories and computer simulation are being used to gain an understanding of the fundamental features of protein folding. A major obstacle in the computation of protein structures is the multiple-minima problem arising from the existence of many local minima in the multidimensional energy landscape of the protein. This problem has been surmounted for small open-chain and cyclic peptides, and for regular-repeating sequences of models of fibrous proteins. Progress is being made in resolving this problem for globular proteins.
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Extreme Mechanics: Self-Folding Origami
Santangelo, Christian D.
2017-03-01
Origami has emerged as a tool for designing three-dimensional structures from flat films. Because they can be fabricated by lithographic or roll-to-roll processing techniques, they have great potential for the manufacture of complicated geometries and devices. This article discusses the mechanics of origami and kirigami with a view toward understanding how to design self-folding origami structures. Whether an origami structure can be made to fold autonomously depends strongly on the geometry and kinematics of the origami fold pattern. This article collects some of the results on origami rigidity into a single framework, and discusses how these aspects affect the foldability of origami. Despite recent progress, most problems in origami and origami design remain completely open.
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Periodic and stochastic thermal modulation of protein folding kinetics.
Platkov, Max; Gruebele, Martin
2014-07-21
Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.
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Reader, J.S.; van Nuland, N.A.J.; Thompson, G.S.; Ferguson, S.J.; Dobson, C.M.; Radford, S.E.
2001-01-01
The folding of apo-pseudoazurin, a 123-residue, predominantly -sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using farand near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to
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The nature of folded states of globular proteins.
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.
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Probabilistic analysis for identifying the driving force of protein folding
Tokunaga, Yoshihiko; Yamamori, Yu; Matubayasi, Nobuyuki
2018-03-01
Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.
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Bergasa-Caceres, Fernando; Rabitz, Herschel A.
2013-06-01
A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.
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Conformational dynamics of a protein in the folded and the unfolded state
Energy Technology Data Exchange (ETDEWEB)
Fitter, Joerg
2003-08-01
In a quasielastic neutron scattering experiment, the picosecond dynamics of {alpha}-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D{sub 2}O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 A; unfolded state, 1.8 A). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.
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Thermodynamic properties of an extremely rapid protein folding reaction.
Schindler, T; Schmid, F X
1996-12-24
The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to
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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.
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A highly compliant protein native state with a spontaneous-like mechanical unfolding pathway
DEFF Research Database (Denmark)
Heiðarsson, Pétur Orri; Valpapuram, Immanuel; Camilloni, Carlo
2012-01-01
The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate...... states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force...... of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal...
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Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E; Lieberman, Andrew P; Osawa, Yoichi
2014-11-01
In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. © 2014 by the Society for Experimental Biology and Medicine.
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Transferable coarse-grained potential for de novo protein folding and design.
Directory of Open Access Journals (Sweden)
Ivan Coluzza
Full Text Available Protein folding and design are major biophysical problems, the solution of which would lead to important applications especially in medicine. Here we provide evidence of how a novel parametrization of the Caterpillar model may be used for both quantitative protein design and folding. With computer simulations it is shown that, for a large set of real protein structures, the model produces designed sequences with similar physical properties to the corresponding natural occurring sequences. The designed sequences require further experimental testing. For an independent set of proteins, previously used as benchmark, the correct folded structure of both the designed and the natural sequences is also demonstrated. The equilibrium folding properties are characterized by free energy calculations. The resulting free energy profiles not only are consistent among natural and designed proteins, but also show a remarkable precision when the folded structures are compared to the experimentally determined ones. Ultimately, the updated Caterpillar model is unique in the combination of its fundamental three features: its simplicity, its ability to produce natural foldable designed sequences, and its structure prediction precision. It is also remarkable that low frustration sequences can be obtained with such a simple and universal design procedure, and that the folding of natural proteins shows funnelled free energy landscapes without the need of any potentials based on the native structure.
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Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.
Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe
2016-01-12
We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.
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An update of the DEF database of protein fold class predictions
DEFF Research Database (Denmark)
Reczko, Martin; Karras, Dimitris; Bohr, Henrik
1997-01-01
An update is given on the Database of Expected Fold classes (DEF) that contains a collection of fold-class predictions made from protein sequences and a mail server that provides new predictions for new sequences. To any given sequence one of 49 fold-classes is chosen to classify the structure re...... related to the sequence with high accuracy. The updated predictions system is developed using data from the new version of the 3D-ALI database of aligned protein structures and thus is giving more reliable and more detailed predictions than the previous DEF system.......An update is given on the Database of Expected Fold classes (DEF) that contains a collection of fold-class predictions made from protein sequences and a mail server that provides new predictions for new sequences. To any given sequence one of 49 fold-classes is chosen to classify the structure...
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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.
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Mechanical Models of Fault-Related Folding
Energy Technology Data Exchange (ETDEWEB)
Johnson, A. M.
2003-01-09
The subject of the proposed research is fault-related folding and ground deformation. The results are relevant to oil-producing structures throughout the world, to understanding of damage that has been observed along and near earthquake ruptures, and to earthquake-producing structures in California and other tectonically-active areas. The objectives of the proposed research were to provide both a unified, mechanical infrastructure for studies of fault-related foldings and to present the results in computer programs that have graphical users interfaces (GUIs) so that structural geologists and geophysicists can model a wide variety of fault-related folds (FaRFs).
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Thermodynamics of protein folding: a random matrix formulation.
Shukla, Pragya
2010-10-20
The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies. © 2010 IOP Publishing Ltd
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Stretching and folding mechanism in foams
International Nuclear Information System (INIS)
Tufaile, Alberto; Pedrosa Biscaia Tufaile, Adriana
2008-01-01
We have described the stretching and folding of foams in a vertical Hele-Shaw cell containing air and a surfactant solution, from a sequence of upside-down flips. Besides the fractal dimension of the foam, we have observed the logistic growth for the soap film length. The stretching and folding mechanism is present during the foam formation, and this mechanism is observed even after the foam has reached its respective maximum fractal dimension. Observing the motion of bubbles inside the foam, large bubbles present power spectrum associated with random walk motion in both directions, while the small bubbles are scattered like balls in a Galton board
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Stretching and folding mechanism in foams
Energy Technology Data Exchange (ETDEWEB)
Tufaile, Alberto [Escola de Artes, Ciencias e Humanidades, Soft Matter Laboratory, Universidade de Sao Paulo, 03828-000 Sao Paulo, SP (Brazil)], E-mail: tufaile@usp.br; Pedrosa Biscaia Tufaile, Adriana [Escola de Artes, Ciencias e Humanidades, Soft Matter Laboratory, Universidade de Sao Paulo, 03828-000 Sao Paulo, SP (Brazil)
2008-10-13
We have described the stretching and folding of foams in a vertical Hele-Shaw cell containing air and a surfactant solution, from a sequence of upside-down flips. Besides the fractal dimension of the foam, we have observed the logistic growth for the soap film length. The stretching and folding mechanism is present during the foam formation, and this mechanism is observed even after the foam has reached its respective maximum fractal dimension. Observing the motion of bubbles inside the foam, large bubbles present power spectrum associated with random walk motion in both directions, while the small bubbles are scattered like balls in a Galton board.
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Some physical approaches to protein folding
Bascle, J.; Garel, T.; Orland, H.
1993-02-01
To understand how a protein folds is a problem which has important biological implications. In this article, we would like to present a physics-oriented point of view, which is twofold. First of all, we introduce simple statistical mechanics models which display, in the thermodynamic limit, folding and related transitions. These models can be divided into (i) crude spin glass-like models (with their Mattis analogs), where one may look for possible correlations between the chain self-interactions and the folded structure, (ii) glass-like models, where one emphasizes the geometrical competition between one- or two-dimensional local order (mimicking α helix or β sheet structures), and the requirement of global compactness. Both models are too simple to predict the spatial organization of a realistic protein, but are useful for the physicist and should have some feedback in other glassy systems (glasses, collapsed polymers .... ). These remarks lead us to the second physical approach, namely a new Monte-Carlo method, where one grows the protein atom-by-atom (or residue-by-residue), using a standard form (CHARMM .... ) for the total energy. A detailed comparison with other Monte-Carlo schemes, or Molecular Dynamics calculations, is then possible; we will sketch such a comparison for poly-alanines. Our twofold approach illustrates some of the difficulties one encounters in the protein folding problem, in particular those associated with the existence of a large number of metastable states. Le repliement des protéines est un problème qui a de nombreuses implications biologiques. Dans cet article, nous présentons, de deux façons différentes, un point de vue de physicien. Nous introduisons tout d'abord des modèles simples de mécanique statistique qui exhibent, à la limite thermodynamique, des transitions de repliement. Ces modèles peuvent être divisés en (i) verres de spin (éventuellement à la Mattis), où l'on peut chercher des corrélations entre les
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Experimental investigation into the mechanism of folding
Kuenen, Ph.H.; Sitter, de L.U.
1938-01-01
The investigation of geological structures due to folding led de Sitter to form an opinion on the mechanical problems involved (Bibl. 7). His principal contention is that in simple cases the relative movements of particles with respect to eachother during deformation leading to a fold, have been
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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.
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CASP10-BCL::Fold efficiently samples topologies of large proteins.
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.
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Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study.
Strickland, Mallory L; Boylan, Helen M
2010-07-01
The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.
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Modulation of the maladaptive stress response to manage diseases of protein folding.
Directory of Open Access Journals (Sweden)
Daniela Martino Roth
2014-11-01
Full Text Available Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD, Niemann-Pick type C1 disease (NPC1, Alzheimer's disease (AD, and cystic fibrosis (CF. Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR, through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.
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On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1.
Wu, Chun; Shea, Joan-Emma
2010-11-18
FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures.
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Multiple molecule effects on the cooperativity of protein folding transitions in simulations
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.
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A Particle Swarm Optimization-Based Approach with Local Search for Predicting Protein Folding.
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.
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Peptide folding in the presence of interacting protein crowders
Energy Technology Data Exchange (ETDEWEB)
Bille, Anna, E-mail: anna.bille@thep.lu.se; Irbäck, Anders, E-mail: anders@thep.lu.se [Computational Biology and Biological Physics, Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, SE-223 62 Lund (Sweden); Mohanty, Sandipan, E-mail: s.mohanty@fz-juelich.de [Jülich Supercomputing Centre, Institute for Advanced Simulation, Forschungszentrum Jülich, D-52425 Jülich (Germany)
2016-05-07
Using Monte Carlo methods, we explore and compare the effects of two protein crowders, BPTI and GB1, on the folding thermodynamics of two peptides, the compact helical trp-cage and the β-hairpin-forming GB1m3. The thermally highly stable crowder proteins are modeled using a fixed backbone and rotatable side-chains, whereas the peptides are free to fold and unfold. In the simulations, the crowder proteins tend to distort the trp-cage fold, while having a stabilizing effect on GB1m3. The extent of the effects on a given peptide depends on the crowder type. Due to a sticky patch on its surface, BPTI causes larger changes than GB1 in the melting properties of the peptides. The observed effects on the peptides stem largely from attractive and specific interactions with the crowder surfaces, and differ from those seen in reference simulations with purely steric crowder particles.
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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.
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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.
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Protein folding and misfolding shining light by infrared spectroscopy
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...
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Reliable protein folding on non-funneled energy landscapes: the free energy reaction path
Lois, Gregg; Blawzdziewicz, Jerzy; O'Hern, Corey S.
2008-01-01
A theoretical framework is developed to study the dynamics of protein folding. The key insight is that the search for the native protein conformation is influenced by the rate r at which external parameters, such as temperature, chemical denaturant or pH, are adjusted to induce folding. A theory based on this insight predicts that (1) proteins with non-funneled energy landscapes can fold reliably to their native state, (2) reliable folding can occur as an equilibrium or out-of-equilibrium pro...
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Kinetic partitioning mechanism of HDV ribozyme folding
Energy Technology Data Exchange (ETDEWEB)
Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing, E-mail: wbzhang@whu.edu.cn [Department of Physics, Wuhan University, Wuhan, Hubei 430072 (China)
2014-01-14
RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.
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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.
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A replica exchange Monte Carlo algorithm for protein folding in the HP model
Directory of Open Access Journals (Sweden)
Shmygelska Alena
2007-09-01
neighbourhood significantly outperforms current state-of-the-art methods for protein structure prediction in the HP model on 2D and 3D lattices. This is particularly noteworthy, since so far, the state-of-the-art methods for 2D and 3D HP protein folding – in particular, the pruned-enriched Rosenbluth method (PERM and, to some extent, Ant Colony Optimisation (ACO – were based on chain growth mechanisms. To the best of our knowledge, this is the first application of REMC to HP protein folding on the cubic lattice, and the first extension of the pull move neighbourhood to a 3D lattice.
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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...
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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.
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New insights into structural determinants of prion protein folding and stability.
Benetti, Federico; Legname, Giuseppe
2015-01-01
Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.
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Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation.
Madain, Alia; Abu Dalhoum, Abdel Latif; Sleit, Azzam
2018-06-01
The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic-hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H-H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H's at even positions and the other side ignores H's at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H-H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.
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Suhanovsky, Margaret M.; Teschke, Carolyn M.
2013-01-01
The bacteriophage P22 coat protein has the common HK97-like fold but with a genetically inserted domain (I-domain). The role of the I-domain, positioned at the outermost surface of the capsid, is unknown. We hypothesize that the I-domain may act as an intramolecular chaperone because the coat protein folds independently, and many folding mutants are localized to the I-domain. The function of the I-domain was investigated by generating the coat protein core without its I-domain and the isolated I-domain. The core coat protein shows a pronounced folding defect. The isolated I-domain folds autonomously and has a high thermodynamic stability and fast folding kinetics in the presence of a peptidyl prolyl isomerase. Thus, the I-domain provides thermodynamic stability to the full-length coat protein so that it can fold reasonably efficiently while still allowing the HK97-like core to retain the flexibility required for conformational switching during procapsid assembly and maturation. PMID:24126914
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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.
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Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis
Krishnappa, Laxmi; Monteferrante, Carmine G; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten
The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold
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DEFF Research Database (Denmark)
Amatori, Andrea; Ferkinghoff-Borg, Jesper; Tiana, Guido
2008-01-01
The thermodynamics of proteins designed on three common folds (SH3, chymotrypsin inhibitor 2 [CI2], and protein G) is studied with a simplified C alpha, model and compared with the thermodynamics of proteins designed on random-generated folds. The model allows to design sequences to fold within a...
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Protein Folding Free Energy Landscape along the Committor - the Optimal Folding Coordinate.
Krivov, Sergei V
2018-06-06
Recent advances in simulation and experiment have led to dramatic increases in the quantity and complexity of produced data, which makes the development of automated analysis tools very important. A powerful approach to analyze dynamics contained in such data sets is to describe/approximate it by diffusion on a free energy landscape - free energy as a function of reaction coordinates (RC). For the description to be quantitatively accurate, RCs should be chosen in an optimal way. Recent theoretical results show that such an optimal RC exists; however, determining it for practical systems is a very difficult unsolved problem. Here we describe a solution to this problem. We describe an adaptive nonparametric approach to accurately determine the optimal RC (the committor) for an equilibrium trajectory of a realistic system. In contrast to alternative approaches, which require a functional form with many parameters to approximate an RC and thus extensive expertise with the system, the suggested approach is nonparametric and can approximate any RC with high accuracy without system specific information. To avoid overfitting for a realistically sampled system, the approach performs RC optimization in an adaptive manner by focusing optimization on less optimized spatiotemporal regions of the RC. The power of the approach is illustrated on a long equilibrium atomistic folding simulation of HP35 protein. We have determined the optimal folding RC - the committor, which was confirmed by passing a stringent committor validation test. It allowed us to determine a first quantitatively accurate protein folding free energy landscape. We have confirmed the recent theoretical results that diffusion on such a free energy profile can be used to compute exactly the equilibrium flux, the mean first passage times, and the mean transition path times between any two points on the profile. We have shown that the mean squared displacement along the optimal RC grows linear with time as for
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Mallik, Saurav; Das, Smita; Kundu, Sudip
2016-01-01
Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.
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Quality control mechanisms of protein biogenesis: proteostasis dies hard
Directory of Open Access Journals (Sweden)
Timothy Jan Bergmann
2016-10-01
Full Text Available The biosynthesis of proteins entails a complex series of chemical reactions that transform the information stored in the nucleic acid sequence into a polypeptide chain that needs to properly fold and reach its functional location in or outside the cell. It is of no surprise that errors might occur that alter the polypeptide sequence leading to a non-functional proteins or that impede delivery of proteins at the appropriate site of activity. In order to minimize such mistakes and guarantee the synthesis of the correct amount and quality of the proteome, cells have developed folding, quality control, degradation and transport mechanisms that ensure and tightly regulate protein biogenesis. Genetic mutations, harsh environmental conditions or attack by pathogens can subvert the cellular quality control machineries and perturb cellular proteostasis leading to pathological conditions. This review summarizes basic concepts of the flow of information from DNA to folded and active proteins and to the variable fidelity (from incredibly high to quite sloppy characterizing these processes. We will give particular emphasis on events that maintain or recover the homeostasis of the endoplasmic reticulum (ER, a major site of proteins synthesis and folding in eukaryotic cells. Finally, we will report on how cells can adapt to stressful conditions, how perturbation of ER homeostasis may result in diseases and how these can be treated.
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DEFF Research Database (Denmark)
Ramnarayan, K.; Bohr, Henrik; Jalkanen, Karl J.
2008-01-01
We present different means of classifying protein structure. One is made rigorous by mathematical knot invariants that coincide reasonably well with ordinary graphical fold classification and another classification is by packing analysis. Furthermore when constructing our mathematical fold...... classifications, we utilize standard neural network methods for predicting protein fold classes from amino acid sequences. We also make an analysis of the redundancy of the structural classifications in relation to function and ligand binding. Finally we advocate the use of combining the measurement of the VA...
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Goyal, Megha; Chaudhuri, Tapan K
2015-07-01
Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.
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How Many Protein Sequences Fold to a Given Structure? A Coevolutionary Analysis.
Tian, Pengfei; Best, Robert B
2017-10-17
Quantifying the relationship between protein sequence and structure is key to understanding the protein universe. A fundamental measure of this relationship is the total number of amino acid sequences that can fold to a target protein structure, known as the "sequence capacity," which has been suggested as a proxy for how designable a given protein fold is. Although sequence capacity has been extensively studied using lattice models and theory, numerical estimates for real protein structures are currently lacking. In this work, we have quantitatively estimated the sequence capacity of 10 proteins with a variety of different structures using a statistical model based on residue-residue co-evolution to capture the variation of sequences from the same protein family. Remarkably, we find that even for the smallest protein folds, such as the WW domain, the number of foldable sequences is extremely large, exceeding the Avogadro constant. In agreement with earlier theoretical work, the calculated sequence capacity is positively correlated with the size of the protein, or better, the density of contacts. This allows the absolute sequence capacity of a given protein to be approximately predicted from its structure. On the other hand, the relative sequence capacity, i.e., normalized by the total number of possible sequences, is an extremely tiny number and is strongly anti-correlated with the protein length. Thus, although there may be more foldable sequences for larger proteins, it will be much harder to find them. Lastly, we have correlated the evolutionary age of proteins in the CATH database with their sequence capacity as predicted by our model. The results suggest a trade-off between the opposing requirements of high designability and the likelihood of a novel fold emerging by chance. Published by Elsevier Inc.
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Dry molten globule intermediates and the mechanism of protein unfolding.
Baldwin, Robert L; Frieden, Carl; Rose, George D
2010-10-01
New experimental results show that either gain or loss of close packing can be observed as a discrete step in protein folding or unfolding reactions. This finding poses a significant challenge to the conventional two-state model of protein folding. Results of interest involve dry molten globule (DMG) intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the DMG state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form DMGs as the first step of unfolding, suggesting that such an intermediate may well be commonplace in both folding and unfolding. Data from the literature show that peptide amide protons are protected in the DMG, indicating that backbone structure is intact despite loss of side-chain close packing. Other complementary evidence shows that secondary structure formation provides a major source of compaction during folding. In our model, the major free-energy barrier separating unfolded from native states usually occurs during the transition between the unfolded state and the DMG. The absence of close packing at this barrier provides an explanation for why phi-values, derived from a Brønsted-Leffler plot, depend primarily on structure at the mutational site and not on specific side-chain interactions. The conventional two-state folding model breaks down when there are DMG intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding. 2010 Wiley-Liss, Inc.
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Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function
International Nuclear Information System (INIS)
Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.
2008-01-01
T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family
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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.
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Self-organization and mismatch tolerance in protein folding: General theory and an application
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.
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Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.
2016-06-01
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.
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Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.
Finka, Andrija; Goloubinoff, Pierre
2013-09-01
In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.
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Co-evolutionary constraints of globular proteins correlate with their folding rates.
Mallik, Saurav; Kundu, Sudip
2015-08-04
Folding rates (lnkf) of globular proteins correlate with their biophysical properties, but relationship between lnkf and patterns of sequence evolution remains elusive. We introduce 'relative co-evolution order' (rCEO) as length-normalized average primary chain separation of co-evolving pairs (CEPs), which negatively correlates with lnkf. In addition to pairs in native 3D contact, indirectly connected and structurally remote CEPs probably also play critical roles in protein folding. Correlation between rCEO and lnkf is stronger in multi-state proteins than two-state proteins, contrasting the case of contact order (co), where stronger correlation is found in two-state proteins. Finally, rCEO, co and lnkf are fitted into a 3D linear correlation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Directory of Open Access Journals (Sweden)
Laura E Rosen
Full Text Available An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I(core and I(core+1 hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH fragments have very similar stabilities, suggesting that the presence of additional residues in the I(core+1 fragment does not affect the folding or structure as compared to I(core. NMR experiments provide additional evidence that only the I(core intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I(core+1 intermediate. We determine that the ttRNH I(core+1 intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which I(core is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.
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Equilibrium amide hydrogen exchange and protein folding kinetics
International Nuclear Information System (INIS)
Bai Yawen
1999-01-01
The classical Linderstrom-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (I↔U↔N). On the other hand, in an on-pathway three-state system (U↔I↔N), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments
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An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.
Directory of Open Access Journals (Sweden)
Marcin Michalik
Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.
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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.
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Bioinformatics analysis identify novel OB fold protein coding genes in C. elegans.
Directory of Open Access Journals (Sweden)
Daryanaz Dargahi
Full Text Available BACKGROUND: The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5-25%. Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n=46 compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n=344 or fruit fly D. melanogaster (n=84. Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies. METHODOLOGY/PRINCIPAL FINDINGS: This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans. CONCLUSIONS/SIGNIFICANCE: This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.
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Sampling-based exploration of folded state of a protein under kinematic and geometric constraints
Yao, Peggy; Zhang, Liangjun; Latombe, Jean-Claude
2011-01-01
Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable
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Protein folding on the ribosome studied using NMR spectroscopy
Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John
2013-01-01
NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462
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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.
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Folding Membrane Proteins by Deep Transfer Learning
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.
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Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis
Directory of Open Access Journals (Sweden)
Qian Zhao
2018-01-01
Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead
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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.
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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.
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2015-01-01
Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351
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PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.
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.
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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/.
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Generic framework for mining cellular automata models on protein-folding simulations.
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.
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Folded membrane dialyzer with mechanically sealed edges
Energy Technology Data Exchange (ETDEWEB)
Markley, F.W.
A semipermeable membrane is folded in accordion fashion to form a stack of pleats and the edges are sealed so as to isolate the opposite surfaces of the membrane. The stack is contained within a case that provides ports for flow of blood in contact with one surface of the membrane through channels formed by the pleats and also provides ports for flow of a dialysate through channels formed by the pleats in contact with the other surface of the membrane. The serpentine side edges of the membrane are sealed by a solidified plastic material, whereas effective mechanical means are provided to seal the end edges of the folded membrane. The mechanical means include a clamping strip which biases case sealing flanges into a sealed relationship with end portions of the membrane near the end edges, which portions extend from the stack and between the sealing flanges.
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An expanded view of the protein folding landscape of PDZ domains
DEFF Research Database (Denmark)
Hultqvist, Greta; Pedersen, Søren W; Chi, Celestine N.
2012-01-01
state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding...
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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
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Directory of Open Access Journals (Sweden)
Jin Wang
2011-04-01
Full Text Available Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change - protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.
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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.
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Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.
Sato, Yoshimi; Kojima, Rieko; Okumura, Masaki; Hagiwara, Masatoshi; Masui, Shoji; Maegawa, Ken-ichi; Saiki, Masatoshi; Horibe, Tomohisa; Suzuki, Mamoru; Inaba, Kenji
2013-01-01
The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.
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Sampling-based exploration of folded state of a protein under kinematic and geometric constraints
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.
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Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation
Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting
2017-07-01
To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project
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Two states or not two states: Single-molecule folding studies of protein L
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.
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A Bystander Mechanism Explains the Specific Phenotype of a Broadly Expressed Misfolded Protein.
Directory of Open Access Journals (Sweden)
Lauren Klabonski
2016-12-01
Full Text Available Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like C.elegans protein DAF-28. DAF-28(R37C is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype-deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations.
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Theory and simulation of explicit solvent effects on protein folding in vitro and in vivo
England, Jeremy L.
The aim of this work is to develop theoretical tools for understanding what happens to water that is confined in amphipathic cavities, and for testing the consequences of this understanding for protein folding in vitro and in vivo. We begin in the first chapter with a brief review of the theoretical and simulation literature on the hydrophobic effect and the aqueous solvation of charged species that also puts forward a simple theoretical framework within which various solvation phenomena reported in past studies may be unified. Subsequently, in the second chapter we also review past computational and theoretical work on the specific question of how chaperonin complexes assist the folding of their substrates. With the context set, we turn in Chapter 3 to the case of an open system with water trapped between hydrophobic plates that experiences a uniform electric field normal to and between the plates. Classic bulk theory of electrostriction in polarizable fluids tells us that the electric field should cause an increase in local water density as it rises, yet some simulations have suggested the opposite. We present a mean-field Potts model we have developed to explain this discrepancy, and show how such a simple, coarse-grained lattice description can capture the fundamental consequences of the fact that external electric fields can frustrate the hydrogen bond network in confined water. Chapter 4 continues to pursue the issue of solvent evacuation between hydrophobic plates, but focuses on the impact of chemical denaturants on hydrophobic effects using molecular dynamics simulations of hydrophobic dewetting. We find that while urea and guanidinium have similar qualitative effects at the bulk level, they seem to differ in the microscopic mechanism by which they denature proteins, although both inhibit the onset of dewetting. Lastly, Chapters 5 and 6 examine the potential importance of solvent-mediated forces to protein folding in vivo. Chapter 5 develops a Landau
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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.
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Shaheen, Alaa
2018-05-05
The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.
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Zhang, Dawei; Lazim, Raudah; Mun Yip, Yew
2017-09-01
We conducted an all-atom ab initio folding of FSD-EY, a protein with a ββα configuration using non-polarizable (AMBER) and polarizable force fields (PHB designed by Gao et al.) in implicit solvent. The effect of reducing the polarization effect integrated into the force field by the PHB model, termed the PHB0.7 was also examined in the folding of FSD-EY. This model incorporates into the force field 70% of the original polarization effect to minimize the likelihood of over-stabilizing the backbone hydrogen bonds. Precise folding of the β-sheet of FSD-EY was further achieved by relaxing the REMD structure obtained in explicit water.
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Structure of the thioredoxin-fold domain of human phosducin-like protein 2
International Nuclear Information System (INIS)
Lou, Xiaochu; Bao, Rui; Zhou, Cong-Zhao; Chen, Yuxing
2009-01-01
The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence
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Identification of a key structural element for protein folding within beta-hairpin turns.
Kim, Jaewon; Brych, Stephen R; Lee, Jihun; Logan, Timothy M; Blaber, Michael
2003-05-09
Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.
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Protein folding includes oligomerization – examples from the endoplasmic reticulum and cytosol
Christis, C.; Lubsen, N.H.; Braakman, I.
2008-01-01
A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma
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General Protein Data Bank-Based Collective Variables for Protein Folding.
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.
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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.
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Force generation by titin folding.
Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós
2017-07-01
Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.
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A multi-directional rapidly exploring random graph (mRRG) for protein folding
Nath, Shuvra Kanti; Thomas, Shawna; Ekenna, Chinwe; Amato, Nancy M.
2012-01-01
Modeling large-scale protein motions, such as those involved in folding and binding interactions, is crucial to better understanding not only how proteins move and interact with other molecules but also how proteins misfold, thus causing many devastating diseases. Robotic motion planning algorithms, such as Rapidly Exploring Random Trees (RRTs), have been successful in simulating protein folding pathways. Here, we propose a new multi-directional Rapidly Exploring Random Graph (mRRG) specifically tailored for proteins. Unlike traditional RRGs which only expand a parent conformation in a single direction, our strategy expands the parent conformation in multiple directions to generate new samples. Resulting samples are connected to the parent conformation and its nearest neighbors. By leveraging multiple directions, mRRG can model the protein motion landscape with reduced computational time compared to several other robotics-based methods for small to moderate-sized proteins. Our results on several proteins agree with experimental hydrogen out-exchange, pulse-labeling, and F-value analysis. We also show that mRRG covers the conformation space better as compared to the other computation methods. Copyright © 2012 ACM.
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In vivo labelling of proteins associated with folded chromosomes of yeast
International Nuclear Information System (INIS)
Litske Petersen, J.G.; Pinon, R.
1980-01-01
Proteins associated with the pre-replicative (g 1 ) and post-replicative (g 2 ) folded chromosomes of Saccharomyces cerevisiae can be labelled in vivo by growing cells in acetate vegetative medium containing [ 35 S]methionine. In both sporulating (MATa/MATα) and non-sporulating (MATa/MATa, MATα/MATα) diploids proteins associated with the resting stage genome (g 0 ) can be labelled with [ 35 S]methionine during nitrogen starvation and in sporulation medium. In addition, in MATa/MATα diploids proteins associated with the meiotic replication form (r) can also be labelled. SDS-polyacrylamide gel electrophoresis and autoradiography of the labelled proteins from the various folded genome forms showed that the g 1 and g 2 patterns are, with the exception of one polypeptide band, essentially identical. Several differences distinguished the r and g 0 patterns from those of the g 1 and g 2 structures. At least four polypeptide bands distinguish the r and g 0 patterns. No significant differences were observed between the g 0 proteins of sporulating and non-sporulating diploids. (author)
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Shannon, G.; Wei, C.; Pohorille, A.
2017-01-01
Considering the range of functions proteins perform, it is surprising they fold into a relatively small set of structures or "folds" that facilitate such function. One explanation is that only a minority were fit enough to emerge from Darwinian selection during the early evolution of life. Alternatively, perhaps only a fraction of all possible folds were trialed. Understanding proto-catalyst selection will aid understanding of the origins and early evolution of life. To investigate which explanation is correct, we study a protein evolved in vitro to bind ATP by Jack Szostak (Fig. 1). This protein adopts a fold which is absent from nature. We are testing whether this fold would have possessed the capability to evolve that would have been essential to survive natural selection on early Earth. Folds that couldn't improve their fitness and evolve to perform new functions would have been replaced by rivals that could. To determine whether the fold is evolvable, we are attempting to change the function of the protein by rationally redesigning to bind GTP. Two design strategies in the region of the nucleobase have been implemented to provide hydrogen bonding partners for the ligand i) an insertion ii) a MET to ASN mutation. Redesigns are being studied computationally at Ames Research Center including free energy of binding calculations. Binding affinities of promising redesigns are to be validated by experimental collaborators at ForteBio using Super Streptavidin Biosensors. If the fold is found to be non-evolvable, this may suggest that many structures were trialed, but the majority were pruned on the basis of their evolvability. Alternatively, if the fold is demonstrated to be evolvable, it would be difficult to explain its absence from nature without considering the possibility that the fold simply wasn't sampled on early Earth. This would not only further our understanding of the origins of life on Earth but also suggest a common phe-nomenon of proto
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Energy Technology Data Exchange (ETDEWEB)
Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul [Department of Physics, University at Buffalo, Buffalo, New York 14260 (United States)
2015-01-21
The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.
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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.
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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.
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Fast mapping of global protein folding states by multivariate NMR:
DEFF Research Database (Denmark)
Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel
2010-01-01
To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method......-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat....
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Bordner, Andrew J.; Zorman, Barry; Abagyan, Ruben
2011-10-01
Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host-guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.
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Can Natural Proteins Designed with ‘Inverted’ Peptide Sequences Adopt Native-Like Protein Folds?
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
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Adaptive local learning in sampling based motion planning for protein folding.
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.
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FoldEco: A Model for Proteostasis in E. coli
Directory of Open Access Journals (Sweden)
Evan T. Powers
2012-03-01
Full Text Available To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide questions and shows how to convert these questions to experimentally measurable quantities, such as changes in protein concentrations with chaperone or protease levels, which can then be used to improve our current understanding of proteostasis and refine the model. A web version of FoldEco is available at http://foldeco.scripps.edu.
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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.
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Effect of the geometry of confining media on the stability and folding rate of α -helix proteins
Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad
2018-05-01
Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding
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Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein
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
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Wen, Fu-Lai; Wang, Yu-Chiun; Shibata, Tatsuo
2017-06-20
During embryonic development, epithelial sheets fold into complex structures required for tissue and organ functions. Although substantial efforts have been devoted to identifying molecular mechanisms underlying epithelial folding, far less is understood about how forces deform individual cells to sculpt the overall sheet morphology. Here we describe a simple and general theoretical model for the autonomous folding of monolayered epithelial sheets. We show that active modulation of intracellular mechanics along the basal-lateral as well as the apical surfaces is capable of inducing fold formation in the absence of buckling instability. Apical modulation sculpts epithelia into shallow and V-shaped folds, whereas basal-lateral modulation generates deep and U-shaped folds. These characteristic tissue shapes remain unchanged when subject to mechanical perturbations from the surroundings, illustrating that the autonomous folding is robust against environmental variabilities. At the cellular scale, how cells change shape depends on their initial aspect ratios and the modulation mechanisms. Such cell deformation characteristics are verified via experimental measurements for a canonical folding process driven by apical modulation, indicating that our theory could be used to infer the underlying folding mechanisms based on experimental data. The mechanical principles revealed in our model could potentially guide future studies on epithelial folding in diverse systems. Copyright © 2017. Published by Elsevier Inc.
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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
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Geierhaas, Christian D; Salvatella, Xavier; Clarke, Jane; Vendruscolo, Michele
2008-03-01
It has been suggested that Phi-values, which allow structural information about transition states (TSs) for protein folding to be obtained, are most reliably interpreted when divided into three classes (high, medium and low). High Phi-values indicate almost completely folded regions in the TS, intermediate Phi-values regions with a detectable amount of structure and low Phi-values indicate mostly unstructured regions. To explore the extent to which this classification can be used to characterise in detail the structure of TSs for protein folding, we used Phi-values divided into these classes as restraints in molecular dynamics simulations. This type of procedure is related to that used in NMR spectroscopy to define the structure of native proteins from the measurement of inter-proton distances derived from nuclear Overhauser effects. We illustrate this approach by determining the TS ensembles of five proteins and by showing that the results are similar to those obtained by using as restraints the actual numerical Phi-values measured experimentally. Our results indicate that the simultaneous consideration of a set of low-resolution Phi-values can provide sufficient information for characterising the architecture of a TS for folding of a protein.
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Introducing the Levinthal's Protein Folding Paradox and Its Solution
Martínez, Leandro
2014-01-01
The protein folding (Levinthal's) paradox states that it would not be possible in a physically meaningful time to a protein to reach the native (functional) conformation by a random search of the enormously large number of possible structures. This paradox has been solved: it was shown that small biases toward the native conformation result…
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Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R
2006-11-28
We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.
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Co-Transcriptional Folding and Regulation Mechanisms of Riboswitches
Directory of Open Access Journals (Sweden)
Sha Gong
2017-07-01
Full Text Available Riboswitches are genetic control elements within non-coding regions of mRNA. These self-regulatory elements have been found to sense a range of small metabolites, ions, and other physical signals to exert regulatory control of transcription, translation, and splicing. To date, more than a dozen riboswitch classes have been characterized that vary widely in size and secondary structure. Extensive experiments and theoretical studies have made great strides in understanding the general structures, genetic mechanisms, and regulatory activities of individual riboswitches. As the ligand-dependent co-transcriptional folding and unfolding dynamics of riboswitches are the key determinant of gene expression, it is important to investigate the thermodynamics and kinetics of riboswitches both in the presence and absence of metabolites under the transcription. This review will provide a brief summary of the studies about the regulation mechanisms of the pbuE, SMK, yitJ, and metF riboswitches based on the ligand-dependent co-transcriptional folding of the riboswitches.
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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.
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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
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From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.
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.
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Improving decoy databases for protein folding algorithms
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.
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Maitip, Jakkrawut; Trueman, Holly E; Kaehler, Benjamin D; Huttley, Gavin A; Chantawannakul, Panuwan; Sutherland, Tara D
2015-04-01
Multiple gene duplication events in the precursor of the Aculeata (bees, ants, hornets) gave rise to four silk genes. Whilst these homologs encode proteins with similar amino acid composition and coiled coil structure, the retention of all four homologs implies they each are important. In this study we identified, produced and characterized the four silk proteins from Apis dorsata, the giant Asian honeybee. The proteins were readily purified, allowing us to investigate the folding behavior of solutions of individual proteins in comparison to mixtures of all four proteins at concentrations where they assemble into their native coiled coil structure. In contrast to solutions of any one protein type, solutions of a mixture of the four proteins formed coiled coils that were stable against dilution and detergent denaturation. The results are consistent with the formation of a heteromeric coiled coil protein complex. The mechanism of silk protein coiled coil formation and evolution is discussed in light of these results. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Impaired folding and subunit assembly as disease mechanism
DEFF Research Database (Denmark)
Bross, P; Andresen, B S; Gregersen, N
1998-01-01
folding is a common effect of missense mutations occurring in genetic diseases, (ii) increasing the level of available chaperones may augment the level of functional mutant protein in vivo, and (iii) one mutation may have multiple effects. The interplay between the chaperones assisting folding......Rapid progress in DNA technology has entailed the possibility of readily detecting mutations in disease genes. In contrast to this, techniques to characterize the effects of mutations are still very time consuming. It has turned out that many of the mutations detected in disease genes are missense...... mutations. Characterization of the effect of these mutations is particularly important in order to establish that they are disease causing and to estimate their severity. We use the experiences with investigation of medium-chain acyl-CoA dehydrogenase deficiency as an example to illustrate that (i) impaired...
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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.
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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.)
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Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding.
Jacob, Maik H; D'Souza, Roy N; Schwarzlose, Thomas; Wang, Xiaojuan; Huang, Fang; Haas, Elisha; Nau, Werner M
2018-04-26
Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.
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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.
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Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.
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.
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Long range correlations and folding angle with applications to α-helical proteins
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.
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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.
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Towse, Clare-Louise; Akke, Mikael; Daggett, Valerie
2017-04-27
Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing data set of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identifies deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, α-helices have lower entropy on average than do β-sheets, and both are lower than coil regions.
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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.
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International Nuclear Information System (INIS)
Weiss, M.A.; Shoelson, S.E.; Nguyen, D.T.; O'Shea, E.; Karplus, M.; Khait, I.; Neuringer, L.J.; Inouye, K.; Frank, B.H.; Beckage, M.
1989-01-01
The aromatic 1 H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25 → Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constrains in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures
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Directory of Open Access Journals (Sweden)
Flower Ann M
2002-11-01
Full Text Available Abstract Background The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. Results In the current work, we tested this hypothesis using a mutant form of λ repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant λ repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase. Conclusions These results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.
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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.
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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...
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An essential nonredundant role for mycobacterial DnaK in native protein folding.
Directory of Open Access Journals (Sweden)
Allison Fay
2014-07-01
Full Text Available Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.
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Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M
2016-01-01
The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.
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The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain.
Directory of Open Access Journals (Sweden)
Søren W. Pedersen
Full Text Available Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.
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DEFF Research Database (Denmark)
Lyngsø, C.; Kjaerulff, S.; Muller, S.
2010-01-01
in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality...
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Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.
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.
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Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin
2012-01-01
Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740
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TMFoldWeb: a web server for predicting transmembrane protein fold class.
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.
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Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.
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.
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FRAN and RBF-PSO as two components of a hyper framework to recognize protein folds.
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.
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Unique Features of Halophilic Proteins.
Arakawa, Tsutomu; Yamaguchi, Rui; Tokunaga, Hiroko; Tokunaga, Masao
2017-01-01
Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.
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Synchrotron radiation circular dichroism spectroscopy study of recombinant T β4 folding
Huang, Yung-Chin; Chu, Hsueh-Liang; Chen, Peng-Jen; Chang, Chia-Ching
Thymosin beta 4 (T β4) is a 43-amino acid small peptide, has been demonstrated that it can promote cardiac repair, wound repair, tissue protection, and involve in the proliferation of blood cell precursor stem cells of bone marrow. Moreover, T β4 has been identified as a multifunction intrinsically disordered protein, which is lacking the stable tertiary structure. Owing to the small size and disordered character, the T β4 protein degrades rapidly and the storage condition is critical. Therefore, it is not easy to reveal its folding mechanism of native T β4. However, recombinant T β4 protein (rT β4), which fused with a 5-kDa peptide in its amino-terminal, is stable and possesses identical function of T β4. Therefore, rT β4 can be used to study its folding mechanism. By using over-critical folding process, stable folding intermediates of rT β4 can be obtained. Structure analysis of folding intermediates by synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies indicate that rT β4 is a random coli major protein and its hydrophobic region becomes compact gradually. Moreover, the rT β4 folding is a two state transition. Thermal denaturation analysis indicates that rT β4 lacks stable tertiary structure. These results indicated that rT β4, similar to T β4, is an intrinsically disordered protein. Research is supported by MOST, Taiwan. MOST 103-2112-M-009-011-MY3. Corresponding author: Chia-Ching Chang; ccchang01@faculty.nctu.edu.tw.
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Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.
1999-01-01
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
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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.
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Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin
2015-03-01
This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.
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Neuromuscular compensation mechanisms in vocal fold paralysis and paresis.
Dewan, Karuna; Vahabzadeh-Hagh, Andrew; Soofer, Donna; Chhetri, Dinesh K
2017-07-01
Vocal fold paresis and paralysis are common conditions. Treatment options include augmentation laryngoplasty and voice therapy. The optimal management for this condition is unclear. The objective of this study was to assess possible neuromuscular compensation mechanisms that could potentially be used in the treatment of vocal fold paresis and paralysis. In vivo canine model. In an in vivo canine model, we examined three conditions: 1) unilateral right recurrent laryngeal nerve (RLN) paresis and paralysis, 2) unilateral superior laryngeal nerve (SLN) paralysis, and 3) unilateral vagal nerve paresis and paralysis. Phonatory acoustics and aerodynamics were measured in each of these conditions. Effective compensation was defined as improved acoustic and aerodynamic profile. The most effective compensation for all conditions was increasing RLN activation and decreasing glottal gap. Increasing RLN activation increased the percentage of possible phonatory conditions that achieved phonation onset. SLN activation generally led to decreased number of total phonation onset conditions within each category. Differential effects of SLN (cricothyroid [CT] muscle) activation were seen. Ipsilateral SLN activation could compensate for RLN paralysis; normal CT compensated well in unilateral SLN paralysis; and in vagal paresis/paralysis, contralateral SLN and RLN displayed antagonistic relationships. Methods to improve glottal closure should be the primary treatment for large glottal gaps. Neuromuscular compensation is possible for paresis. This study provides insights into possible compensatory mechanisms in vocal fold paresis and paralysis. NA Laryngoscope, 127:1633-1638, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.
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The Complexity of Folding Self-Folding Origami
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.
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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
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International Nuclear Information System (INIS)
Giesen, Alexander W.; Homans, Steve W.; Brown, Jonathan Miles
2003-01-01
We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N i -H N i , N i -C' i-1 , H N i - C' i-1 ) in two tensor frames and only backbone H N -H N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 A with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms
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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.
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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.
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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.
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Frehner, Marcel; Reif, Daniel; Grasemann, Bernhard
2012-06-01
This paper compares kinematical and mechanical techniques for the palinspastic reconstruction of folded cross sections in collision orogens. The studied area and the reconstructed NE-SW trending, 55.5 km long cross section is located in the High Folded Zone of the Zagros fold-and-thrust belt in the Kurdistan region of Iraq. The present-day geometry of the cross section has been constructed from field as well as remote sensing data. In a first step, the structures and the stratigraphy are simplified and summarized in eight units trying to identify the main geometric and mechanical parameters. In a second step, the shortening is kinematically estimated using the dip domain method to 11%-15%. Then the same cross section is used in a numerical finite element model to perform dynamical unfolding simulations taking various rheological parameters into account. The main factor allowing for an efficient dynamic unfolding is the presence of interfacial slip conditions between the mechanically strong units. Other factors, such as Newtonian versus power law viscous rheology or the presence of a basement, affect the numerical simulations much less strongly. If interfacial slip is accounted for, fold amplitudes are reduced efficiently during the dynamical unfolding simulations, while welded layer interfaces lead to unrealistic shortening estimates. It is suggested that interfacial slip and decoupling of the deformation along detachment horizons is an important mechanical parameter that controlled the folding processes in the Zagros High Folded Zone.
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Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy
2012-01-01
Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950
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MFIB: a repository of protein complexes with mutual folding induced by binding.
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.
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The mechanics of fault-bend folding and tear-fault systems in the Niger Delta
Benesh, Nathan Philip
This dissertation investigates the mechanics of fault-bend folding using the discrete element method (DEM) and explores the nature of tear-fault systems in the deep-water Niger Delta fold-and-thrust belt. In Chapter 1, we employ the DEM to investigate the development of growth structures in anticlinal fault-bend folds. This work was inspired by observations that growth strata in active folds show a pronounced upward decrease in bed dip, in contrast to traditional kinematic fault-bend fold models. Our analysis shows that the modeled folds grow largely by parallel folding as specified by the kinematic theory; however, the process of folding over a broad axial surface zone yields a component of fold growth by limb rotation that is consistent with the patterns observed in natural folds. This result has important implications for how growth structures can he used to constrain slip and paleo-earthquake ages on active blind-thrust faults. In Chapter 2, we expand our DEM study to investigate the development of a wider range of fault-bend folds. We examine the influence of mechanical stratigraphy and quantitatively compare our models with the relationships between fold and fault shape prescribed by the kinematic theory. While the synclinal fault-bend models closely match the kinematic theory, the modeled anticlinal fault-bend folds show robust behavior that is distinct from the kinematic theory. Specifically, we observe that modeled structures maintain a linear relationship between fold shape (gamma) and fault-horizon cutoff angle (theta), rather than expressing the non-linear relationship with two distinct modes of anticlinal folding that is prescribed by the kinematic theory. These observations lead to a revised quantitative relationship for fault-bend folds that can serve as a useful interpretation tool. Finally, in Chapter 3, we examine the 3D relationships of tear- and thrust-fault systems in the western, deep-water Niger Delta. Using 3D seismic reflection data and new
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Kinematics, structural mechanics, and design of origami structures with smooth folds
Peraza Hernandez, Edwin Alexander
model for the structural mechanics of origami continuum bodies with smooth folds is presented. Such a model entails the integration of the presented kinematic model and existing plate theories in order to obtain a structural representation for folds having non-zero thickness and comprised of arbitrary materials. The model is validated against finite element analysis. The last contribution addresses the design and analysis of active material-based self-folding structures that morph via simultaneous folding towards a given three-dimensional goal shape starting from a planar configuration. Implementation examples including shape memory alloy (SMA)-based self-folding structures are provided.
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Self-folding mechanics of graphene tearing and peeling from a substrate
He, Ze-Zhou; Zhu, Yin-Bo; Wu, Heng-An
2018-06-01
Understanding the underlying mechanism in the tearing and peeling processes of graphene is crucial for the further hierarchical design of origami-like folding and kirigami-like cutting of graphene. However, the complex effects among bending moduli, adhesion, interlayer interaction, and local crystal structure during origami-like folding and kirigami-like cutting remain unclear, resulting in challenges to the practical applications of existing theoretical and experimental findings as well as to potential manipulations of graphene in metamaterials and nanodevices. Toward this end, classical molecular dynamics (MD) simulations are performed with synergetic theoretical analysis to explore the tearing and peeling of self-folded graphene from a substrate driven by external force and by thermal activation. It is found that the elastic energy localized at the small folding ridge plays a significant role in the crack trajectory. Due to the extremely small bending modulus of monolayer graphene, its taper angle when pulled by an external force follows a scaling law distinct from that in case of bilayer graphene. With the increase in the initial width of the folding ridge, the self-folded graphene, motivated by thermal fluctuations, can be self-assembled by spontaneous self-tearing and peeling from a substrate. Simultaneously, the scaling law between the taper angle and adhesive energy is independent of the motivations for thermal activation-induced self-assembly and external force tearing, providing effective insights into the underlying physics for graphene-based origami-like folding and kirigami-like cutting.
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The Role of Short-Chain Conjugated Poly-(R-3-Hydroxybutyrate (cPHB in Protein Folding
Directory of Open Access Journals (Sweden)
Rosetta N. Reusch
2013-05-01
Full Text Available Poly-(R-3-hydroxybutyrate (PHB, a linear polymer of R-3-hydroxybutyrate (R-3HB, is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues, which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues which is conjugated to proteins (cPHB, primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA of Escherichia coli.
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Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A
2018-04-27
Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.
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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.
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NoFold: RNA structure clustering without folding or alignment.
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.
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From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.
Farinha, Carlos M; Canato, Sara
2017-01-01
CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.
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Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type
Directory of Open Access Journals (Sweden)
Fujiwara Kazuo
2012-08-01
Full Text Available Abstract Background A large number of studies have been carried out to obtain amino acid propensities for α-helices and β-sheets. The obtained propensities for α-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the β-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects β-sheet propensity. Results We calculated amino acid propensities for α-helices and β-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that α-helix propensities do not differ significantly by fold, but β-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for α-helix, but such is not the case for the β-sheet propensities. We also found some fold dependence on amino acid frequency in β-strands. Folds with a high Ser, Thr and Asn content at exposed sites in β-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = −0.90 and to have flat β-sheets. At buried sites in β-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = −0.93. "All-β" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas "α/β" proteins tend to have a higher content of Val, Ile and Leu. Conclusions The α-helix propensities are similar for all folds and for exposed and buried residues. However, β-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in β-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding
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Jethva, Prashant N; Udgaonkar, Jayant B
2017-09-07
Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.
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Multiple routes and milestones in the folding of HIV-1 protease monomer.
Directory of Open Access Journals (Sweden)
Massimiliano Bonomi
Full Text Available Proteins fold on a time scale incompatible with a mechanism of random search in conformational space thus indicating that somehow they are guided to the native state through a funneled energetic landscape. At the same time the heterogeneous kinetics suggests the existence of several different folding routes. Here we propose a scenario for the folding mechanism of the monomer of HIV-1 protease in which multiple pathways and milestone events coexist. A variety of computational approaches supports this picture. These include very long all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of clusters found in multiple high-temperature unfolding simulations and a complete characterization of free-energy surfaces carried out using a structure-based potential at atomistic resolution and a combination of metadynamics and parallel tempering. Our results confirm that the monomer in solution is stable toward unfolding and show that at least two unfolding pathways exist. In our scenario, the formation of a hydrophobic core is a milestone in the folding process which must occur along all the routes that lead this protein towards its native state. Furthermore, the ensemble of folding pathways proposed here substantiates a rational drug design strategy based on inhibiting the folding of HIV-1 protease.
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Balakrishnan, Swati; Sarma, Siddhartha P
2017-08-22
Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b 5 . Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b 5 . The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a C m higher than that of apo-cytochrome b 5 and are more stable to chaotropic agents than apo-cytochrome b 5 . The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.
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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.
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Mechanics of the scrolling and folding of graphene
Li, Hao; Li, Ming; Kang, Zhan
2018-06-01
The competition between the out-of-plane rigidity and the van der Waals interaction leads to the scrolled and folded structural configurations of graphene. These configuration changes, as compared with the initially planar geometry, significantly affect the electronic, optical and mechanical properties of graphene, promising exciting applications in graphene-nanoelectronics. We propose a finite-deformation theoretical model, in which no presumed assumptions on the geometries of deformed configurations are required. Both the predicted deformed profiles and the critical conditions show great agreements with molecular dynamics simulations results when compared with existing studies with simple geometrical assumptions. Moreover, MD simulations are performed to explore the morphology transitions between different configurations. It is observed that the folded configuration is energetically favorable for a short graphene sheet, while a long graphene sheet tends to scroll. Of particular interest, we observe the morphology transition from a Fermat scroll to the Archimedean scroll for the bi-scrolled graphene. These findings are useful for understanding the stability of graphene and may provide guidance to the design of programmable graphene-nanoelectronics.
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Mechanics of the scrolling and folding of graphene.
Li, Hao; Li, Ming; Kang, Zhan
2018-06-15
The competition between the out-of-plane rigidity and the van der Waals interaction leads to the scrolled and folded structural configurations of graphene. These configuration changes, as compared with the initially planar geometry, significantly affect the electronic, optical and mechanical properties of graphene, promising exciting applications in graphene-nanoelectronics. We propose a finite-deformation theoretical model, in which no presumed assumptions on the geometries of deformed configurations are required. Both the predicted deformed profiles and the critical conditions show great agreements with molecular dynamics simulations results when compared with existing studies with simple geometrical assumptions. Moreover, MD simulations are performed to explore the morphology transitions between different configurations. It is observed that the folded configuration is energetically favorable for a short graphene sheet, while a long graphene sheet tends to scroll. Of particular interest, we observe the morphology transition from a Fermat scroll to the Archimedean scroll for the bi-scrolled graphene. These findings are useful for understanding the stability of graphene and may provide guidance to the design of programmable graphene-nanoelectronics.
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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/
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RNA folding: structure prediction, folding kinetics and ion electrostatics.
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.
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Tavenor, Nathan Albert
Protein-based supramolecular polymers (SMPs) are a class of biomaterials which draw inspiration from and expand upon the many examples of complex protein quaternary structures observed in nature: collagen, microtubules, viral capsids, etc. Designing synthetic supramolecular protein scaffolds both increases our understanding of natural superstructures and allows for the creation of novel materials. Similar to small-molecule SMPs, protein-based SMPs form due to self-assembly driven by intermolecular interactions between monomers, and monomer structure determines the properties of the overall material. Using protein-based monomers takes advantage of the self-assembly and highly specific molecular recognition properties encodable in polypeptide sequences to rationally design SMP architectures. The central hypothesis underlying our work is that alpha-helical coiled coils, a well-studied protein quaternary folding motif, are well-suited to SMP design through the addition of synthetic linkers at solvent-exposed sites. Through small changes in the structures of the cross-links and/or peptide sequence, we have been able to control both the nanoscale organization and the macroscopic properties of the SMPs. Changes to the linker and hydrophobic core of the peptide can be used to control polymer rigidity, stability, and dimensionality. The gaps in knowledge that this thesis sought to fill on this project were 1) the relationship between the molecular structure of the cross-linked polypeptides and the macroscopic properties of the SMPs and 2) a means of creating materials exhibiting multi-dimensional net or framework topologies. Separate from the above efforts on supramolecular architectures was work on improving backbone modification strategies for an alpha-helix in the context of a complex protein tertiary fold. Earlier work in our lab had successfully incorporated unnatural building blocks into every major secondary structure (beta-sheet, alpha-helix, loops and beta
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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.
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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
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International Nuclear Information System (INIS)
Roder, H.; Wuethrich, K.
1986-01-01
A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1 H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1 H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes
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Method of generating ploynucleotides encoding enhanced folding variants
Energy Technology Data Exchange (ETDEWEB)
Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.
2017-05-02
The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.
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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
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Coarsely resolved topography along protein folding pathways
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.
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DEFF Research Database (Denmark)
Kragelund, Birthe Brandt; Osmark, Peter; Neergaard, Thomas B.
1999-01-01
The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24...... probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding...... step and that a sequential framework model can describe the protein folding reaction....
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The role of atomic level steric effects and attractive forces in protein folding.
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.
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BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints
Fischer, Axel W.; Alexander, Nathan S.; Woetzel, Nils; Karakaş, Mert; Weiner, Brian E.; Meiler, Jens
2016-01-01
For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The protein-size-normalized root-mean-square-deviation (RMSD100) value of the most accurate model is better than 8 Å for twenty-seven, better than 6 Å for twenty-two, and better than 4 Å for fifteen out of twenty-nine proteins, demonstrating the algorithm’s ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data. PMID:25820805
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Single-molecule folding mechanism of an EF-hand neuronal calcium sensor
DEFF Research Database (Denmark)
Heiðarsson, Pétur Orri; Otazo, Mariela R.; Bellucci, Luca
2013-01-01
EF-hand calcium sensors respond structurally to changes in intracellular Ca2+ concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors...... of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy...
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Combinatorial pattern discovery approach for the folding trajectory analysis of a beta-hairpin.
Directory of Open Access Journals (Sweden)
Laxmi Parida
2005-06-01
Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity c in RO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the
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Combinatorial Pattern Discovery Approach for the Folding Trajectory Analysis of a beta-Hairpin.
Directory of Open Access Journals (Sweden)
2005-06-01
Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity cinRO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the
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Vocal fold ion transport and mucin expression following acrolein exposure.
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.
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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.
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The ModFOLD4 server for the quality assessment of 3D protein models
McGuffin, Liam J.; Buenavista, Maria T.; Roche, Daniel B.
2013-01-01
Once you have generated a 3D model of a protein,\\ud how do you know whether it bears any resemblance\\ud to the actual structure? To determine the usefulness\\ud of 3D models of proteins, they must be assessed in\\ud terms of their quality by methods that predict their\\ud similarity to the native structure. The ModFOLD4\\ud server is the latest version of our leading independent\\ud server for the estimation of both the global and\\ud local (per-residue) quality of 3D protein models. The\\ud server ...
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Gianni, Stefano; Jemth, Per
2014-07-01
The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.
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Polymer collapse, protein folding, and the percolation threshold.
Meirovitch, Hagai
2002-01-15
We study the transition of polymers in the dilute regime from a swollen shape at high temperatures to their low-temperature structures. The polymers are modeled by a single self-avoiding walk (SAW) on a lattice for which l of the monomers (the H monomers) are self-attracting, i.e., if two nonbonded H monomers become nearest neighbors on the lattice they gain energy of interaction (epsilon = -/epsilon/); the second type of monomers, denoted P, are neutral. This HP model was suggested by Lau and Dill (Macromolecules 1989, 22, 3986-3997) to study protein folding, where H and P are the hydrophobic and polar amino acid residues, respectively. The model is simulated on the square and simple cubic (SC) lattices using the scanning method. We show that the ground state and the sharpness of the transition depend on the lattice, the fraction g of the H monomers, as well as on their arrangement along the chain. In particular, if the H monomers are distributed at random and g is larger than the site percolation threshold of the lattice, a collapsed transition is very likely to occur. This conclusion, drawn for the lattice models, is also applicable to proteins where an effective lattice with coordination number between that of the SC lattice and the body centered cubic lattice is defined. Thus, the average fraction of hydrophobic amino acid residues in globular proteins is found to be close to the percolation threshold of the effective lattice.
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Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P
2012-03-01
Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. Copyright © 2011 Elsevier B.V. All rights reserved.
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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.
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ModFOLD6: an accurate web server for the global and local quality estimation of 3D protein models.
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.
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Folding and Function of Proteorhodopsins in Photoenergy Transducing Membranes
Energy Technology Data Exchange (ETDEWEB)
Spudich, John L. [University of Texas Medical School, Houston, TX (United States). Health Science Center, Dept. of Biochemistry and Molecular Biology
2012-08-10
The overall research objectives are to develop proteorhodopsin (PR) proteins as a model system for α-helical membrane protein insertion and folding, and to advance understanding of the diversity and mechanisms of PRs, a large family of photoenergy transducers (~4000 identified) abundant in the world’s oceans. Specific aims are: (1) To develop a high-efficiency genetic selection procedure for light-driven proton-pumping in E. coli cells. Such a procedure would provide a positive selection method for proper folding and function of PRs in the E. coli membrane. (2) Characterize flash-induced absorption changes and photocurrents in PR variants in organisms from various environments, and their expression level and function when expressed in E. coli. Subaims are to: (a) elucidate the relationship of the transport mechanism to mechanisms of other microbial rhodopsins, some of which like PRs function as ion transporters and some of which use light energy to activate signaling pathways (sensory rhodopsins); and (b) identify important residues and chemical events in light-driven proton transport by PRs. In addition to their importance to the energy of the biosphere PRs have attracted interest for their potential for use in making photoenergy-transducing membranes for bioengineering applications.
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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.
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Avital-Shmilovici, Michal; Whittaker, Jonathan; Weiss, Michael A; Kent, Stephen B H
2014-08-22
Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly(B8) is replaced by Ser(B8). Modular total chemical synthesis was used to prepare the wild-type [Gly(B8)]proinsulin molecule and three analogs: [D-Ala(B8)]proinsulin, [L-Ala(B8)]proinsulin, and the clinical mutant [L-Ser(B8)]proinsulin. The protein diastereomer [D-Ala(B8)]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [L-Ser(B8)]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [L-Ser(B8)]proinsulin did not fold well under the physiological conditions investigated, once folded the [L-Ser(B8)]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad
2017-09-01
The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.
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Folding and membrane insertion of the pore-forming peptide gramicidin occur as a concerted process.
Hicks, Matthew R; Damianoglou, Angeliki; Rodger, Alison; Dafforn, Timothy R
2008-11-07
Many antibiotic peptides function by binding and inserting into membranes. Understanding this process provides an insight into the fundamentals of both membrane protein folding and antibiotic peptide function. For the first time, in this work, flow-aligned linear dichroism (LD) is used to study the folding of the antibiotic peptide gramicidin. LD provides insight into the combined processes of peptide folding and insertion and has the advantage over other similar techniques of being insensitive to off-membrane aggregation events. By combining LD data with conventional measurements of protein fluorescence and circular dichroism, the mechanism of gramicidin insertion is elucidated. The mechanism consists of five separately assignable steps that include formation of a water-insoluble gramicidin aggregate, dissociation from the aggregate, partitioning of peptide to the membrane surface, oligomerisation on the surface and concerted insertion and folding of the peptide to the double-helical form of gramicidin. Measurement of the rates of each step shows that although changes in the fluorescence signal cease 10 s after the initiation of the process, the insertion of the peptide into the membrane is actually not complete for a further 60 min. This last membrane insertion phase is only apparent by measurement of LD and circular dichroism signal changes. In summary, this study demonstrates the importance of multi-technique approaches, including LD, in studies of membrane protein folding.
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Current Understanding and Future Directions for Vocal Fold Mechanobiology
Li, Nicole Y.K.; Heris, Hossein K.; Mongeau, Luc
2013-01-01
The vocal folds, which are located in the larynx, are the main organ of voice production for human communication. The vocal folds are under continuous biomechanical stress similar to other mechanically active organs, such as the heart, lungs, tendons and muscles. During speech and singing, the vocal folds oscillate at frequencies ranging from 20 Hz to 3 kHz with amplitudes of a few millimeters. The biomechanical stress associated with accumulated phonation is believed to alter vocal fold cell activity and tissue structure in many ways. Excessive phonatory stress can damage tissue structure and induce a cell-mediated inflammatory response, resulting in a pathological vocal fold lesion. On the other hand, phonatory stress is one major factor in the maturation of the vocal folds into a specialized tri-layer structure. One specific form of vocal fold oscillation, which involves low impact and large amplitude excursion, is prescribed therapeutically for patients with mild vocal fold injuries. Although biomechanical forces affect vocal fold physiology and pathology, there is little understanding of how mechanical forces regulate these processes at the cellular and molecular level. Research into vocal fold mechanobiology has burgeoned over the past several years. Vocal fold bioreactors are being developed in several laboratories to provide a biomimic environment that allows the systematic manipulation of physical and biological factors on the cells of interest in vitro. Computer models have been used to simulate the integrated response of cells and proteins as a function of phonation stress. The purpose of this paper is to review current research on the mechanobiology of the vocal folds as it relates to growth, pathogenesis and treatment as well as to propose specific research directions that will advance our understanding of this subject. PMID:24812638
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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/.
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Cross-tropopause Transport In Tropopause Folds: Mechanisms and Sensitivity To Model Resolution
Gray, S. L.
The rate and processes of transfer of mass and chemical species between the strato- sphere and troposphere (stratosphere-troposphere exchange) are currently uncertain. In the midlatitudes exchange appears to be dominated by processes associated with tropopause folds and cut-off lows. The development of a tropopause fold is a reversible process and thus irreversible processes must occur for the permanent transfer of ma- terial across the tropopause boundary. Proposed processes include turbulent mixing, quasi-isentropic mixing, convectively breaking gravity waves, deep convection and radiative heating. Numerical models run at typical climate or regional-scale resolutions are unable to re- solve the fine-scale features observed in tropopause folds. It is hypothesised that both the rate of exchange and its partitioning into different processes, as derived from nu- merical model simulations, are sensitive to model resolution. This hypothesis is tested through simulations of a tropopause folding event associated with a vigorous surface cold front which tracked across the British Isles. Climate to high-mesoscale resolution simulations incorporating passive tracers are performed using the mesoscale version of the Met Office Unified Model. The mechanism by which the parametrized convec- tion leads to exchange is the subject of further examination.
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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.
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Electrostatic mechanism of nucleosomal array folding revealed by computer simulation.
Sun, Jian; Zhang, Qing; Schlick, Tamar
2005-06-07
Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended "beads-on-a-string" conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA-nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt.
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Molecular mechanics work station for protein conformational studies
International Nuclear Information System (INIS)
Fine, R.; Levinthal, C.; Schoenborn, B.; Dimmier, G.; Rankowitz, C.
1984-01-01
Interest in computational problems in Biology has intensified over the last few years, partly due to the development of techniques for the rapid cloning, sequencing, and mutagenesis of genes from organisims ranging from E. coli to Man. The central dogma of molecular biology; that DNA codes for mRNA which codes for protein, has been understood in a linear programming sense since the genetic code was cracked. But what is not understood at present is how a protein, once assembled as a long sequence of amino acids, folds back on itself to produce a three-dimensional structure which is unique to that protein and which dictates its chemical and biological activity. This folding process is purely physics, and involves the time evolution of a system of several thousand atoms which interact with each other and with atoms from the surrounding solvent. Molecular dynamics simulations on smaller molecules suggest that approaches which treat the protein as a classical ensemble of atoms interacting with each other via an empirical Hamiltonian can yield the kind of predictive results one would like when applied to proteins
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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.
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Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan
2014-10-14
The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.
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Natural triple beta-stranded fibrous folds.
Mitraki, Anna; Papanikolopoulou, Katerina; Van Raaij, Mark J
2006-01-01
A distinctive family of beta-structured folds has recently been described for fibrous proteins from viruses. Virus fibers are usually involved in specific host-cell recognition. They are asymmetric homotrimeric proteins consisting of an N-terminal virus-binding tail, a central shaft or stalk domain, and a C-terminal globular receptor-binding domain. Often they are entirely or nearly entirely composed of beta-structure. Apart from their biological relevance and possible gene therapy applications, their shape, stability, and rigidity suggest they may be useful as blueprints for biomechanical design. Folding and unfolding studies suggest their globular C-terminal domain may fold first, followed by a "zipping-up" of the shaft domains. The C-terminal domains appear to be important for registration because peptides corresponding to shaft domains alone aggregate into nonnative fibers and/or amyloid structures. C-terminal domains can be exchanged between different fibers and the resulting chimeric proteins are useful as a way to solve structures of unknown parts of the shaft domains. The following natural triple beta-stranded fibrous folds have been discovered by X-ray crystallography: the triple beta-spiral, triple beta-helix, and T4 short tail fiber fold. All have a central longitudinal hydrophobic core and extensive intermonomer polar and nonpolar interactions. Now that a reasonable body of structural and folding knowledge has been assembled about these fibrous proteins, the next challenge and opportunity is to start using this information in medical and industrial applications such as gene therapy and nanotechnology.
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Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O
2012-12-15
Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured
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Competition between folding and glycosylation in the endoplasmic reticulum
DEFF Research Database (Denmark)
Holst, B; Bruun, A W; Kielland-Brandt, Morten
1996-01-01
Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...
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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.
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Vendruscolo, M; Najmanovich, R; Domany, E
2000-02-01
We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading.
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Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism
International Nuclear Information System (INIS)
Ahluwalia, Unnati; Deep, Shashank
2012-01-01
Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.
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Directory of Open Access Journals (Sweden)
Daniel Bölinger
2010-04-01
Full Text Available Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered alpha-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip.
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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
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WW Domain Folding Complexity Revealed by Infrared Spectroscopy
Davis, Caitlin M.; Dyer, R. Brian
2014-01-01
Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescen...
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Design of an Efficient Turbulent Micro-Mixer for Protein Folding Experiments
Inguva, Venkatesh; Perot, Blair
2015-11-01
Protein folding studies require the development of micro-mixers that require less sample, mix at faster rates, and still provide a high signal to noise ratio. Chaotic to marginally turbulent micro-mixers are promising candidates for this application. In this study, various turbulence and unsteadiness generation concepts are explored that avoid cavitation. The mixing enhancements include flow turning regions, flow splitters, and vortex shedding. The relative effectiveness of these different approaches for rapid micro-mixing is discussed. Simulations found that flow turning regions provided the best mixing profile. Experimental validation of the optimal design is verified through laser confocal microscopy experiments. This work is support by the National Science Foundation.
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Structural basis of the 9-fold symmetry of centrioles.
Kitagawa, Daiju; Vakonakis, Ioannis; Olieric, Natacha; Hilbert, Manuel; Keller, Debora; Olieric, Vincent; Bortfeld, Miriam; Erat, Michèle C; Flückiger, Isabelle; Gönczy, Pierre; Steinmetz, Michel O
2011-02-04
The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles. Copyright © 2011 Elsevier Inc. All rights reserved.
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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.
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Winstanley, Henry F.; Abeln, Sanne; Deane, Charlotte M.
Motivation: At present there exists no age estimate for the different protein structures found in nature. It has become clear from occurrence studies that different folds arose at different points in evolutionary time. An estimation of the age of different folds would be a starting point for many
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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...
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Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation
Fluitt, Aaron Michael
Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.
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Energy Technology Data Exchange (ETDEWEB)
Hansen, Alexandar L.; Bouvignies, Guillaume; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)
2013-03-15
A {sup 13}C{sup {alpha}} chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-{sup 13}C]-glucose is the sole carbon source is exploited, producing proteins with {sup 13}C at the C{sup {alpha}} position, while the majority of residues remain unlabeled at CO or C{sup {beta}}. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state {sup 13}C{sup {alpha}} chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.
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International Nuclear Information System (INIS)
Oshima, Hiraku; Kinoshita, Masahiro
2015-01-01
In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient
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Mechanism of Action of Secreted Newt Anterior Gradient Protein.
Directory of Open Access Journals (Sweden)
Kathrin S Grassme
Full Text Available Anterior gradient (AG proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.
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Mondal, Abhisek; Datta, Saumen
2017-06-01
Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge-based potential. Proteins 2017; 85:1046-1055. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function.
Directory of Open Access Journals (Sweden)
Xinxin Liu
Full Text Available Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3 expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001. Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (p<0.05 and a reduction in transepithelial electrical resistance (TEER by 180.0% (p<0.001. While the expression of tight junctional protein did not change in acrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (p<0.05. Taken together, these data provide evidence that acute acrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium.
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Positive therapy of andrographolide in vocal fold leukoplakia.
Xu, Jue; Xue, Tao; Bao, Ying; Wang, Dong-Hai; Ma, Bing-Liang; Yin, Chen-Yi; Yang, Guang-Hui; Ren, Gang; Lan, Long-Jiang; Wang, Jian-Qiu; Zhang, Xiao-Lan; Zhao, Yu-Qin
2014-01-01
Vocal fold leukoplakia is a premalignant precursor of squamous cell carcinoma. Although many efforts have been contributed to therapy of this disease, none exhibits a satisfactory result. The aims of this study were to investigate the effectiveness and feasibility of andrographolide therapy in vocal fold leukoplakia and to explore the preliminary mechanism underlying. Forty-one eligible patients were enrolled in the study. The patients were treated for 10-minute exposures of 5 ml (25mg/ml) andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Electronic laryngoscope was used to observe the condition of vocal fold leukoplakia during the treatment. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months. Toxic reactions of treatments were evaluated on the basis of the standards of the United States MD Anderson Cancer Center. Moreover, laryngeal carcinoma cell line Hep2 was applied to explore the mechanism of effect of andrographolide. Anti-proliferative effect on Hep2, cell nuclear morphology, express of mitogen-activated protein kinases (MAPK) and pro-apoptotic protein were detected after andrographolide treatment. We found that andrographolide exhibited significant curative effects on treatments, which were accompanied by thinning of the lesion of leukoplakia, reduction in the whitish surface area, and return of pink or red epithelium. A complete response up to 85% was observed, and no toxic side effect events occurred during the study. No patient with a complete response had a recurrence in the follow-up. Moreover, cellular experiments in Hep2 indicated that andrographolide activated MAPK pathway and caspase cascade, and finally induced apoptosis in laryngeal carcinoma cell. The advantages of andrographolide are connected with minimally invasive and localized character of the treatment and no damage of collagenous tissue structures, which are more convenient and less painful
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Surfing the free energy landscape of flavodoxin folding
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
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Energy Technology Data Exchange (ETDEWEB)
Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)
2010-09-21
Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.
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Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A
2011-04-28
Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded
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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
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New variants of known folds: do they bring new biology?
International Nuclear Information System (INIS)
Koonin, Eugene V.
2010-01-01
New distinct versions of known protein folds provide a powerful means of protein-function prediction that complements sequence and genomic context analysis. New distinct versions of known protein folds provide a powerful means of protein-function prediction that complements sequence and genomic context analysis. These structures do not supplant direct biochemical experiments, but are indispensable for the complete characterization of proteins
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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.
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Complete fold annotation of the human proteome using a novel structural feature space.
Middleton, Sarah A; Illuminati, Joseph; Kim, Junhyong
2017-04-13
Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.
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Multiple scales and phases in discrete chains with application to folded proteins
Sinelnikova, A.; Niemi, A. J.; Nilsson, Johan; Ulybyshev, M.
2018-05-01
Chiral heteropolymers such as large globular proteins can simultaneously support multiple length scales. The interplay between the different scales brings about conformational diversity, determines the phase properties of the polymer chain, and governs the structure of the energy landscape. Most importantly, multiple scales produce complex dynamics that enable proteins to sustain live matter. However, at the moment there is incomplete understanding of how to identify and distinguish the various scales that determine the structure and dynamics of a complex protein. Here we address this impending problem. We develop a methodology with the potential to systematically identify different length scales, in the general case of a linear polymer chain. For this we introduce and analyze the properties of an order parameter that can both reveal the presence of different length scales and can also probe the phase structure. We first develop our concepts in the case of chiral homopolymers. We introduce a variant of Kadanoff's block-spin transformation to coarse grain piecewise linear chains, such as the C α backbone of a protein. We derive analytically, and then verify numerically, a number of properties that the order parameter can display, in the case of a chiral polymer chain. In particular, we propose that in the case of a chiral heteropolymer the order parameter can reveal traits of several different phases, contingent on the length scale at which it is scrutinized. We confirm that this is the case with crystallographic protein structures in the Protein Data Bank. Thus our results suggest relations between the scales, the phases, and the complexity of folding pathways.
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Bonvin, A.M.J.J.; Houben, K.; Guenneugues, M.N.L.; Kaptein, R.; Boelens, R.
2001-01-01
The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein
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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
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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)
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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
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WW domain folding complexity revealed by infrared spectroscopy.
Davis, Caitlin M; Dyer, R Brian
2014-09-02
Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.
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Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A
2018-02-21
Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.
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Proteomic Data From Human Cell Cultures Refine Mechanisms of Chaperone-Mediated Protein homeostasis
Finka, Andrija; Goloubinoff, Andrija Finka and Pierre
2013-01-01
In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here “the chaperome,” which can prevent formation of potentially harmful misfolded protein conformers and use the...
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Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1
International Nuclear Information System (INIS)
Helander, Sara; Montecchio, Meri; Lemak, Alexander; Farès, Christophe; Almlöf, Jonas; Li, Yanjun; Yee, Adelinda; Arrowsmith, Cheryl H.; Dhe-Paganon, Sirano; Sunnerhagen, Maria
2014-01-01
Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25 1–73 , a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains
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Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1
Energy Technology Data Exchange (ETDEWEB)
Helander, Sara; Montecchio, Meri [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Lemak, Alexander [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Farès, Christophe [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Almlöf, Jonas [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Li, Yanjun [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Yee, Adelinda [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Arrowsmith, Cheryl H. [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Dhe-Paganon, Sirano [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Sunnerhagen, Maria, E-mail: maria.sunnerhagen@liu.se [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)
2014-04-25
Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.
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Zhou, Tao; Dang, Ying; Zheng, Yong-Hui
2014-03-01
The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins
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Molecular mechanisms for protein-encoded inheritance
Wiltzius, Jed J. W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David
2013-01-01
Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds. PMID:19684598
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Karmi, Ola; Marjault, Henri-Baptiste; Pesce, Luca; Carloni, Paolo; Onuchic, Jose' N; Jennings, Patricia A; Mittler, Ron; Nechushtai, Rachel
2018-02-12
NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT's backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins' [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs' [2Fe-2S] cluster and the implication of the latter to the NEET proteins' cellular and systemic function in health and disease.
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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.
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Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function.
Liu, Xinxin; Zheng, Wei; Sivasankar, M Preeti
2016-01-01
Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3) expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001). Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (pacrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (pacrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium.
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Energy Technology Data Exchange (ETDEWEB)
Knott, Michael [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Best, Robert B., E-mail: robertbe@helix.nih.gov [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520 (United States)
2014-05-07
Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.
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International Nuclear Information System (INIS)
Knott, Michael; Best, Robert B.
2014-01-01
Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD
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International Nuclear Information System (INIS)
Teschke, Carolyn M.; Parent, Kristin N.
2010-01-01
The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine. DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.
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Topologies to geometries in protein folding: Hierarchical and nonhierarchical scenarios
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.
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Zhou, Rui; Maisuradze, Gia G; Suñol, David; Todorovski, Toni; Macias, Maria J; Xiao, Yi; Scheraga, Harold A; Czaplewski, Cezary; Liwo, Adam
2014-12-23
To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecond-timescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single- and double-exponential kinetics and their correlations with two- and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.
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Energy Technology Data Exchange (ETDEWEB)
Kozlov, Guennadi [McGill University, Department of Biochemistry (Canada); Ekiel, Irena [National Research Council of Canada, Biomolecular NMR Group, Sector of Pharmaceutical Biotechnology, Biotechnology Research Institute (Canada); Beglova, Natalia [McGill University, Department of Biochemistry (Canada); Yee, Adelinda; Dharamsi, Akil; Engel, Asaph [University of Toronto, Department of Medical Biophysics (Canada); Siddiqui, Nadeem; Nong, Andrew; Gehring, Kalle [McGill University, Department of Biochemistry (Canada)
2000-07-15
The tertiary fold of the elongation factor, aEF-1{beta}, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1{beta} was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1{beta} structure revealed close similarity to its human analogue, eEF-1{beta}. In agreement with studies on EF-Ts and human EF-1{beta}, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1{alpha}. aEF-1{beta} was also found to bind calcium in the groove between helix {alpha}2 and strand {beta}4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.
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International Nuclear Information System (INIS)
Kozlov, Guennadi; Ekiel, Irena; Beglova, Natalia; Yee, Adelinda; Dharamsi, Akil; Engel, Asaph; Siddiqui, Nadeem; Nong, Andrew; Gehring, Kalle
2000-01-01
The tertiary fold of the elongation factor, aEF-1β, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1β was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1β structure revealed close similarity to its human analogue, eEF-1β. In agreement with studies on EF-Ts and human EF-1β, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1α. aEF-1β was also found to bind calcium in the groove between helix α2 and strand β4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation
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Comparing the Folding and Misfolding Energy Landscapes of Phosphoglycerate Kinase
Agocs, Gergely; Szabo, Bence T.; Koehler, Gottfried; Osvath, Szabolcs
2012-01-01
Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the prop...
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Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.
2015-01-01
The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823
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Factors that affect coseismic folds in an overburden layer
Zeng, Shaogang; Cai, Yongen
2018-03-01
Coseismic folds induced by blind thrust faults have been observed in many earthquake zones, and they have received widespread attention from geologists and geophysicists. Numerous studies have been conducted regarding fold kinematics; however, few have studied fold dynamics quantitatively. In this paper, we establish a conceptual model with a thrust fault zone and tectonic stress load to study the factors that affect coseismic folds and their formation mechanisms using the finite element method. The numerical results show that the fault dip angle is a key factor that controls folding. The greater the dip angle is, the steeper the fold slope. The second most important factor is the overburden thickness. The thicker the overburden is, the more gradual the fold. In this case, folds are difficult to identify in field surveys. Therefore, if a fold can be easily identified with the naked eye, the overburden is likely shallow. The least important factors are the mechanical parameters of the overburden. The larger the Young's modulus of the overburden is, the smaller the displacement of the fold and the fold slope. Strong horizontal compression and vertical extension in the overburden near the fault zone are the main mechanisms that form coseismic folds.
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Scheraga, H A; Paine, G H
1986-01-01
We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.
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Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.
Hu, Xiaotang; Li, Hongbin
2014-10-01
Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Directory of Open Access Journals (Sweden)
Tyo Keith EJ
2012-03-01
Full Text Available Abstract Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor or a larger protein (α-amylase. Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a degradation of protein/recycling amino acids, (b overall transcription/translation repression, and (c oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
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Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.
Directory of Open Access Journals (Sweden)
Florence Guillière
Full Text Available While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.
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Folding of non-Euclidean curved shells
Bende, Nakul; Evans, Arthur; Innes-Gold, Sarah; Marin, Luis; Cohen, Itai; Santangelo, Christian; Hayward, Ryan
2015-03-01
Origami-based folding of 2D sheets has been of recent interest for a variety of applications ranging from deployable structures to self-folding robots. Though folding of planar sheets follows well-established principles, folding of curved shells involves an added level of complexity due to the inherent influence of curvature on mechanics. In this study, we use principles from differential geometry and thin shell mechanics to establish fundamental rules that govern folding of prototypical creased shells. In particular, we show how the normal curvature of a crease line controls whether the deformation is smooth or discontinuous, and investigate the influence of shell thickness and boundary conditions. We show that snap-folding of shells provides a route to rapid actuation on time-scales dictated by the speed of sound. The simple geometric design principles developed can be applied at any length-scale, offering potential for bio-inspired soft actuators for tunable optics, microfluidics, and robotics. This work was funded by the National Science Foundation through EFRI ODISSEI-1240441 with additional support to S.I.-G. through the UMass MRSEC DMR-0820506 REU program.
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Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.
2014-01-01
Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080
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Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.
Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael
2017-04-15
Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain
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Analysis of protein folds using protein contact networks
Indian Academy of Sciences (India)
is a well-recognized classification system of proteins, which is based on manual in- ... can easily correspond to the information in the 2D matrix. ..... [7] U K Muppirala and Zhijun Li, Protein Engineering, Design & Selection 19, 265 (2006).
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Energy Technology Data Exchange (ETDEWEB)
K Kucera; L Harrison; M Cappello; Y Modis
2011-12-31
Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.
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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)
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Guiding the folding pathway of DNA origami.
Dunn, Katherine E; Dannenberg, Frits; Ouldridge, Thomas E; Kwiatkowska, Marta; Turberfield, Andrew J; Bath, Jonathan
2015-09-03
DNA origami is a robust assembly technique that folds a single-stranded DNA template into a target structure by annealing it with hundreds of short 'staple' strands. Its guiding design principle is that the target structure is the single most stable configuration. The folding transition is cooperative and, as in the case of proteins, is governed by information encoded in the polymer sequence. A typical origami folds primarily into the desired shape, but misfolded structures can kinetically trap the system and reduce the yield. Although adjusting assembly conditions or following empirical design rules can improve yield, well-folded origami often need to be separated from misfolded structures. The problem could in principle be avoided if assembly pathway and kinetics were fully understood and then rationally optimized. To this end, here we present a DNA origami system with the unusual property of being able to form a small set of distinguishable and well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape, thus allowing us to probe the assembly process. The obtained high yield of well-folded origami structures confirms the existence of efficient folding pathways, while the shape distribution provides information about individual trajectories through the folding landscape. We find that, similarly to protein folding, the assembly of DNA origami is highly cooperative; that reversible bond formation is important in recovering from transient misfoldings; and that the early formation of long-range connections can very effectively enforce particular folds. We use these insights to inform the design of the system so as to steer assembly towards desired structures. Expanding the rational design process to include the assembly pathway should thus enable more reproducible synthesis, particularly when targeting more complex structures. We anticipate that this expansion will be essential if DNA origami is to continue its
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Periodic folding of viscous sheets
Ribe, Neil M.
2003-09-01
The periodic folding of a sheet of viscous fluid falling upon a rigid surface is a common fluid mechanical instability that occurs in contexts ranging from food processing to geophysics. Asymptotic thin-layer equations for the combined stretching-bending deformation of a two-dimensional sheet are solved numerically to determine the folding frequency as a function of the sheet’s initial thickness, the pouring speed, the height of fall, and the fluid properties. As the buoyancy increases, the system bifurcates from “forced” folding driven kinematically by fluid extrusion to “free” folding in which viscous resistance to bending is balanced by buoyancy. The systematics of the numerically predicted folding frequency are in good agreement with laboratory experiments.
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Investigating homology between proteins using energetic profiles.
Wrabl, James O; Hilser, Vincent J
2010-03-26
Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may
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Investigating homology between proteins using energetic profiles.
Directory of Open Access Journals (Sweden)
James O Wrabl
2010-03-01
Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved
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History, rare, and multiple events of mechanical unfolding of repeat proteins
Sumbul, Fidan; Marchesi, Arin; Rico, Felix
2018-03-01
Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.
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An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.
Atkinson, Holly J; Babbitt, Patricia C
2009-10-01
The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.
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From genes to protein mechanics on a chip.
Otten, Marcus; Ott, Wolfgang; Jobst, Markus A; Milles, Lukas F; Verdorfer, Tobias; Pippig, Diana A; Nash, Michael A; Gaub, Hermann E
2014-11-01
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.
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Wierenga, P.A.; Egmond, M.R.; Voragen, A.G.J.; Jongh, H.H.J.de
2006-01-01
Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends
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Unraveling metamaterial properties in zigzag-base folded sheets.
Eidini, Maryam; Paulino, Glaucio H
2015-09-01
Creating complex spatial objects from a flat sheet of material using origami folding techniques has attracted attention in science and engineering. In the present work, we use the geometric properties of partially folded zigzag strips to better describe the kinematics of known zigzag/herringbone-base folded sheet metamaterials such as Miura-ori. Inspired by the kinematics of a one-degree of freedom zigzag strip, we introduce a class of cellular folded mechanical metamaterials comprising different scales of zigzag strips. This class of patterns combines origami folding techniques with kirigami. Using analytical and numerical models, we study the key mechanical properties of the folded materials. We show that our class of patterns, by expanding on the design space of Miura-ori, is appropriate for a wide range of applications from mechanical metamaterials to deployable structures at small and large scales. We further show that, depending on the geometry, these materials exhibit either negative or positive in-plane Poisson's ratios. By introducing a class of zigzag-base materials in the current study, we unify the concept of in-plane Poisson's ratio for similar materials in the literature and extend it to the class of zigzag-base folded sheet materials.
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The natural history of biocatalytic mechanisms.
Nath, Neetika; Mitchell, John B O; Caetano-Anollés, Gustavo
2014-05-01
Phylogenomic analysis of the occurrence and abundance of protein domains in proteomes has recently showed that the α/β architecture is probably the oldest fold design. This holds important implications for the origins of biochemistry. Here we explore structure-function relationships addressing the use of chemical mechanisms by ancestral enzymes. We test the hypothesis that the oldest folds used the most mechanisms. We start by tracing biocatalytic mechanisms operating in metabolic enzymes along a phylogenetic timeline of the first appearance of homologous superfamilies of protein domain structures from CATH. A total of 335 enzyme reactions were retrieved from MACiE and were mapped over fold age. We define a mechanistic step type as one of the 51 mechanistic annotations given in MACiE, and each step of each of the 335 mechanisms was described using one or more of these annotations. We find that the first two folds, the P-loop containing nucleotide triphosphate hydrolase and the NAD(P)-binding Rossmann-like homologous superfamilies, were α/β architectures responsible for introducing 35% (18/51) of the known mechanistic step types. We find that these two oldest structures in the phylogenomic analysis of protein domains introduced many mechanistic step types that were later combinatorially spread in catalytic history. The most common mechanistic step types included fundamental building blocks of enzyme chemistry: "Proton transfer," "Bimolecular nucleophilic addition," "Bimolecular nucleophilic substitution," and "Unimolecular elimination by the conjugate base." They were associated with the most ancestral fold structure typical of P-loop containing nucleotide triphosphate hydrolases. Over half of the mechanistic step types were introduced in the evolutionary timeline before the appearance of structures specific to diversified organisms, during a period of architectural diversification. The other half unfolded gradually after organismal diversification and during a
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Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo
Directory of Open Access Journals (Sweden)
Baik L. Seong
2011-03-01
Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.
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An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.
Directory of Open Access Journals (Sweden)
Holly J Atkinson
2009-10-01
Full Text Available The group of proteins that contain a thioredoxin (Trx fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.
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Adapted to roar: functional morphology of tiger and lion vocal folds.
Directory of Open Access Journals (Sweden)
Sarah A Klemuk
Full Text Available Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice.
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Jeffery, Kathleen A; Pelaez, Nancy; Anderson, Trevor R
2018-01-01
To keep biochemistry instruction current and relevant, it is crucial to expose students to cutting-edge scientific research and how experts reason about processes governed by thermodynamics and kinetics such as protein folding and dynamics. This study focuses on how experts explain their research into this topic with the intention of informing instruction. Previous research has modeled how expert biologists incorporate research methods, social or biological context, and analogies when they talk about their research on mechanisms. We used this model as a guiding framework to collect and analyze interview data from four experts. The similarities and differences that emerged from analysis indicate that all experts integrated theoretical knowledge with their research context, methods, and analogies when they explained how phenomena operate, in particular by mapping phenomena to mathematical models; they explored different processes depending on their explanatory aims, but readily transitioned between different perspectives and explanatory models; and they explained thermodynamic and kinetic concepts of relevance to protein folding in different ways that aligned with their particular research methods. We discuss how these findings have important implications for teaching and future educational research. © 2018 K. A. Jeffery et al. CBE—Life Sciences Education © 2018 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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A Self-Assisting Protein Folding Model for Teaching Structural Molecular Biology.
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.
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Folding pathways explored with artificial potential functions
International Nuclear Information System (INIS)
Ulutaş, B; Bozma, I; Haliloglu, T
2009-01-01
This paper considers the generation of trajectories to a given protein conformation and presents a novel approach based on artificial potential functions—originally proposed for multi-robot navigation. The artificial potential function corresponds to a simplified energy model, but with the novelty that—motivated by work on robotic navigation—a nonlinear compositional scheme of constructing the energy model is adapted instead of an additive formulation. The artificial potential naturally gives rise to a dynamic system for the protein structure that ensures collision-free motion to an equilibrium point. In cases where the equilibrium point is the native conformation, the motion trajectory corresponds to the folding pathway. This framework is used to investigate folding in a variety of protein structures, and the results are compared with those of other approaches including experimental studies
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Energy Technology Data Exchange (ETDEWEB)
Bonvin, Alexandre M.J.J.; Houben, Klaartje; Guenneugues, Marc; Kaptein, Robert; Boelens, Rolf [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)
2001-11-15
The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small {alpha}/{beta} protein chymotrypsin inhibitor 2. Dihedral angle restraints for the {phi} and {psi} angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond {sup 3hb}J{sub NC'} coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.
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Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers
Mannini, Benedetta; Cascella, Roberta; Zampagni, Mariagioia; Van Waarde-Verhagen, Maria; Meehan, Sarah; Roodveldt, Cintia; Campioni, Silvia; Boninsegna, Matilde; Penco, Amanda; Relini, Annalisa; Kampinga, Harm H.; Dobson, Christopher M.; Wilson, Mark R.; Cecchi, Cristina; Chiti, Fabrizio
2012-01-01
Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded
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DEFF Research Database (Denmark)
Risør, Michael Wulff
2014-01-01
Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils......, is a general hallmark. They also include the α1-antitrypsin deficiency, where disease-causing mutations in the serine protease inhibitor, α1-antitrypsin (α1AT), leads to accumulation of the aberrant protein in the liver of these patients. The native metastable structure of α1AT constitutes a molecular trap...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding...
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Protein degradation and protection against misfolded or damaged proteins
Goldberg, Alfred L.
2003-12-01
The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.
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Fragment-based quantum mechanical calculation of protein-protein binding affinities.
Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao
2018-04-29
The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.
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Thermostability in endoglucanases is fold-specific
2011-01-01
Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533
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Thermostability in endoglucanases is fold-specific
Directory of Open Access Journals (Sweden)
Wolt Jeffrey D
2011-02-01
Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.
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Ca-Dependent Folding of Human Calumenin
Mazzorana, Marco; Hussain, Rohanah; Sorensen, Thomas
2016-01-01
Human calumenin (hCALU) is a six EF-hand protein belonging to the CREC family. As other members of the family, it is localized in the secretory pathway and regulates the activity of SERCA2a and of the ryanodine receptor in the endoplasmic reticulum (ER). We have studied the effects of Ca2+ binding to the protein and found it to attain a more compact structure upon ion binding. Circular Dichroism (CD) measurements suggest a major rearrangement of the protein secondary structure, which reversibly switches from disordered at low Ca2+ concentrations to predominantly alpha-helical when Ca2+ is added. SAXS experiments confirm the transition from an unfolded to a compact structure, which matches the structural prediction of a trilobal fold. Overall our experiments suggest that calumenin is a Ca2+ sensor, which folds into a compact structure, capable of interacting with its molecular partners, when Ca2+ concentration within the ER reaches the millimolar range. PMID:26991433
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Directory of Open Access Journals (Sweden)
Dallakyan Sargis
2008-08-01
Full Text Available Abstract Background Gram-negative bacteria use periplasmic-binding proteins (bPBP to transport nutrients through the periplasm. Despite immense diversity within the recognized substrates, all members of the family share a common fold that includes two domains that are separated by a conserved hinge. The hinge allows the protein to cycle between open (apo and closed (ligated conformations. Conformational changes within the proteins depend on a complex interplay of mechanical and thermodynamic response, which is manifested as an increase in thermal stability and decrease of flexibility upon ligand binding. Results We use a distance constraint model (DCM to quantify the give and take between thermodynamic stability and mechanical flexibility across the bPBP family. Quantitative stability/flexibility relationships (QSFR are readily evaluated because the DCM links mechanical and thermodynamic properties. We have previously demonstrated that QSFR is moderately conserved across a mesophilic/thermophilic RNase H pair, whereas the observed variance indicated that different enthalpy-entropy mechanisms allow similar mechanical response at their respective melting temperatures. Our predictions of heat capacity and free energy show marked diversity across the bPBP family. While backbone flexibility metrics are mostly conserved, cooperativity correlation (long-range couplings also demonstrate considerable amount of variation. Upon ligand removal, heat capacity, melting point, and mechanical rigidity are, as expected, lowered. Nevertheless, significant differences are found in molecular cooperativity correlations that can be explained by the detailed nature of the hydrogen bond network. Conclusion Non-trivial mechanical and thermodynamic variation across the family is explained by differences within the underlying H-bond networks. The mechanism is simple; variation within the H-bond networks result in altered mechanical linkage properties that directly affect
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Directory of Open Access Journals (Sweden)
Sreenivas Gannavaram
2011-01-01
Full Text Available In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1 and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2 specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.
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Rationally designed synthetic protein hydrogels with predictable mechanical properties.
Wu, Junhua; Li, Pengfei; Dong, Chenling; Jiang, Heting; Bin Xue; Gao, Xiang; Qin, Meng; Wang, Wei; Bin Chen; Cao, Yi
2018-02-12
Designing synthetic protein hydrogels with tailored mechanical properties similar to naturally occurring tissues is an eternal pursuit in tissue engineering and stem cell and cancer research. However, it remains challenging to correlate the mechanical properties of protein hydrogels with the nanomechanics of individual building blocks. Here we use single-molecule force spectroscopy, protein engineering and theoretical modeling to prove that the mechanical properties of protein hydrogels are predictable based on the mechanical hierarchy of the cross-linkers and the load-bearing modules at the molecular level. These findings provide a framework for rationally designing protein hydrogels with independently tunable elasticity, extensibility, toughness and self-healing. Using this principle, we demonstrate the engineering of self-healable muscle-mimicking hydrogels that can significantly dissipate energy through protein unfolding. We expect that this principle can be generalized for the construction of protein hydrogels with customized mechanical properties for biomedical applications.
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Directory of Open Access Journals (Sweden)
Haina Qin
2013-10-01
Full Text Available Paradoxically, aggregation of specific proteins is characteristic of many human diseases and aging, yet aggregates have been found to be unnecessary for initiating pathogenesis. Here we determined the NMR topology and dynamics of a helical mutant in a membrane environment transformed from the 125-residue cytosolic all-β MSP by the ALS-causing P56S mutation. Unexpectedly, despite its low hydrophobicity, the P56S major sperm protein (MSP domain becomes largely embedded in the membrane environment with high backbone rigidity. Furthermore it is composed of five helices with amphiphilicity comparable to those of the partly-soluble membrane toxin mellitin and α-synuclein causing Parkinson's disease. Consequently, the mechanism underlying this chameleon transformation becomes clear: by disrupting the specific tertiary interaction network stabilizing the native all-β MSP fold to release previously-locked amphiphilic segments, the P56S mutation acts to convert the classic MSP fold into a membrane-active protein that is fundamentally indistinguishable from mellitin and α-synuclein which are disordered in aqueous solution but spontaneously partition into membrane interfaces driven by hydrogen-bond energetics gained from forming α-helix in the membrane environments. As segments with high amphiphilicity exist in all proteins, our study successfully resolves the paradox by deciphering that the proteins with a higher tendency to aggregate have a stronger potential to partition into membranes through the same mechanism as α-synuclein to initially attack membranes to trigger pathogenesis without needing aggregates. This might represent the common first step for various kinds of aggregated proteins to trigger familiar, sporadic and aging diseases. Therefore the homeostasis of aggregated proteins in vivo is the central factor responsible for a variety of human diseases including aging. The number and degree of the membrane attacks by aggregated proteins may
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Recoverable and Programmable Collapse from Folding Pressurized Origami Cellular Solids.
Li, S; Fang, H; Wang, K W
2016-09-09
We report a unique collapse mechanism by exploiting the negative stiffness observed in the folding of an origami solid, which consists of pressurized cells made by stacking origami sheets. Such a collapse mechanism is recoverable, since it only involves rigid folding of the origami sheets and it is programmable by pressure control and the custom design of the crease pattern. The collapse mechanism features many attractive characteristics for applications such as energy absorption. The reported results also suggest a new branch of origami study focused on its nonlinear mechanics associated with folding.
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Mechanical stretching of proteins-a theoretical survey of the Protein Data Bank
International Nuclear Information System (INIS)
Sulkowska, Joanna I; Cieplak, Marek
2007-01-01
The mechanical stretching of single proteins has been studied experimentally for about 50 proteins, yielding a variety of force patterns and peak forces. Here we perform a theoretical survey of proteins of known native structure and map out the landscape of possible dynamical behaviours under stretching at constant speed. We consider 7510 proteins comprising not more than 150 amino acids and 239 longer proteins. The model used is constructed based on the native geometry. It is solved by methods of molecular dynamics and validated by comparing the theoretical predictions to experimental results. We characterize the distribution of peak forces and investigate correlations with the system size and with the structure classification as characterized by the CATH scheme. Despite the presence of such correlations, proteins with the same CATH index may belong to different classes of dynamical behaviour. We identify proteins with the biggest forces and show that they belong to few topology classes. We determine which protein segments act as mechanical clamps and show that, in most cases, they correspond to long stretches of parallel β-strands, but other mechanisms are also possible. (topical review)
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Energy Technology Data Exchange (ETDEWEB)
Cuello, Susana; Madrid, Yolanda; Luque-Garcia, Jose L.; Camara, Carmen [Complutense University of Madrid, Department of Analytical Chemistry, Faculty of Chemistry, Madrid (Spain); Ramos, Sonia [Institute of Food Science, Technology and Nutrition, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid (Spain)
2012-08-15
Understanding the molecular mechanisms underlying MeHg toxicity and the way in which this molecule interacts with living organisms is a critical point since MeHg represents a well-known risk to ecosystems and human health. We used a quantitative proteomic approach based on stable isotopic labeling by amino acids in cell culture in combination with SDS-PAGE and nanoflow LC-ESI-LTQ for analyzing the differential protein expression of hepatic cells associated to MeHg exposure. Seventy-eight proteins were found de-regulated by more than 1.5-fold. We identified a number of proteins involved in different essential biological processes including apoptosis, mitochondrial dysfunction, cellular trafficking and energy production. Among these proteins, we found several molecules whose de-regulation has been already related to MeHg exposure, thus confirming the usefulness of our discovery approach, and new ones that helped to gain a deeper insight into the biomolecular mechanisms related to MeHg-induced toxicity. Overexpression of several HSPs and the proteasome 26S subunit itself showed the proteasome system as a molecular target of toxic MeHg. As for the interaction networks, the top ranked was the nucleic acid metabolism, where many of the identified de-regulated proteins are involved. (orig.)
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Functional studies on the phosphatidychloride transfer protein
Brouwer, A.P.M. de
2002-01-01
The phosphatidylcholine transfer protein (PC-TP) has been studied for over 30 years now. Despite extensive research concerning the biochemical, biophysical and structural properties of PC-TP, the function of this protein is still elusive. We have studied in vitro the folding and the mechanism of PC
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Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R; Poulsen, Heidi I; Jensen, Peter H; Knudsen, Jens; Kragelund, Birthe B; Poulsen, Flemming M
2005-04-01
Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues. (c) 2005 Wiley-Liss, Inc.
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Energy Landscapes: From Protein Folding to Molecular Assembly
Databases National Security Education Center (NSEC) Center for Nonlinear Studies Engineering Institute assembly is very common in biology and in nanotechnology. Simple examples of self-assembly are the folding efflux pump machinery, ATP synthase, the ribosome, and many others. In nanotechnology, self-assembly has
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Interaction of β-sheet folds with a gold surface.
Directory of Open Access Journals (Sweden)
Martin Hoefling
Full Text Available The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111 surface using computational molecular dynamics (MD simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN. We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance.
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Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold
International Nuclear Information System (INIS)
Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta; Benach, Jordi; Riera, Antoni; Pous, Joan; Macias, Maria J.
2015-01-01
Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily
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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.
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The protein side of the central dogma: permanence and change.
Morange, Michel
2006-01-01
There are two facets to the central dogma proposed by Francis Crick in 1957. One concerns the relation between the sequence of nucleotides and the sequence of amino acids, the second is devoted to the relation between the sequence of amino acids and the native three-dimensional structure of proteins. 'Folding is simply a function of the order of the amino acids,' i.e. no information is required for the proper folding of a protein other than the information contained in its sequence. This protein side of the central dogma was elaborated in a scientific context in which the characteristics and functions of proteins, and the mechanisms of protein folding, were seen very differently. This context, which made the folding problem a simple one, supported the bold proposition of Francis Crick. The protein side of the central dogma was not challenged by the discovery of prions if one adopts the definition of information given by Francis Crick. It might have been challenged by the discovery that regulatory enzymes exist in different conformations, and the evidence for the existence of chaperones assisting protein folding. But it was not, and folding remains what it was for Francis Crick, 'simply a function of the order of amino acids'. But the meaning of 'function' has dramatically changed. It is no longer the result of simple physicochemical laws, but that of a long evolutionary process which has optimized protein folding. Molecular mechanistic explanations have to be allied with evolutionary explanations, in a way characteristic of present biology.
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β-sheet-like formation during the mechanical unfolding of prion protein
Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.
2015-09-01
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.
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β-sheet-like formation during the mechanical unfolding of prion protein
Energy Technology Data Exchange (ETDEWEB)
Tao, Weiwei; Cao, Penghui; Park, Harold S., E-mail: parkhs@bu.edu [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Yoon, Gwonchan [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Department of Mechanical Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Eom, Kilho [Biomechanics Laboratory, College of Sport Science, Sungkyunkwan University, Suwon 16419 (Korea, Republic of)
2015-09-28
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.
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β-sheet-like formation during the mechanical unfolding of prion protein
International Nuclear Information System (INIS)
Tao, Weiwei; Cao, Penghui; Park, Harold S.; Yoon, Gwonchan; Eom, Kilho
2015-01-01
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP C , whose misfolded form PrP Sc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220
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Directory of Open Access Journals (Sweden)
A.J. Parodi
1998-05-01
Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.
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Nonlinear vs. linear biasing in Trp-cage folding simulations
Energy Technology Data Exchange (ETDEWEB)
Spiwok, Vojtěch, E-mail: spiwokv@vscht.cz; Oborský, Pavel; Králová, Blanka [Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, Prague 6 166 28 (Czech Republic); Pazúriková, Jana [Institute of Computer Science, Masaryk University, Botanická 554/68a, 602 00 Brno (Czech Republic); Křenek, Aleš [Institute of Computer Science, Masaryk University, Botanická 554/68a, 602 00 Brno (Czech Republic); Center CERIT-SC, Masaryk Univerzity, Šumavská 416/15, 602 00 Brno (Czech Republic)
2015-03-21
Biased simulations have great potential for the study of slow processes, including protein folding. Atomic motions in molecules are nonlinear, which suggests that simulations with enhanced sampling of collective motions traced by nonlinear dimensionality reduction methods may perform better than linear ones. In this study, we compare an unbiased folding simulation of the Trp-cage miniprotein with metadynamics simulations using both linear (principle component analysis) and nonlinear (Isomap) low dimensional embeddings as collective variables. Folding of the mini-protein was successfully simulated in 200 ns simulation with linear biasing and non-linear motion biasing. The folded state was correctly predicted as the free energy minimum in both simulations. We found that the advantage of linear motion biasing is that it can sample a larger conformational space, whereas the advantage of nonlinear motion biasing lies in slightly better resolution of the resulting free energy surface. In terms of sampling efficiency, both methods are comparable.
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Golf-course and funnel energy landscapes: Protein folding concepts in martensites
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.
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Asymmetric hindwing foldings in rove beetles.
Saito, Kazuya; Yamamoto, Shuhei; Maruyama, Munetoshi; Okabe, Yoji
2014-11-18
Foldable wings of insects are the ultimate deployable structures and have attracted the interest of aerospace engineering scientists as well as entomologists. Rove beetles are known to fold their wings in the most sophisticated ways that have right-left asymmetric patterns. However, the specific folding process and the reason for this asymmetry remain unclear. This study reveals how these asymmetric patterns emerge as a result of the folding process of rove beetles. A high-speed camera was used to reveal the details of the wing-folding movement. The results show that these characteristic asymmetrical patterns emerge as a result of simultaneous folding of overlapped wings. The revealed folding mechanisms can achieve not only highly compact wing storage but also immediate deployment. In addition, the right and left crease patterns are interchangeable, and thus each wing internalizes two crease patterns and can be folded in two different ways. This two-way folding gives freedom of choice for the folding direction to a rove beetle. The use of asymmetric patterns and the capability of two-way folding are unique features not found in artificial structures. These features have great potential to extend the design possibilities for all deployable structures, from space structures to articles of daily use.
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My 65 years in protein chemistry.
Scheraga, Harold A
2015-05-01
This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein-protein interactions and to nucleic acids and to protein-nucleic acid interactions. The ultimate results led to an understanding
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Energy Technology Data Exchange (ETDEWEB)
Chandonia, John-Marc; Kim, Sung-Hou
2006-03-15
Background: Determining the complete repertoire of proteinstructures for all soluble, globular proteins in a single organism hasbeen one of the major goals of several structural genomics projects inrecent years. Results: We report that this goal has nearly been reachedfor several "minimal organisms"--parasites or symbionts with reducedgenomes--for which over 95 percent of the soluble, globular proteins maynow be assigned folds, overall 3-D backbone structures. We analyze thestructures of these proteins as they relate to cellular functions, andcompare conservation off old usage between functional categories. We alsocompare patterns in the conservation off olds among minimal organisms andthose observed between minimal organisms and other bacteria. Conclusion:We find that proteins performing essential cellular functions closelyrelated to transcription and translation exhibit a higher degree ofconservation in fold usage than proteins in other functional categories.Folds related to transcription and translation functional categories werealso over represented in minimal organisms compared to otherbacteria.
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NAPS: Network Analysis of Protein Structures
Chakrabarty, Broto; Parekh, Nita
2016-01-01
Traditionally, protein structures have been analysed by the secondary structure architecture and fold arrangement. An alternative approach that has shown promise is modelling proteins as a network of non-covalent interactions between amino acid residues. The network representation of proteins provide a systems approach to topological analysis of complex three-dimensional structures irrespective of secondary structure and fold type and provide insights into structure-function relationship. We have developed a web server for network based analysis of protein structures, NAPS, that facilitates quantitative and qualitative (visual) analysis of residue–residue interactions in: single chains, protein complex, modelled protein structures and trajectories (e.g. from molecular dynamics simulations). The user can specify atom type for network construction, distance range (in Å) and minimal amino acid separation along the sequence. NAPS provides users selection of node(s) and its neighbourhood based on centrality measures, physicochemical properties of amino acids or cluster of well-connected residues (k-cliques) for further analysis. Visual analysis of interacting domains and protein chains, and shortest path lengths between pair of residues are additional features that aid in functional analysis. NAPS support various analyses and visualization views for identifying functional residues, provide insight into mechanisms of protein folding, domain-domain and protein–protein interactions for understanding communication within and between proteins. URL:http://bioinf.iiit.ac.in/NAPS/. PMID:27151201
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Energy Technology Data Exchange (ETDEWEB)
Mostaert, Anika S; Jarvis, Suzanne P [Centre for Research on Adaptive Nanostructures and Nanodevices, Trinity College Dublin, Dublin 2 (Ireland)
2007-01-31
While biological systems are notorious for their complexity, nature sometimes displays mechanisms that are elegant in their simplicity. We have recently identified such a mechanism at work to enhance the mechanical properties of certain natural adhesives. The mechanism is simple because it utilizes a non-specific protein folding and subsequent aggregation process, now thought to be generic for any polypeptide under appropriate conditions. This non-specific folding forms proteinaceous crossed {beta}-sheet amyloid fibrils, which are usually associated with neurodegenerative diseases. Here we show evidence for the beneficial mechanical characteristics of these fibrils discovered in natural adhesives. We suggest that amyloid protein quaternary structures should be considered as a possible generic mechanism for mechanical strength in a range of natural adhesives and other natural materials due to their many beneficial mechanical features and apparent ease of self-assembly.
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International Nuclear Information System (INIS)
Mostaert, Anika S; Jarvis, Suzanne P
2007-01-01
While biological systems are notorious for their complexity, nature sometimes displays mechanisms that are elegant in their simplicity. We have recently identified such a mechanism at work to enhance the mechanical properties of certain natural adhesives. The mechanism is simple because it utilizes a non-specific protein folding and subsequent aggregation process, now thought to be generic for any polypeptide under appropriate conditions. This non-specific folding forms proteinaceous crossed β-sheet amyloid fibrils, which are usually associated with neurodegenerative diseases. Here we show evidence for the beneficial mechanical characteristics of these fibrils discovered in natural adhesives. We suggest that amyloid protein quaternary structures should be considered as a possible generic mechanism for mechanical strength in a range of natural adhesives and other natural materials due to their many beneficial mechanical features and apparent ease of self-assembly
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Paul, Fabian; Noé, Frank; Weikl, Thomas R
2018-03-27
Unstructured proteins and peptides typically fold during binding to ligand proteins. A challenging problem is to identify the mechanism and kinetics of these binding-induced folding processes in experiments and atomistic simulations. In this Article, we present a detailed picture for the folding of the inhibitor peptide PMI into a helix during binding to the oncoprotein fragment 25-109 Mdm2 obtained from atomistic, explicit-water simulations and Markov state modeling. We find that binding-induced folding of PMI is highly parallel and can occur along a multitude of pathways. Some pathways are induced-fit-like with binding occurring prior to PMI helix formation, while other pathways are conformational-selection-like with binding after helix formation. On the majority of pathways, however, binding is intricately coupled to folding, without clear temporal ordering. A central feature of these pathways is PMI motion on the Mdm2 surface, along the binding groove of Mdm2 or over the rim of this groove. The native binding groove of Mdm2 thus appears as an asymmetric funnel for PMI binding. Overall, binding-induced folding of PMI does not fit into the classical picture of induced fit or conformational selection that implies a clear temporal ordering of binding and folding events. We argue that this holds in general for binding-induced folding processes because binding and folding events in these processes likely occur on similar time scales and do exhibit the time-scale separation required for temporal ordering.
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Free energy landscape and multiple folding pathways of an H-type RNA pseudoknot.
Directory of Open Access Journals (Sweden)
Yunqiang Bian
Full Text Available How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA.
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Protein folding pathology in domestic animals.
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
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Vielberg, Marie-Theres; Bauer, Verena C; Groll, Michael
2018-03-02
The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O γ , Asp17O δ1 and Lys32N ε , it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH 2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Numerical construction of the p(fold) (committor) reaction coordinate for a Markov process.
Krivov, Sergei V
2011-10-06
To simplify the description of a complex multidimensional dynamical process, one often projects it onto a single reaction coordinate. In protein folding studies, the folding probability p(fold) is an optimal reaction coordinate which preserves many important properties of the dynamics. The construction of the coordinate is difficult. Here, an efficient numerical approach to construct the p(fold) reaction coordinate for a Markov process (satisfying the detailed balance) is described. The coordinate is obtained by optimizing parameters of a chosen functional form to make a generalized cut-based free energy profile the highest. The approach is illustrated by constructing the p(fold) reaction coordinate for the equilibrium folding simulation of FIP35 protein reported by Shaw et al. (Science 2010, 330, 341-346). © 2011 American Chemical Society
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Structural anatomy of telomere OB proteins.
Horvath, Martin P
2011-10-01
Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.
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The impact of intraglottal vortices on vocal fold dynamics
Erath, Byron; Pirnia, Alireza; Peterson, Sean
2016-11-01
During voiced speech a critical pressure is produced in the lungs that separates the vocal folds and creates a passage (the glottis) for airflow. As air passes through the vocal folds the resulting aerodynamic loading, coupled with the tissue properties of the vocal folds, produces self-sustained oscillations. Throughout each cycle a complex flow field develops, characterized by a plethora of viscous flow phenomena. Air passing through the glottis creates a jet, with periodically-shed vortices developing due to flow separation and the Kelvin-Helmholtz instability in the shear layer. These vortices have been hypothesized to be a crucial mechanism for producing vocal fold vibrations. In this study the effect of vortices on the vocal fold dynamics is investigated experimentally by passing a vortex ring over a flexible beam with the same non-dimensional mechanical properties as the vocal folds. Synchronized particle image velocimetry data are acquired in tandem with the beam dynamics. The resulting impact of the vortex ring loading on vocal fold dynamics is discussed in detail. This work was supported by the National Science Foundation Grant CBET #1511761.
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DEFF Research Database (Denmark)
Granados, Alba; Brunskog, Jonas; Misztal, M. K.
2015-01-01
When vocal folds vibrate at normal speaking frequencies, collisions occurs. The numerics and formulations behind a position-based continuum model of contact is an active field of research in the contact mechanics community. In this paper, a frictionless three-dimensional finite element model...
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Can misfolded proteins be beneficial? The HAMLET case.
Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina
2009-01-01
By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.
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International Nuclear Information System (INIS)
Wedin, R.E.; Delepierre, M.; Dobson, C.M.; Poulsen, F.M.
1982-01-01
The individual rates of solvent exchange of the six tryptophan indole NH hydrogens of lysozyme in 2 H 2 O have been measured over a wide range of temperatures by using 1 H NMR. Two distinct mechanisms for exchange have been identified, one characterized by a high activation energy and the other by a much lower activation energy. The high-energy process has been shown to be associated directly with the cooperative thermal unfolding of the protein and is the dominant mechanism for exchange of the most slowly exchanging hydrogen even 15 0 C below the denaturation temperature. Rate constants and activation energies for the folding and unfolding reactions were obtained from the experimental exchange rates. At low temperatures, a lower activation energy mechanism is dominant for all hydrogens, and this can be associated with local fluctuations in the protein structure which allow access of solvent. The relative exchange rates and activation energies can only qualitatively be related to the different environments of the residues in the crystal structure. There is provisional evidence that a mechanism intermediate between these two extremes may be significant for some hydrogens under restricted conditions
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Axons Pull on the Brain, But Tension Does Not Drive Cortical Folding
Xu, Gang; Knutsen, Andrew K.; Dikranian, Krikor; Kroenke, Christopher D.; Bayly, Philip V.; Taber, Larry A.
2011-01-01
During human brain development, the cerebral cortex undergoes substantial folding, leading to its characteristic highly convoluted form. Folding is necessary to accommodate the expansion of the cerbral cortex; abnormal cortical folding is linked to various neurological disorders, including schizophrenia, epilepsy, autism and mental retardation. Although this process requires mechanical forces, the specific force-generating mechanisms that drive folding remain unclear. The two most widely accepted hypotheses are (1) folding is caused by differential growth of the cortex and (2) folding is caused by mechanical tension generated in axons. Direct evidence supporting either theory, however, is lacking. Here we show that axons are indeed under considerable tension in the developing ferret brain, but the patterns of tissue stress are not consistent with a causal role for axonal tension. In particular, microdissection assays reveal that significant tension exists along axons aligned circumferentially in subcortical white matter tracts, as well as those aligned radially inside developing gyri (outward folds). Contrary to previous speculation, however, axonal tension is not directed across developing gyri, suggesting that axon tension does not drive folding. On the other hand, using computational (finite element) models, we show that differential cortical growth accompanied by remodeling of the subplate leads to outward folds and stress fields that are consistent with our microdissection experiments, supporting a mechanism involving differential growth. Local perturbations, such as temporal differences in the initiation of cortical growth, can ensure consistent folding patterns. This study shows that a combination of experimental and computational mechanics can be used to evaluate competing hypotheses of morphogenesis, and illuminate the biomechanics of cortical folding. PMID:20590291
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Modeling the mechanism of CLN025 beta-hairpin formation
McKiernan, Keri A.; Husic, Brooke E.; Pande, Vijay S.
2017-09-01
Beta-hairpins are substructures found in proteins that can lend insight into more complex systems. Furthermore, the folding of beta-hairpins is a valuable test case for benchmarking experimental and theoretical methods. Here, we simulate the folding of CLN025, a miniprotein with a beta-hairpin structure, at its experimental melting temperature using a range of state-of-the-art protein force fields. We construct Markov state models in order to examine the thermodynamics, kinetics, mechanism, and rate-determining step of folding. Mechanistically, we find the folding process is rate-limited by the formation of the turn region hydrogen bonds, which occurs following the downhill hydrophobic collapse of the extended denatured protein. These results are presented in the context of established and contradictory theories of the beta-hairpin folding process. Furthermore, our analysis suggests that the AMBER-FB15 force field, at this temperature, best describes the characteristics of the full experimental CLN025 conformational ensemble, while the AMBER ff99SB-ILDN and CHARMM22* force fields display a tendency to overstabilize the native state.
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Energy Technology Data Exchange (ETDEWEB)
Dantas, Gautam; Watters, Alexander L.; Lunde, Bradley; Eletr, Ziad; Isern, Nancy G.; Roseman, Toby; Lipfert, Jan; Doniach, Sebastian; Tompa, Martin; Kuhlman, Brian; Stoddard, Barry L.; Varani, Gabriele; Baker, David
2006-10-06
We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mistranslated and accumulates at high levels in E. coli. We used circular dichroism spectroscopy, size-exclusion chromatography, small-angle x-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting CFr protein adopts a compact, extremely-stable, obligate, symmetric, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery, and the rarity of correspondingly stable fragments in natural proteins implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mistranslated CFr sub-units to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of stable protein sub-structures.
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Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim
2006-04-01
The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.
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Insights into the role of elastin in vocal fold health and disease
Moore, Jaime
2011-01-01
Elastic fibers are large, complex and surprisingly poorly understood extracellular matrix (ECM) macromolecules. The elastin fiber, generated from a single human gene - elastin (ELN), is a self assembling integral protein that endows critical mechanic proprieties to elastic tissues and organs such as the skin, lungs, and arteries. The biology of elastic fibers is complex because they have multiple components, a tightly regulated developmental deposition, a multi-step hierarchical assembly and unique biomechanical functions. Elastin is present in vocal folds, where it plays a pivotal role in the quality of phonation. This review article provides an overview of the genesis of elastin and its wide- ranging structure and function. Specific distribution within the vocal fold lamina propria across the lifespan in normal and pathological states and its contribution to vocal fold biomechanics will be examined. Elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. The properties of various elastin– based materials will be discussed and their current and future applications evaluated. A new level of understanding of the biomechanical properties of vocal fold elastin composites and their molecular basis should lead to new strategies for elastic fiber repair and regeneration in aging and disease. PMID:21708449
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Jornot, L; Junod, A F
1997-01-01
Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a
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Non-cylindrical fold growth in the Zagros fold and thrust belt (Kurdistan, NE-Iraq)
Bartl, Nikolaus; Bretis, Bernhard; Grasemann, Bernhard; Lockhart, Duncan
2010-05-01
The Zagros mountains extends over 1800 km from Kurdistan in N-Iraq to the Strait of Hormuz in Iran and is one of the world most promising regions for the future hydrocarbon exploration. The Zagros Mountains started to form as a result of the collision between the Eurasian and Arabian Plates, whose convergence began in the Late Cretaceous as part of the Alpine-Himalayan orogenic system. Geodetic and seismological data document that both plates are still converging and that the fold and thrust belt of the Zagros is actively growing. Extensive hydrocarbon exploration mainly focuses on the antiforms of this fold and thrust belt and therefore the growth history of the folds is of great importance. This work investigates by means of structural field work and quantitative geomorphological techniques the progressive fold growth of the Permam, Bana Bawi- and Safeen- Anticlines located in the NE of the city of Erbil in the Kurdistan region of Northern Iraq. This part of the Zagros fold and thrust belt belongs to the so-called Simply Folded Belt, which is dominated by gentle to open folding. Faults or fault related folds have only minor importance. The mechanical anisotropy of the formations consisting of a succession of relatively competent (massive dolomite and limestone) and incompetent (claystone and siltstone) sediments essentially controls the deformation pattern with open to gentle parallel folding of the competent layers and flexural flow folding of the incompetent layers. The characteristic wavelength of the fold trains is around 10 km. Due to faster erosion of the softer rock layers in the folded sequence, the more competent lithologies form sharp ridges with steeply sloping sides along the eroded flanks of the anticlines. Using an ASTER digital elevation model in combination with geological field data we quantified 250 drainage basins along the different limbs of the subcylindrical Permam, Bana Bawi- and Safeen- Anticlines. Geomorphological indices of the drainage
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Protein kinesis: The dynamics of protein trafficking and stability
Energy Technology Data Exchange (ETDEWEB)
NONE
1995-12-31
The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.
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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
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The Multiple-Minima Problem in Protein Folding
Scheraga, Harold A.
1991-10-01
The conformational energy surface of a polypeptide or protein has many local minima, and conventional energy minimization procedures reach only a local minimum (near the starting point of the optimization algorithm) instead of the global minimum (the multiple-minima problem). Several procedures have been developed to surmount this problem, the most promising of which are: (a) build up procedure, (b) optimization of electrostatics, (c) Monte Carlo-plus-energy minimization, (d) electrostatically-driven Monte Carlo, (e) inclusion of distance restraints, (f) adaptive importance-sampling Monte Carlo, (g) relaxation of dimensionality, (h) pattern-recognition, and (i) diffusion equation method. These procedures have been applied to a variety of polypeptide structural problems, and the results of such computations are presented. These include the computation of the structures of open-chain and cyclic peptides, fibrous proteins and globular proteins. Present efforts are being devoted to scaling up these procedures from small polypeptides to proteins, to try to compute the three-dimensional structure of a protein from its amino sequence.
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Protein tyrosine phosphatases: regulatory mechanisms.
den Hertog, J.; Ostman, A.; Bohmer, F.D.
2008-01-01
Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and
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Self-folding graphene-polymer bilayers
Energy Technology Data Exchange (ETDEWEB)
Deng, Tao [Institute of Microelectronics, Tsinghua University, Beijing 100084 (China); Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Yoon, ChangKyu [Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Jin, Qianru [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Li, Mingen [Department of Physics, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Liu, Zewen [Institute of Microelectronics, Tsinghua University, Beijing 100084 (China); Gracias, David H., E-mail: dgracias@jhu.edu [Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218 (United States)
2015-05-18
In order to incorporate the extraordinary intrinsic thermal, electrical, mechanical, and optical properties of graphene with three dimensional (3D) flexible substrates, we introduce a solvent-driven self-folding approach using graphene-polymer bilayers. A polymer (SU-8) film was spin coated atop chemically vapor deposited graphene films on wafer substrates and graphene-polymer bilayers were patterned with or without metal electrodes using photolithography, thin film deposition, and etching. After patterning, the bilayers were released from the substrates and they self-folded to form fully integrated, curved, and folded structures. In contrast to planar graphene sensors on rigid substrates, we assembled curved and folded sensors that are flexible and they feature smaller form factors due to their 3D geometry and large surface areas due to their multiple rolled architectures. We believe that this approach could be used to assemble a range of high performance 3D electronic and optical devices of relevance to sensing, diagnostics, wearables, and energy harvesting.
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Garg, Dushyant K; Tomar, Rachana; Dhoke, Reema R; Srivastava, Ankit; Kundu, Bishwajit
2015-05-01
Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, L-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.
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Pharmacological chaperoning: a primer on mechanism and pharmacology.
Leidenheimer, Nancy J; Ryder, Katelyn G
2014-05-01
Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast
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New force replica exchange method and protein folding pathways probed by force-clamp technique.
Kouza, Maksim; Hu, Chin-Kun; Li, Mai Suan
2008-01-28
We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes
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Wang, Fei; Gong, Haoran; Chen, Xi; Chen, C. Q.
2016-09-01
Origami structures enrich the field of mechanical metamaterials with the ability to convert morphologically and systematically between two-dimensional (2D) thin sheets and three-dimensional (3D) spatial structures. In this study, an in-plane design method is proposed to approximate curved surfaces of interest with generalized Miura-ori units. Using this method, two combination types of crease lines are unified in one reprogrammable procedure, generating multiple types of cylindrical structures. Structural completeness conditions of the finite-thickness counterparts to the two types are also proposed. As an example of the design method, the kinematics and elastic properties of an origami-based circular cylindrical shell are analysed. The concept of Poisson’s ratio is extended to the cylindrical structures, demonstrating their auxetic property. An analytical model of rigid plates linked by elastic hinges, consistent with numerical simulations, is employed to describe the mechanical response of the structures. Under particular load patterns, the circular shells display novel mechanical behaviour such as snap-through and limiting folding positions. By analysing the geometry and mechanics of the origami structures, we extend the design space of mechanical metamaterials and provide a basis for their practical applications in science and engineering.
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The review on tessellation origami inspired folded structure
Chu, Chai Chen; Keong, Choong Kok
2017-10-01
Existence of folds enhances the load carrying capacity of a folded structure which makes it suitable to be used for application where large open space is required such as large span roof structures and façade. Folded structure is closely related to origami especially the tessellation origami. Tessellation origami provides a folded configuration with facetted surface as a result from repeated folding pattern. Besides that, tessellation origami has flexible folding mechanism that produced a variety of 3-dimensional folded configurations. Despite the direct relationship between fold in origami and folded structure, the idea of origami inspired folded structure is not properly reviewed in the relevant engineering field. Hence, this paper aims to present the current studies from related discipline which has direct relation with application of tessellation origami in folded structure. First, tessellation origami is properly introduced and defined. Then, the review covers the topic on the origami tessellation design suitable for folded structure, its modeling and simulation method, and existing studies and applications of origami as folded structure is presented. The paper also includes the discussion on the current issues related to each topic.
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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.
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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.
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de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter
2016-06-08
The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.
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Thermodynamics of protein folding using a modified Wako-Saitô-Muñoz-Eaton model.
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.
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Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.
Directory of Open Access Journals (Sweden)
Nathan V Welham
2011-03-01
Full Text Available The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match. The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.
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Pohl, Gábor; Beke, Tamás; Borbély, János; Perczel, András
2006-11-15
Because of their great flexibility and strength resistance, both spider silks and silkworm silks are of increasing scientific and commercial interest. Despite numerous spectroscopic and theoretical studies, several structural properties at the atomic level have yet to be identified. The present theoretical investigation focuses on these issues by studying three silk-like model peptides: (AG)(64), [(AG)(4)EG](16), and [(AG)(4)PEG](16), using a Lego-type approach to construct these polypeptides. On the basis of these examples it is shown that thermoneutral isodesmic reactions and ab initio calculations provide a capable method to investigate structural properties of repetitive polypeptides. The most probable overall fold schema of these molecules with respect to the type of embedded hairpin structures were determined at the ab initio level of theory (RHF/6-311++G(d,p)//RHF/3-21G). Further on, analysis is carried out on the possible hairpin and turn regions and on their effect on the global fold. In the case of the (AG)(64) model peptide, the optimal beta-sheet/turn ratio was also determined, which provided good support for experimental observations. In addition, lateral shearing of a hairpin "folding unit" was investigated at the quantum chemical level to explain the mechanical properties of spider silk. The unique mechanical characteristics of silk bio-compounds are now investigated at the atomic level.
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Quantifying the mechanisms of domain gain in animal proteins.
Buljan, Marija; Frankish, Adam; Bateman, Alex
2010-01-01
Protein domains are protein regions that are shared among different proteins and are frequently functionally and structurally independent from the rest of the protein. Novel domain combinations have a major role in evolutionary innovation. However, the relative contributions of the different molecular mechanisms that underlie domain gains in animals are still unknown. By using animal gene phylogenies we were able to identify a set of high confidence domain gain events and by looking at their coding DNA investigate the causative mechanisms. Here we show that the major mechanism for gains of new domains in metazoan proteins is likely to be gene fusion through joining of exons from adjacent genes, possibly mediated by non-allelic homologous recombination. Retroposition and insertion of exons into ancestral introns through intronic recombination are, in contrast to previous expectations, only minor contributors to domain gains and have accounted for less than 1% and 10% of high confidence domain gain events, respectively. Additionally, exonization of previously non-coding regions appears to be an important mechanism for addition of disordered segments to proteins. We observe that gene duplication has preceded domain gain in at least 80% of the gain events. The interplay of gene duplication and domain gain demonstrates an important mechanism for fast neofunctionalization of genes.
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Structure and elevator mechanism of the Na(+)-citrate transporter CitS
Lolkema, Juke S; Slotboom, Dirk Jan
2016-01-01
The recently determined crystal structure of the bacterial Na(+)-citrate symporter CitS provides unexpected structural and mechanistic insights. The protein has a fold that has not been seen in other proteins, but the oligomeric state, domain organization and proposed transport mechanism strongly
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Energy Technology Data Exchange (ETDEWEB)
frydman, judith
2018-03-23
Archaea offer a potentially cost effective and renewable source of energy. The methanogen M. maripaludis, a fast growing archaea that obtains energy by sequestering H2 and reducing CO2 to methane by the methanogenic pathway, is an attractive source for biofuel production. More recently, it has also been suggested that the methanogenesis pathway could be run in reverse, to produce H2 growing the organism in formate. A multi-level understanding of archaeal protein homeostasis, should be instrumental for improving the functionality and design of the enzyme pathways and complexes involved in energy production and storage. One additional importance consequence of a better understanding of archaeal protein homeostasis will be to increase their stress resistance, since their utilization for the efficient large-scale production of methane (and eventually also of H2) requires that the organisms are resistance to a range of growth conditions. This proposal was focused on understanding how archaea achieve protein folding and assembly and maintain protein homeostasis, which are essential for function and viability. We hypothesize that the homo-oligomeric ring shaped chaperonin from M. maripaludis, Mm-Cpn, is central to achaeal protein homeostasis and assists folding of a wide spectrum of metabolic, structural and regulatory archaeal proteins. Through a combination of biochemistry, systems biology, computational and structural biology, we have been testing this hypothesis through two complementary efforts: (i) identify the archaeal substrate repertoire of Mm-Cpn, and (ii) define mechanistic and structural principles of Mm-Cpn mediated protein folding.
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Predicting Folding Sequences Based on the Maximum Rock Strength and Mechanical Equilibrium
Cubas, N.; Souloumiac, P.; Maillot, B.; Leroy, Y. M.
2007-12-01
The objective is to propose and validate simple procedures, compared to the finite-element method, to select and optimize the dominant mode of folding in fold-and-thrust belts and accretionary wedges, and to determine its stress distribution. Mechanical equilibrium as well as the constraints due to the limited rock strength of the bulk material and of major discontinuities, such as décollements, are accounted for. The first part of the proposed procedure, which is at the core of the external approach of classical limit analysis, consists in estimating the least upper bound on the tectonic force by minimisation of the internal dissipation and part of the external work. The new twist to the method is that the optimization is also done with respect to the geometry of the evolving fold. If several folding events are possible, the dominant mode is the one leading to the least upper bound. The second part of the procedure is based on the Equilibrium Element Method, which is an application of the internal approach of limit analysis. The optimum stress field, obtained by spatial discretisation of the fold, provides the best lower bound on the tectonic force. The difference between the two bounds defines an error estimate of the exact unknown tectonic force. To show the merits of the proposed procedure, its first part is applied to predict the life span of a thrust within an accretionary prism, from its onset, its development with a relief build up and its arrest because of the onset of a more favorable new thrust (Cubas et al., 2007). This life span is sensitive to the friction angles over the ramp and the décollement. It is shown how the normal sequence of thrusting in a supercritical wedge is ended with the first out-of sequence event. The second part of the procedure provides the stress state over each thrust showing that the active back thrust is a narrow fan which dip is sensitive to the friction angle over the ramp and the amount of relief build up (Souloumiac et
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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
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Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases
DEFF Research Database (Denmark)
Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah
2016-01-01
’s disease (PD), Alzheimer’s disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other......In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post......-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially...
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Farley, Peter C.
2013-01-01
This article describes a novel approach to teaching novice Biochemistry students visual literacy skills and understanding of some aspects of protein structure using the internet resource FoldIt and a worksheet based on selected Introductory Puzzles from this computer game. In responding to a questionnaire, students indicated that they (94%)…
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Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim
2006-01-01
The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. PMID:16531499
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Protein mislocalization: mechanisms, functions and clinical applications in cancer
Wang, Xiaohong; Li, Shulin
2014-01-01
The changes from normal cells to cancer cells are primarily regulated by genome instability, which foster hallmark functions of cancer through multiple mechanisms including protein mislocalization. Mislocalization of these proteins, including oncoproteins, tumor suppressors, and other cancer-related proteins, can interfere with normal cellular function and cooperatively drive tumor development and metastasis. This review describes the cancer-related effects of protein subcellular mislocalization, the related mislocalization mechanisms, and the potential application of this knowledge to cancer diagnosis, prognosis, and therapy. PMID:24709009
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DEFF Research Database (Denmark)
Naqvi, Mohsin M; Heiðarsson, Pétur Orri; Otazo, Mariela R
2015-01-01
, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process...... in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated...... by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions...
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Heavy metals and metalloids as a cause for protein misfolding and aggregation.
Tamás, Markus J; Sharma, Sandeep K; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp
2014-02-25
While the toxicity of metals and metalloids, like arsenic, cadmium, mercury, lead and chromium, is undisputed, the underlying molecular mechanisms are not entirely clear. General consensus holds that proteins are the prime targets; heavy metals interfere with the physiological activity of specific, particularly susceptible proteins, either by forming a complex with functional side chain groups or by displacing essential metal ions in metalloproteins. Recent studies have revealed an additional mode of metal action targeted at proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit the in vitro refolding of chemically denatured proteins, to interfere with protein folding in vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded proteins with motile backbone and side chains are considerably more prone to engage in stable, pluridentate metal complexes than native proteins with their well-defined 3D structure. By interfering with the folding process, heavy metal ions and metalloids profoundly affect protein homeostasis and cell viability. This review describes how heavy metals impede protein folding and promote protein aggregation, how cells regulate quality control systems to protect themselves from metal toxicity and how metals might contribute to protein misfolding disorders.
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Moparthi, Satish Babu; Carlsson, Uno; Vincentelli, Renaud; Jonsson, Bengt-Harald; Hammarström, Per; Wenger, Jérôme
2016-01-01
Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of β-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes. PMID:27328749
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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.
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Folding and insertion thermodynamics of the transmembrane WALP peptide
Bereau, T.; Bennett, W.F.D.; Pfaendtner, J.; Deserno, M.; Karttunen, M.E.J.
2015-01-01
The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)$_n$(L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural
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Folding and insertion thermodynamics of the transmembrane WALP peptide
Bereau, T.; Bennett, W.F.D. Drew; Pfaendtner, J.; Deserno, M.; Karttunen, M.
2015-01-01
The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural
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Protein Structure Prediction by Protein Threading
Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong
The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.
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Liao, Yi-Ting; Manson, Anthony C.; DeLyser, Michael R.; Noid, William G.; Cremer, Paul S.
2017-01-01
We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air–water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer–water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins. PMID:28228526
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Leliveld, S Rutger; Stitz, Lothar; Korth, Carsten
2008-06-10
A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.
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A collaborative visual analytics suite for protein folding research.
Harvey, William; Park, In-Hee; Rübel, Oliver; Pascucci, Valerio; Bremer, Peer-Timo; Li, Chenglong; Wang, Yusu
2014-09-01
Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind important biochemical processes such as protein folding and molecular interaction. With the rapidly increasing power of modern computers, large-scale MD simulation experiments can be performed regularly, generating huge amounts of MD data. An important question is how to analyze and interpret such massive and complex data. One of the (many) challenges involved in analyzing MD simulation data computationally is the high-dimensionality of such data. Given a massive collection of molecular conformations, researchers typically need to rely on their expertise and prior domain knowledge in order to retrieve certain conformations of interest. It is not easy to make and test hypotheses as the data set as a whole is somewhat "invisible" due to its high dimensionality. In other words, it is hard to directly access and examine individual conformations from a sea of molecular structures, and to further explore the entire data set. There is also no easy and convenient way to obtain a global view of the data or its various modalities of biochemical information. To this end, we present an interactive, collaborative visual analytics tool for exploring massive, high-dimensional molecular dynamics simulation data sets. The most important utility of our tool is to provide a platform where researchers can easily and effectively navigate through the otherwise "invisible" simulation data sets, exploring and examining molecular conformations both as a whole and at individual levels. The visualization is based on the concept of a topological landscape, which is a 2D terrain metaphor preserving certain topological and geometric properties of the high dimensional protein energy landscape. In addition to facilitating easy exploration of conformations, this 2D terrain metaphor also provides a platform where researchers can visualize and analyze various properties (such as contact density) overlayed on the
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Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases
Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah; Abdelhameed, Ali Saber; Khan, Rizwan Hasan
2016-11-01
In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially misfolded intermediates aggregate via the interaction with the complementary intermediates and consequently enhance oligomers formation that grows into fibrils and proto-fibrils. The amyloid fibrils for example, accumulate in the brain and central nervous system (CNS) as amyloid deposits in the Parkinson's disease (PD), Alzheimer's disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other neurodegenerative diseases. Treatment of such misfolding maladies is considered as one of the most important challenges of the 21st century. Currently, several treatments strategies have been and are being discovered. These therapeutic interventions partly reversed or prevented the pathological state. More recently, a new approach was discovered, which employs nanobodies that targets multisteps in fibril formation pathway that may possibly completely cure these misfolding diseases. Keeping the above views in mind in the current review, we have comprehensively discussed the different mechanisms underlying protein misfolding thereby leading to diseases conditions and their therapeutic interventions.
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Self-folding miniature elastic electric devices
International Nuclear Information System (INIS)
Miyashita, Shuhei; Meeker, Laura; Rus, Daniela; Tolley, Michael T; Wood, Robert J
2014-01-01
Printing functional materials represents a considerable impact on the access to manufacturing technology. In this paper we present a methodology and validation of print-and-self-fold miniature electric devices. Polyvinyl chloride laminated sheets based on metalized polyester film show reliable self-folding processes under a heat application, and it configures 3D electric devices. We exemplify this technique by fabricating fundamental electric devices, namely a resistor, capacitor, and inductor. Namely, we show the development of a self-folded stretchable resistor, variable resistor, capacitive strain sensor, and an actuation mechanism consisting of a folded contractible solenoid coil. Because of their pre-defined kinematic design, these devices feature elasticity, making them suitable as sensors and actuators in flexible circuits. Finally, an RLC circuit obtained from the integration of developed devices is demonstrated, in which the coil based actuator is controlled by reading a capacitive strain sensor. (paper)
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A bidirectional shape memory alloy folding actuator
International Nuclear Information System (INIS)
Paik, Jamie K; Wood, Robert J
2012-01-01
This paper presents a low-profile bidirectional folding actuator based on annealed shape memory alloy sheets applicable for meso- and microscale systems. Despite the advantages of shape memory alloys—high strain, silent operation, and mechanical simplicity—their application is often limited to unidirectional operation. We present a bidirectional folding actuator that produces two opposing 180° motions. A laser-patterned nickel alloy (Inconel 600) heater localizes actuation to the folding sections. The actuator has a thin ( < 1 mm) profile, making it appropriate for use in robotic origami. Various design parameters and fabrication variants are described and experimentally explored in the actuator prototype. (paper)
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Protein Misfolding Cyclic Amplification of Infectious Prions.
Moda, Fabio
2017-01-01
Transmissible spongiform encephalopathies, or prion diseases, are a group of incurable disorders caused by the accumulation of an abnormally folded prion protein (PrP Sc ) in the brain. According to the "protein-only" hypothesis, PrP Sc is the infectious agent able to propagate the disease by acting as a template for the conversion of the correctly folded prion protein (PrP C ) into the pathological isoform. Recently, the mechanism of PrP C conversion has been mimicked in vitro using an innovative technique named protein misfolding cyclic amplification (PMCA). This technology represents a great tool for studying diverse aspects of prion biology in the field of basic research and diagnosis. Moreover, PMCA can be expanded for the study of the misfolding process associated to other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal lobar degeneration. © 2017 Elsevier Inc. All rights reserved.
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Mechanical strength of 17,134 model proteins and cysteine slipknots.
Directory of Open Access Journals (Sweden)
Mateusz Sikora
2009-10-01
Full Text Available A new theoretical survey of proteins' resistance to constant speed stretching is performed for a set of 17,134 proteins as described by a structure-based model. The proteins selected have no gaps in their structure determination and consist of no more than 250 amino acids. Our previous studies have dealt with 7510 proteins of no more than 150 amino acids. The proteins are ranked according to the strength of the resistance. Most of the predicted top-strength proteins have not yet been studied experimentally. Architectures and folds which are likely to yield large forces are identified. New types of potent force clamps are discovered. They involve disulphide bridges and, in particular, cysteine slipknots. An effective energy parameter of the model is estimated by comparing the theoretical data on characteristic forces to the corresponding experimental values combined with an extrapolation of the theoretical data to the experimental pulling speeds. These studies provide guidance for future experiments on single molecule manipulation and should lead to selection of proteins for applications. A new class of proteins, involving cysteine slipknots, is identified as one that is expected to lead to the strongest force clamps known. This class is characterized through molecular dynamics simulations.
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A kinetic model of trp-cage folding from multiple biased molecular dynamics simulations.
Directory of Open Access Journals (Sweden)
Fabrizio Marinelli
2009-08-01
Full Text Available Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins.Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For theTrp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap.Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at Calpha-RMSD of 4.4 A from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the Pro12-delta3 and Gly11-alpha3 chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.
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Structural Basis for Target Protein Regcognition by Thiredoxin
DEFF Research Database (Denmark)
Maeda, Kenji
2007-01-01
Ser) and a mutant of an in vitro substrate alpha-amylase/subtilisin inhibitor (BASI) (Cys144Ser), as a reaction intermediate-mimic of Trx-catalyzed disulfide reduction. The resultant structure showed a sequence of BASI residues along a conserved hydrophobic groove constituted of three loop segments...... of Trx-fold proteins glutaredoxin and glutathione transferase. This study suggests that the features of main chain conformation as well as charge property around disulfide bonds in protein substrates are important factors for interaction with Trx. Moreover, this study describes a detailed structural......Thioredoxin (Trx) is an ubiquitous protein disulfide reductase that possesses two redox active cysteines in the conserved active site sequence motif, Trp-CysN-Gly/Pro-Pro-CysC situated in the so called Trx-fold. The lack of insight into the protein substrate recognition mechanism of Trx has to date...
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Understanding curcumin-induced modulation of protein aggregation.
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.
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TALE proteins search DNA using a rotationally decoupled mechanism.
Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M
2016-10-01
Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins used extensively for gene editing. Despite recent progress, however, little is known about their sequence search mechanism. Here, we use single-molecule experiments to study TALE search along DNA. Our results show that TALEs utilize a rotationally decoupled mechanism for nonspecific search, despite remaining associated with DNA templates during the search process. Our results suggest that the protein helical structure enables TALEs to adopt a loosely wrapped conformation around DNA templates during nonspecific search, facilitating rapid one-dimensional (1D) diffusion under a range of solution conditions. Furthermore, this model is consistent with a previously reported two-state mechanism for TALE search that allows these proteins to overcome the search speed-stability paradox. Taken together, our results suggest that TALE search is unique among the broad class of sequence-specific DNA-binding proteins and supports efficient 1D search along DNA.
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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.
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Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.
Meuzelaar, Heleen; Tros, Martijn; Huerta-Viga, Adriana; van Dijk, Chris N; Vreede, Jocelyne; Woutersen, Sander
2014-03-06
Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu - and Arg + are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.
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Single-molecule mechanics of protein-labelled DNA handles
Directory of Open Access Journals (Sweden)
Vivek S. Jadhav
2016-01-01
Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular
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Rate constant and reaction coordinate of Trp-cage folding in explicit water
Juraszek, J.; Bolhuis, P.G.
2008-01-01
We report rate constant calculations and a reaction coordinate analysis of the rate-limiting folding and unfolding process of the Trp-cage mini-protein in explicit solvent using transition interface sampling. Previous transition path sampling simulations revealed that in this (un)folding process the
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International Nuclear Information System (INIS)
Rahaman, Hamidur; Khan, Md. Khurshid Alam; Hassan, Md. Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan
2013-01-01
Highlights: ► Non-coincidence of normalized sigmoidal curves of two different structural properties is consistence with the two-state protein folding/unfolding. ► DSC measurements of denaturation show a two-state behavior of g-cyt-c at pH 6.0. ► Urea-induced denaturation of g-cyt-c is a variable two- state process at pH 6.0. ► GdmCl-induced denaturation of g-cyt-c is a fixed two- state process at pH 6.0. -- Abstract: In practice, the observation of non-coincidence of normalized sigmoidal transition curves measured by two different structural properties constitutes a proof of existence of thermodynamically stable intermediate(s) on the folding ↔ unfolding pathway of a protein. Here we give first experimental evidence that this non-coincidence is also observed for a two-state protein denaturation. Proof of this evidence comes from our studies of denaturation of goat cytochrome-c (g-cyt-c) at pH 6.0. These studies involve differential scanning calorimetry (DSC) measurements in the absence of urea and measurements of urea-induced denaturation curves monitored by observing changes in absorbance at 405, 530, and 695 nm and circular dichroism (CD) at 222, 405, and 416 nm. DSC measurements showed that denaturation of the protein is a two-state process, for calorimetric and van’t Hoff enthalpy changes are, within experimental errors, identical. Normalization of urea-induced denaturation curves monitored by optical properties leads to noncoincident sigmoidal curves. Heat-induced transition of g-cyt-c in the presence of different urea concentrations was monitored by CD at 222 nm and absorption at 405 nm. It was observed that these two different structural probes gave not only identical values of T m (transition temperature), ΔH m (change in enthalpy at T m ) and ΔC p (constant-pressure heat capacity change), but these thermodynamic parameters in the absence of urea are also in agreement with those obtained from DSC measurements
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Directory of Open Access Journals (Sweden)
Gabriele Giachin
Full Text Available Humic substances (HS are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of "salting-out" whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of "prion ecology". These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.
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Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects
Finkelstein, A. V.
1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox
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Papanikolopoulou, Katerina; Teixeira, Susana; Belrhali, Hassan; Forsyth, V Trevor; Mitraki, Anna; van Raaij, Mark J
2004-09-03
Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.
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Intrinsically Disordered Proteins in a Physics-Based World
Directory of Open Access Journals (Sweden)
Jianhan Chen
2010-12-01
Full Text Available Intrinsically disordered proteins (IDPs are a newly recognized class of functional proteins that rely on a lack of stable structure for function. They are highly prevalent in biology, play fundamental roles, and are extensively involved in human diseases. For signaling and regulation, IDPs often fold into stable structures upon binding to specific targets. The mechanisms of these coupled binding and folding processes are of significant importance because they underlie the organization of regulatory networks that dictate various aspects of cellular decision-making. This review first discusses the challenge in detailed experimental characterization of these heterogeneous and dynamics proteins and the unique and exciting opportunity for physics-based modeling to make crucial contributions, and then summarizes key lessons from recent de novo simulations of the structure and interactions of several regulatory IDPs.
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Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization.
Haq, Imran; Irving, James A; Saleh, Aarash D; Dron, Louis; Regan-Mochrie, Gemma L; Motamedi-Shad, Neda; Hurst, John R; Gooptu, Bibek; Lomas, David A
2016-01-01
Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.
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Insights into the fold organization of TIM barrel from interaction energy based structure networks.
Vijayabaskar, M S; Vishveshwara, Saraswathi
2012-01-01
There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.
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Chen, Charles H; Wiedman, Gregory; Khan, Ayesha; Ulmschneider, Martin B
2014-09-01
Unbiased molecular simulation is a powerful tool to study the atomic details driving functional structural changes or folding pathways of highly fluid systems, which present great challenges experimentally. Here we apply unbiased long-timescale molecular dynamics simulation to study the ab initio folding and partitioning of melittin, a template amphiphilic membrane active peptide. The simulations reveal that the peptide binds strongly to the lipid bilayer in an unstructured configuration. Interfacial folding results in a localized bilayer deformation. Akin to purely hydrophobic transmembrane segments the surface bound native helical conformer is highly resistant against thermal denaturation. Circular dichroism spectroscopy experiments confirm the strong binding and thermostability of the peptide. The study highlights the utility of molecular dynamics simulations for studying transient mechanisms in fluid lipid bilayer systems. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014. Published by Elsevier B.V.
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Adaptation of Lactobacillus casei Zhang to Gentamycin Involves an Alkaline Shock Protein
Directory of Open Access Journals (Sweden)
Wenyi Zhang
2017-11-01
Full Text Available Lactobacillus (L. casei Zhang is a koumiss-originated probiotic strain, which was used as a model in a long-term antibiotics-driven evolution experiment to reveal bacterial evolutionary dynamics; and we isolated gentamycin-resistant L. casei Zhang descendents. To decipher the gentamycin resistance mechanism, here we cultivated the parental L. casei Zhang and its descendent cells in an antibiotics-containing environment to compare their global protein expression profiles using the iTRAQ-based proteomic approach. A total of 72 proteins were significantly up-regulated (>2.0-fold, P < 0.05, whilst 32 proteins were significantly down-regulated <−2.0-fold, P < 0.05 in the descendent line. The gentamycin-resistant descendent line showed elevated expression in some carbohydrates, amino acids, and purine metabolic pathways. Several stress-related proteins were also differentially expressed. Among them, one alkaline shock protein, asp23, was up-regulated most in the gentamycin-resistant strain (21.9-fold increase compared with the parental strain. The asp23 gene disruption mutant was significantly more sensitive to gentamycin compared with the wild type, suggesting an important role of this gene in developing the gentamycin-resistant phenotype in L. casei. Our report has described the adaptation of a probiotic strain that has acquired antibiotics resistance through long-term antibiotics exposure at the proteome level, and we revealed a novel mechanism of gentamycin resistance.
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Disulfide bonds in folding and transport of the mouse hepatitis virus glycoproteins
Horzinek, M.C.; Opstelten, D.-J.E.; Groote, P. de; Vennema, H.; Rottier, P.J.M.
1993-01-01
We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum
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Barone, Fabrizio; Giordano, Gerardo
2018-02-01
We present the Extended Folded Pendulum Model (EFPM), a model developed for a quantitative description of the dynamical behavior of a folded pendulum generically oriented in space. This model, based on the Tait-Bryan angular reference system, highlights the relationship between the folded pendulum orientation in the gravitational field and its natural resonance frequency. Tis model validated by tests performed with a monolithic UNISA Folded Pendulum, highlights a new technique of implementation of folded pendulum based tiltmeters.
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Corti, Alessio
2007-01-01
This edited collection of chapters, authored by leading experts, provides a complete and essentially self-contained construction of 3-fold and 4-fold klt flips. A large part of the text is a digest of Shokurov's work in the field and a concise, complete and pedagogical proof of the existence of 3-fold flips is presented. The text includes a ten page glossary and is accessible to students and researchers in algebraic geometry.
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Horesh, Yair; Wexler, Ydo; Lebenthal, Ilana; Ziv-Ukelson, Michal; Unger, Ron
2009-03-04
Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L free energy (MFE) for the folding of each of the L-sized substrings of S. The consecutive windows folding problem can be naively solved in O(NL3) by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2) solution for this problem has been described. Here, we describe and implement an O(NLpsi(L)) engine for the consecutive windows folding problem, where psi(L) is shown to converge to O(1) under the assumption of a standard probabilistic polymer folding model, yielding an O(L) speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5') folding bias, i.e. that the minimal free energy (MFE) of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.
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The Mechanisms of Aberrant Protein Aggregation
Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas
2012-02-01
We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.
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Lee, Hyeon-Cheol; Portnoff, Alyse D; Rocco, Mark A; DeLisa, Matthew P
2014-12-22
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.
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Self-folding polymeric containers for encapsulation and delivery of drugs.
Fernandes, Rohan; Gracias, David H
2012-11-01
Self-folding broadly refers to self-assembly processes wherein thin films or interconnected planar templates curve, roll-up or fold into three dimensional (3D) structures such as cylindrical tubes, spirals, corrugated sheets or polyhedra. The process has been demonstrated with metallic, semiconducting and polymeric films and has been used to curve tubes with diameters as small as 2nm and fold polyhedra as small as 100nm, with a surface patterning resolution of 15nm. Self-folding methods are important for drug delivery applications since they provide a means to realize 3D, biocompatible, all-polymeric containers with well-tailored composition, size, shape, wall thickness, porosity, surface patterns and chemistry. Self-folding is also a highly parallel process, and it is possible to encapsulate or self-load therapeutic cargo during assembly. A variety of therapeutic cargos such as small molecules, peptides, proteins, bacteria, fungi and mammalian cells have been encapsulated in self-folded polymeric containers. In this review, we focus on self-folding of all-polymeric containers. We discuss the mechanistic aspects of self-folding of polymeric containers driven by differential stresses or surface tension forces, the applications of self-folding polymers in drug delivery and we outline future challenges. Copyright © 2012 Elsevier B.V. All rights reserved.
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Physiological Importance and Mechanisms of Protein Hydrolysate Absorption
Zhanghi, Brian M.; Matthews, James C.
Understanding opportunities to maximize the efficient digestion and assimilation by production animals of plant- and animal-derived protein products is critical for farmers, nutritionists, and feed manufacturers to sustain and expand the affordable production of high quality animal products for human consumption. The challenge to nutritionists is to match gastrointestinal tract load to existing or inducible digestive and absorptive capacities. The challenge to feed manufacturers is to develop products that are efficient substrates for digestion, absorption, and/or both events. Ultimately, the efficient absorption of digesta proteins depends on the mediated passage (transport) of protein hydrosylate products as dipeptides and unbound amino acids across the lumen- and blood-facing membranes of intestinal absorptive cells. Data testing the relative efficiency of supplying protein as hydrolysates or specific dipeptides versus as free amino acids, and the response of animals in several physiological states to feeding of protein hydrolysates, are presented and reviewed in this chapter. Next, data describing the transport mechanisms responsible for absorbing protein hydrolysate digestion products, and the known and putative regulation of these mechanisms by their substrates (small peptides) and hormones are presented and reviewed. Several conclusions are drawn regarding the efficient use of protein hydrolysate-based diets for particular physiological states, the economically-practical application of which likely will depend on technological advances in the manufacture of protein hydrolysate products.
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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...
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SDEM modelling of fault-propagation folding
DEFF Research Database (Denmark)
Clausen, O.R.; Egholm, D.L.; Poulsen, Jane Bang
2009-01-01
and variations in Mohr-Coulomb parameters including internal friction. Using SDEM modelling, we have mapped the propagation of the tip-line of the fault, as well as the evolution of the fold geometry across sedimentary layers of contrasting rheological parameters, as a function of the increased offset......Understanding the dynamics and kinematics of fault-propagation-folding is important for evaluating the associated hydrocarbon play, for accomplishing reliable section balancing (structural reconstruction), and for assessing seismic hazards. Accordingly, the deformation style of fault-propagation...... a precise indication of when faults develop and hence also the sequential evolution of secondary faults. Here we focus on the generation of a fault -propagated fold with a reverse sense of motion at the master fault, and varying only the dip of the master fault and the mechanical behaviour of the deformed...
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Engineering and Characterization of a Superfolder Green Fluorescent Protein
International Nuclear Information System (INIS)
Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.
2006-01-01
Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP
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Patra, Aditya Prasad; Sharma, Shobhona; Ainavarapu, Sri Rama Koti
2017-02-10
The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSP rep ), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSP ΔHP ). Our results show that the CSP rep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (∼70 pN). CSP ΔHP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Construction and characterization of a novel vocal fold bioreactor.
Zerdoum, Aidan B; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao
2014-08-01
In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.
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McDonald, Sarah K; Fleming, Karen G
2016-11-08
Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.
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High-resolution structure of a retroviral protease folded as a monomer
International Nuclear Information System (INIS)
Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz
2011-01-01
The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C α deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections
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Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.
2015-01-01
Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692
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A Soluble, Folded Protein without Charged Amino Acid Residues
DEFF Research Database (Denmark)
Højgaard, Casper; Kofoed, Christian; Espersen, Roall
2016-01-01
Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find...
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Effects of Polymer Hydrophobicity on Protein Structure and Aggregation Kinetics in Crowded Milieu.
Breydo, Leonid; Sales, Amanda E; Frege, Telma; Howell, Mark C; Zaslavsky, Boris Y; Uversky, Vladimir N
2015-05-19
We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect.
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Bukhari, Syed Abbas; Caetano-Anollés, Gustavo
2013-01-01
The spatial arrangements of secondary structures in proteins, irrespective of their connectivity, depict the overall shape and organization of protein domains. These features have been used in the CATH and SCOP classifications to hierarchically partition fold space and define the architectural make up of proteins. Here we use phylogenomic methods and a census of CATH structures in hundreds of genomes to study the origin and diversification of protein architectures (A) and their associated topologies (T) and superfamilies (H). Phylogenies that describe the evolution of domain structures and proteomes were reconstructed from the structural census and used to generate timelines of domain discovery. Phylogenies of CATH domains at T and H levels of structural abstraction and associated chronologies revealed patterns of reductive evolution, the early rise of Archaea, three epochs in the evolution of the protein world, and patterns of structural sharing between superkingdoms. Phylogenies of proteomes confirmed the early appearance of Archaea. While these findings are in agreement with previous phylogenomic studies based on the SCOP classification, phylogenies unveiled sharing patterns between Archaea and Eukarya that are recent and can explain the canonical bacterial rooting typically recovered from sequence analysis. Phylogenies of CATH domains at A level uncovered general patterns of architectural origin and diversification. The tree of A structures showed that ancient structural designs such as the 3-layer (αβα) sandwich (3.40) or the orthogonal bundle (1.10) are comparatively simpler in their makeup and are involved in basic cellular functions. In contrast, modern structural designs such as prisms, propellers, 2-solenoid, super-roll, clam, trefoil and box are not widely distributed and were probably adopted to perform specialized functions. Our timelines therefore uncover a universal tendency towards protein structural complexity that is remarkable. PMID:23555236
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Directory of Open Access Journals (Sweden)
Haina Qin
2014-07-01
Full Text Available Paradoxically, aggregation of specific proteins is characteristic of many human diseases and aging, yet aggregates have increasingly been found to be unnecessary for initiating pathogenesis. Here we determined the NMR topology and dynamics of a helical mutant in a membrane environment transformed from the 125-residue cytosolic all-β MSP domain of vesicle-associated membrane protein-associated protein B (VAPB by the ALS-causing P56S mutation. Despite its low hydrophobicity, the P56S major sperm protein (MSP domain becomes largely embedded in the membrane environment with high backbone rigidity. Furthermore it is composed of five helices with amphiphilicity comparable to those of the partly-soluble membrane toxin mellitin and α-synuclein causing Parkinson's disease. Consequently, the mechanism underlying this chameleon transformation becomes clear: by disrupting the specific tertiary interaction network stabilizing the native all-β MSP fold to release previously-locked amphiphilic segments, the P56S mutation acts to convert the classic MSP fold into a membrane-active protein that is fundamentally indistinguishable from mellitin and α-synuclein which are disordered in aqueous solution but spontaneously partition into membrane interfaces driven by hydrogen-bond energetics gained from forming α-helix in the membrane environments. As segments with high amphiphilicity exist in all proteins, our study successfully resolves the paradox by deciphering that the proteins with a higher tendency to aggregate have a stronger potential to partition into membranes through the same mechanism as α-synuclein to initially attack membranes to trigger pathogenesis without needing aggregates. This might represent the common first step for various kinds of aggregated proteins to trigger familiar, sporadic and aging diseases. Therefore the homeostasis of aggregated proteins in vivo is the central factor responsible for a variety of human diseases including aging
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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.
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Histopathologic study of human vocal fold mucosa unphonated over a decade.
Sato, Kiminori; Umeno, Hirohito; Ono, Takeharu; Nakashima, Tadashi
2011-12-01
Mechanotransduction caused by vocal fold vibration could possibly be an important factor in the maintenance of extracellular matrices and layered structure of the human adult vocal fold mucosa as a vibrating tissue after the layered structure has been completed. Vocal fold stellate cells (VFSCs) in the human maculae flavae of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Maculae flavae are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate the VFSCs to accelerate production of extracellular matrices. A human adult vocal fold mucosa unphonated over a decade was investigated histopathologically. Vocal fold mucosa unphonated for 11 years and 2 months of a 64-year-old male with cerebral hemorrhage was investigated by light and electron microscopy. The vocal fold mucosae (including maculae flavae) were atrophic. The vocal fold mucosa did not have a vocal ligament, Reinke's space or a layered structure. The lamina propria appeared as a uniform structure. Morphologically, the VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. Consequently, VFSCs appeared to decrease their level of activity.
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Directory of Open Access Journals (Sweden)
Ziv-Ukelson Michal
2009-03-01
Full Text Available Abstract Background Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L 3 by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2 solution for this problem has been described. Results Here, we describe and implement an O(NLψ(L engine for the consecutive windows folding problem, where ψ(L is shown to converge to O(1 under the assumption of a standard probabilistic polymer folding model, yielding an O(L speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5' folding bias, i.e. that the minimal free energy (MFE of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. Conclusion The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.
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Peripheral Protein Unfolding Drives Membrane Bending.
Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian
2018-06-20
Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.
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Folding and activity of hybrid sequence, disulfide-stabilized peptides
Energy Technology Data Exchange (ETDEWEB)
Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))
1990-08-01
Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.
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Folding and activity of hybrid sequence, disulfide-stabilized peptides
International Nuclear Information System (INIS)
Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.
1990-01-01
Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences
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Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)*
da Palma, Joel Ramos; Cendron, Laura; Seidah, Nabil Georges; Pasquato, Antonella; Kunz, Stefan
2016-01-01
The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process. PMID:26645686
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Multimodal imaging of vocal fold scarring in a rabbit model by multiphoton microscopy
Kazarine, Alexei; Bouhabel, Sarah; Douillette, Annie H.; Kost, Karen; Li-Jessen, Nicole Y. K.; Mongeau, Luc; Wiseman, Paul W.
2017-02-01
Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold's ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.
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The human PDI family: Versatility packed into a single fold
DEFF Research Database (Denmark)
Appenzeller-Herzog, Christian; Ellgaard, Lars
2007-01-01
in promoting oxidative protein folding in the ER has been extended in recent years to include roles in other processes such as ER-associated degradation (ERAD), trafficking, calcium homeostasis, antigen presentation and virus entry. Some of these functions are performed by non-catalytic members of the family...... that lack the active-site cysteines. Regardless of their function, all human PDIs contain at least one domain of approximately 100 amino acid residues with structural homology to thioredoxin. As we learn more about the individual proteins of the family, a complex picture is emerging that emphasizes as much...... their differences as their similarities, and underlines the versatility of the thioredoxin fold. Here, we primarily explore the diversity of cellular functions described for the human PDIs. Udgivelsesdato: 2007-Dec-3...
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DEFF Research Database (Denmark)
Tjernsbekk, M. T.; Tauson, A. H.; Kraugerud, O. F.
2017-01-01
Protein quality was evaluated for mechanically separated chicken meat (MSC) and salmon protein hydrolysate (SPH), and for extruded dog foods where MSC or SPH partially replaced poultry meal (PM). Apparent total tract digestibility (ATTD) of crude protein (CP) and amino acids (AA) in the protein...
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Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.
Zhang, Yongli
2017-07-01
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.
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Structures of two Arabidopsis thaliana major latex proteins represent novel helix-grip folds
Energy Technology Data Exchange (ETDEWEB)
Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, Jr., George N.; Volkman, Brian F.; (MCW); (UW)
2009-06-02
Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.
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Mailen, Russell W.; Dickey, Michael D.; Genzer, Jan; Zikry, Mohammed
2017-11-01
Shape memory polymer (SMP) sheets patterned with black ink hinges change shape in response to external stimuli, such as absorbed thermal energy from an infrared (IR) light. The geometry of these hinges, including size, orientation, and location, and the applied thermal loads significantly influence the final folded shape of the sheet, but these variables have not been fully investigated. We perform a systematic study on SMP sheets to fundamentally understand the effects of single and double hinge geometries, hinge orientation and spacing, initial temperature, heat flux intensity, and pattern width on the folding behavior. We have developed thermo-viscoelastic finite element models to characterize and quantify the stresses, strains, and temperatures as they relate to SMP shape changes. Our predictions indicate that hinge orientation can be used to reduce the total bending angle, which is the angle traversed by the folding face of the sheet. Two parallel hinges increase the total bending angle, and heat conduction between the hinges affects the transient folding response. IR intensity and initial temperatures can also influence the transient folding behavior. These results can provide guidelines to optimize the transient folding response and the three-dimensional folded structure obtained from self-folding polymer origami sheets that can be applied for myriad applications.
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Metal cofactor modulated folding and target recognition of HIV-1 NCp7.
Ren, Weitong; Ji, Dongqing; Xu, Xiulian
2018-01-01
The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.