Sample records for protein folding stability

  1. Role of naturally occurring osmolytes in protein folding and stability.

    PubMed

    Kumar, Raj

    2009-11-01

    Osmolytes are typically accumulated in the intracellular environment at relatively high concentrations when cells/tissues are subjected to stress conditions. Osmolytes are common in a variety of organisms, including microorganisms, plants, and animals. They enhance thermodynamic stability of proteins by providing natively folded conformations without perturbing other cellular processes. By burying the backbone into the core of folded proteins, osmolytes can provide significant stability to proteins. Two properties of osmolytes are particularly important: (i) their ability to impart increased thermodynamic stability to folded proteins; and (ii) their compatibility in the intracellular environment at high concentrations. Under physiological conditions, the cellular compositions of osmolytes may vary significantly. This may lead to different protein folding pathways utilized in cells depending upon the intracellular environment. Proper understanding of the role of osmolytes in cell regulation should allow predicting the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions.

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

    PubMed

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

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

  3. Confinement in nanopores can destabilize α-helix folding proteins and stabilize the β structures

    NASA Astrophysics Data System (ADS)

    Javidpour, Leili; Sahimi, Muhammad

    2011-09-01

    Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an α-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller α-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the β structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the α → β conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight

  4. Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

    PubMed Central

    Dadarlat, Voichita M.; Post, Carol Beth

    2016-01-01

    In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

  5. Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

    PubMed Central

    Bastolla, Ugo

    2014-01-01

    The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

  6. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  7. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.

    PubMed

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability.

  8. Influence of protein fold stability on immunogenicity and its implications for vaccine design

    PubMed Central

    Scheiblhofer, Sandra; Laimer, Josef; Machado, Yoan; Weiss, Richard; Thalhamer, Josef

    2017-01-01

    ABSTRACT Introduction: In modern vaccinology and immunotherapy, recombinant proteins more and more replace whole organisms to induce protective or curative immune responses. Structural stability of proteins is of crucial importance for efficient presentation of antigenic peptides on MHC, which plays a decisive role for triggering strong immune reactions. Areas covered: In this review, we discuss structural stability as a key factor for modulating the potency of recombinant vaccines and its importance for antigen proteolysis, presentation, and stimulation of B and T cells. Moreover, the impact of fold stability on downstream events determining the differentiation of T cells into effector cells is reviewed. We summarize studies investigating the impact of protein fold stability on the outcome of the immune response and provide an overview on computational methods to estimate the effects of point mutations on protein stability. Expert commentary: Based on this information, the rational design of up-to-date vaccines is discussed. A model for predicting immunogenicity of proteins based on their conformational stability at different pH values is proposed. PMID:28290225

  9. Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

    PubMed Central

    Colacino, Stefano; Tiana, Guido; Colombo, Giorgio

    2006-01-01

    Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms. PMID:16857062

  10. Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

    PubMed Central

    Steiner, Thomas; Hess, Petra; Bae, Jae Hyun; Wiltschi, Birgit; Moroder, Luis; Budisa, Nediljko

    2008-01-01

    Background Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the Cγ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cγ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides. Methodology/Principal Findings In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the Cγ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines. Conclusions/Significance We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the Cγ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and

  11. Folding and stability of helical bundle proteins from coarse-grained models.

    PubMed

    Kapoor, Abhijeet; Travesset, Alex

    2013-07-01

    We develop a coarse-grained model where solvent is considered implicitly, electrostatics are included as short-range interactions, and side-chains are coarse-grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side-chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics. Copyright © 2013 Wiley Periodicals, Inc.

  12. Stabilization of a protein conferred by an increase in folded state entropy.

    PubMed

    Dagan, Shlomi; Hagai, Tzachi; Gavrilov, Yulian; Kapon, Ruti; Levy, Yaakov; Reich, Ziv

    2013-06-25

    Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.

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

    NASA Astrophysics Data System (ADS)

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

    2018-01-01

    possible connection between the hydrophobic component of protein stability and the native structural topology. We simulated those same 200 targets again with the Mq A, only. However, this time we evaluated the relative frequency {ϕq } in which each target visits its corresponding native structure along an appropriate simulation time. Due to the presence of the hydrophobic effect in our approach we obtained a strong correlation between the stability and the folding rate (R = 0 . 85). So, as faster a sequence found its target, as larger is the hydrophobic component of its stability. The strong correlation fulfills our last goal. This final finding suggests that the hydrophobic effect could not be a general stabilizing factor for proteins.

  14. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  15. Folding and Stabilization of Native-Sequence-Reversed Proteins

    NASA Astrophysics Data System (ADS)

    Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

    2016-04-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

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

    NASA Astrophysics Data System (ADS)

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

    2018-05-01

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

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

    PubMed

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

    2013-12-13

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

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

    NASA Astrophysics Data System (ADS)

    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

  19. Electrostatic effects on the folding stability of FKBP

    NASA Astrophysics Data System (ADS)

    Batra, Jyotica; Zhou, Huan-Xiang

    2006-11-01

    Charged residues play important roles in the folding of proteins and their interactions with biological targets. We have developed computational models for predicting electrostatic contributions to protein folding and binding stability. To rigorously test and further refine these models, we carried out experimental studies on the effects of charge mutations on the folding stability of FKBP. Two close homologues of FKBP, FKBP12 and FKBP12.6, differ in 18 of 107 positions, and 8 of which involve substitutions of charged residues. These 8 substitutions were introduced on FKBP12 and their effects on the folding stability were measured. The changes in unfolding free energy varied from -0.34 to 0.65 kcal/mol. A double and a triple mutation were introduced to accumulate the stabilization effect of individual substitutions, resulting an increase in stability of about 0.84 kcal/mol. On the other hand, neutralizing one or both partners of a conserved salt bridge reduced the stability by as much as 0.64 kcal/mol. These results suggest that charged residues can modulate the folding stability significantly. To further exploit stabilization effects of charged residues, experiments are now underway to introduce charge mutations that are modeled after a thermophilic FKBP.

  20. Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2013-01-01

    This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

  1. The Role of High-Dimensional Diffusive Search, Stabilization, and Frustration in Protein Folding

    PubMed Central

    Rimratchada, Supreecha; McLeish, Tom C.B.; Radford, Sheena E.; Paci, Emanuele

    2014-01-01

    Proteins are polymeric molecules with many degrees of conformational freedom whose internal energetic interactions are typically screened to small distances. Therefore, in the high-dimensional conformation space of a protein, the energy landscape is locally relatively flat, in contrast to low-dimensional representations, where, because of the induced entropic contribution to the full free energy, it appears funnel-like. Proteins explore the conformation space by searching these flat subspaces to find a narrow energetic alley that we call a hypergutter and then explore the next, lower-dimensional, subspace. Such a framework provides an effective representation of the energy landscape and folding kinetics that does justice to the essential characteristic of high-dimensionality of the search-space. It also illuminates the important role of nonnative interactions in defining folding pathways. This principle is here illustrated using a coarse-grained model of a family of three-helix bundle proteins whose conformations, once secondary structure has formed, can be defined by six rotational degrees of freedom. Two folding mechanisms are possible, one of which involves an intermediate. The stabilization of intermediate subspaces (or states in low-dimensional projection) in protein folding can either speed up or slow down the folding rate depending on the amount of native and nonnative contacts made in those subspaces. The folding rate increases due to reduced-dimension pathways arising from the mere presence of intermediate states, but decreases if the contacts in the intermediate are very stable and introduce sizeable topological or energetic frustration that needs to be overcome. Remarkably, the hypergutter framework, although depending on just a few physically meaningful parameters, can reproduce all the types of experimentally observed curvature in chevron plots for realizations of this fold. PMID:24739172

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

    NASA Astrophysics Data System (ADS)

    Pappu, Rohit V.; Nussinov, Ruth

    2009-03-01

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

  3. Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.

    PubMed

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

    2012-02-07

    Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application. © 2011 American Chemical Society

  4. Protein folding and misfolding: mechanism and principles

    PubMed Central

    Englander, S. Walter; Mayne, Leland; Krishna, Mallela M. G.

    2012-01-01

    Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors

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

    PubMed

    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.

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

    PubMed

    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.

  7. Mutation Bias Favors Protein Folding Stability in the Evolution of Small Populations

    PubMed Central

    Porto, Markus; Bastolla, Ugo

    2010-01-01

    Mutation bias in prokaryotes varies from extreme adenine and thymine (AT) in obligatory endosymbiotic or parasitic bacteria to extreme guanine and cytosine (GC), for instance in actinobacteria. GC mutation bias deeply influences the folding stability of proteins, making proteins on the average less hydrophobic and therefore less stable with respect to unfolding but also less susceptible to misfolding and aggregation. We study a model where proteins evolve subject to selection for folding stability under given mutation bias, population size, and neutrality. We find a non-neutral regime where, for any given population size, there is an optimal mutation bias that maximizes fitness. Interestingly, this optimal GC usage is small for small populations, large for intermediate populations and around 50% for large populations. This result is robust with respect to the definition of the fitness function and to the protein structures studied. Our model suggests that small populations evolving with small GC usage eventually accumulate a significant selective advantage over populations evolving without this bias. This provides a possible explanation to the observation that most species adopting obligatory intracellular lifestyles with a consequent reduction of effective population size shifted their mutation spectrum towards AT. The model also predicts that large GC usage is optimal for intermediate population size. To test these predictions we estimated the effective population sizes of bacterial species using the optimal codon usage coefficients computed by dos Reis et al. and the synonymous to non-synonymous substitution ratio computed by Daubin and Moran. We found that the population sizes estimated in these ways are significantly smaller for species with small and large GC usage compared to species with no bias, which supports our prediction. PMID:20463869

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Global analysis of protein folding using massively parallel design, synthesis and testing

    PubMed Central

    Rocklin, Gabriel J.; Chidyausiku, Tamuka M.; Goreshnik, Inna; Ford, Alex; Houliston, Scott; Lemak, Alexander; Carter, Lauren; Ravichandran, Rashmi; Mulligan, Vikram K.; Chevalier, Aaron; Arrowsmith, Cheryl H.; Baker, David

    2017-01-01

    Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Though these forces are “encoded” in the thousands of known protein structures, “decoding” them is challenging due to the complexity of natural proteins that have evolved for function, not stability. Here we combine computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for over 15,000 de novo designed miniproteins, 1,000 natural proteins, 10,000 point-mutants, and 30,000 negative control sequences, identifying over 2,500 new stable designed proteins in four basic folds. This scale—three orders of magnitude greater than that of previous studies of design or folding—enabled us to systematically examine how sequence determines folding and stability in uncharted protein space. Iteration between design and experiment increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and experiment, and promises to transform computational protein design into a data-driven science. PMID:28706065

  10. Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

    PubMed Central

    Street, Timothy O; Barrick, Doug

    2009-01-01

    The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape-based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C-terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats. PMID:19177351

  11. General mechanism of two-state protein folding kinetics.

    PubMed

    Rollins, Geoffrey C; Dill, Ken A

    2014-08-13

    We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s.

  12. General Mechanism of Two-State Protein Folding Kinetics

    PubMed Central

    Rollins, Geoffrey C.; Dill, Ken A.

    2016-01-01

    We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s. PMID:25056406

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  16. Practical Approaches to Protein Folding and Assembly

    PubMed Central

    Walters, Jad; Milam, Sara L.; Clark, A. Clay

    2009-01-01

    We describe here the use of several spectroscopies, such as fluorescence emission, circular dichroism, and differential quenching by acrylamide, in examining the equilibrium and kinetic folding of proteins. The first section regarding equilibrium techniques provides practical information for determining the conformational stability of a protein. In addition, several equilibrium-folding models are discussed, from two-state monomer to four-state homodimer, providing a comprehensive protocol for interpretation of folding curves. The second section focuses on the experimental design and interpretation of kinetic data, such as burst-phase analysis and exponential fits, used in elucidating kinetic folding pathways. In addition, simulation programs are used routinely to support folding models generated by kinetic experiments, and the fundamentals of simulations are covered. PMID:19289201

  17. Roles of β-Turns in Protein Folding: From Peptide Models to Protein Engineering

    PubMed Central

    Marcelino, Anna Marie C.; Gierasch, Lila M.

    2010-01-01

    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. PMID:18275088

  18. Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli

    PubMed Central

    Pilipczuk, Justyna; Zalewska-Piątek, Beata; Bruździak, Piotr; Czub, Jacek; Wieczór, Miłosz; Olszewski, Marcin; Wanarska, Marta; Nowicki, Bogdan; Augustin-Nowacka, Danuta; Piątek, Rafał

    2017-01-01

    Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol−1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10−17 s−1 to 10−7 s−1. Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae. PMID:28739804

  19. Probing the Folding-Unfolding Transition of a Thermophilic Protein, MTH1880

    PubMed Central

    Jung, Youngjin; Han, Jeongmin; Yun, Ji-Hye; Chang, Iksoo; Lee, Weontae

    2016-01-01

    The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central β-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880. PMID:26766214

  20. Effect of interactions with the chaperonin cavity on protein folding and misfolding†

    PubMed Central

    Sirur, Anshul; Knott, Michael; Best, Robert B.

    2015-01-01

    Recent experimental and computational results have suggested that attractive interactions between a chaperonin and an enclosed substrate can have an important effect on the protein folding rate: it appears that folding may even be slower inside the cavity than under unconfined conditions, in contrast to what we would expect from excluded volume effects on the unfolded state. Here we examine systematically the dependence of the protein stability and folding rate on the strength of such attractive interactions between the chaperonin and substrate, by using molecular simulations of model protein systems in an idealised attractive cavity. Interestingly, we find a maximum in stability, and a rate which indeed slows down at high attraction strengths. We have developed a simple phenomenological model which can explain the variations in folding rate and stability due to differing effects on the free energies of the unfolded state, folded state, and transition state; changes in the diffusion coefficient along the folding coordinate are relatively small, at least for our simplified model. In order to investigate a possible role for these attractive interactions in folding, we have studied a recently developed model for misfolding in multidomain proteins. We find that, while encapsulation in repulsive cavities greatly increases the fraction of misfolded protein, sufficiently strong attractive protein-cavity interactions can strongly reduce the fraction of proteins reaching misfolded traps. PMID:24077053

  1. Evolutionary Strategies for Protein Folding

    NASA Astrophysics Data System (ADS)

    Murthy Gopal, Srinivasa; Wenzel, Wolfgang

    2006-03-01

    The free energy approach for predicting the protein tertiary structure describes the native state of a protein as the global minimum of an appropriate free-energy forcefield. The low-energy region of the free-energy landscape of a protein is extremely rugged. Efficient optimization methods must therefore speed up the search for the global optimum by avoiding high energy transition states, adapt large scale moves or accept unphysical intermediates. Here we investigate an evolutionary strategies(ES) for optimizing a protein conformation in our all-atom free-energy force field([1],[2]). A set of random conformations is evolved using an ES to get a diverse population containing low energy structure. The ES is shown to balance energy improvement and yet maintain diversity in structures. The ES is implemented as a master-client model for distributed computing. Starting from random structures and by using this optimization technique, we were able to fold a 20 amino-acid helical protein and 16 amino-acid beta hairpin[3]. We compare ES to basin hopping method. [1]T. Herges and W. Wenzel,Biophys.J. 87,3100(2004) [2] A. Verma and W. Wenzel Stabilization and folding of beta-sheet and alpha-helical proteins in an all-atom free energy model(submitted)(2005) [3] S. M. Gopal and W. Wenzel Evolutionary Strategies for Protein Folding (in preparation)

  2. Designing pH induced fold switch in proteins

    NASA Astrophysics Data System (ADS)

    Baruah, Anupaul; Biswas, Parbati

    2015-05-01

    This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

  3. Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

    PubMed Central

    Phillips, J J; Javadi, Y; Millership, C; Main, E R G

    2012-01-01

    Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium. PMID:22170589

  4. Probing Protein Fold Space with a Simplified Model

    PubMed Central

    Minary, Peter; Levitt, Michael

    2008-01-01

    We probe the stability and near-native energy landscape of protein fold space using powerful conformational sampling methods together with simple reduced models and statistical potentials. Fold space is represented by a set of 280 protein domains spanning all topological classes and having a wide range of lengths (0-300 residues), amino acid composition, and number of secondary structural elements. The degrees of freedom are taken as the loop torsion angles. This choice preserves the native secondary structure but allows the tertiary structure to change. The proteins are represented by three-point per residue, three-dimensional models with statistical potentials derived from a knowledge-based study of known protein structures. When this space is sampled by a combination of Parallel Tempering and Equi-Energy Monte Carlo, we find that the three-point model captures the known stability of protein native structures with stable energy basins that are near-native (all-α: 4.77 Å, all-β: 2.93 Å, α/β: 3.09 Å, α+β: 4.89 Å on average and within 6 Å for 71.41 %, 92.85 %, 94.29 % and 64.28 % for all-α, all-β, α/β and α+β, classes respectively). Denatured structures also occur and these have interesting structural properties that shed light on the different landscape characteristics of α and β folds. We find that α/β proteins with alternating α and β segments (such as the beta-barrel) are more stable than proteins in other fold classes. PMID:18054792

  5. Selection of stably folded proteins by phage-display with proteolysis.

    PubMed

    Bai, Yawen; Feng, Hanqiao

    2004-05-01

    To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

  6. Intermediates and the folding of proteins L and G

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brown, Scott; Head-Gordon, Teresa

    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 contactsmore » 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.« less

  7. Intermediates and the folding of proteins L and G

    PubMed Central

    Brown, Scott; Head-Gordon, Teresa

    2004-01-01

    We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which 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 β-1 and β-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 β-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. PMID:15044729

  8. Folding superfunnel to describe cooperative folding of interacting proteins.

    PubMed

    Smeller, László

    2016-07-01

    This paper proposes a generalization of the well-known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein-protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein-protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009-1016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Protein folding by NMR.

    PubMed

    Zhuravleva, Anastasia; Korzhnev, Dmitry M

    2017-05-01

    Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease. Copyright © 2016 Elsevier B.V. All

  10. Folding and stability of the isolated Greek key domains of the long-lived human lens proteins γD-crystallin and γS-crystallin

    PubMed Central

    Mills, Ishara A.; Flaugh, Shannon L.; Kosinski-Collins, Melissa S.; King, Jonathan A.

    2007-01-01

    The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric γ-crystallins and oligomeric β-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human γD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human γD-crystallin, as well as the isolated domains of human γS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37°C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human γD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein. PMID:17905830

  11. Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

    PubMed Central

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

    2012-01-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

  12. Effects of tethering a multistate folding protein to a surface

    NASA Astrophysics Data System (ADS)

    Wei, Shuai; Knotts, Thomas A.

    2011-05-01

    Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

  13. How cooperative are protein folding and unfolding transitions?

    PubMed Central

    Malhotra, Pooja

    2016-01-01

    Abstract A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding. PMID:27522064

  14. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  15. Evidence for the principle of minimal frustration in the evolution of protein folding landscapes.

    PubMed

    Tzul, Franco O; Vasilchuk, Daniel; Makhatadze, George I

    2017-02-28

    Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates. However, depending on the optimal living temperature of the organism, proteins also need to modulate their thermodynamic stability. Consequently, the difference in thermodynamic stability should be primarily caused by differences in the unfolding rates. To test this hypothesis experimentally, we performed comprehensive thermodynamic and kinetic analyses of 15 different proteins from the thioredoxin family. Eight of these thioredoxins were extant proteins from psychrophilic, mesophilic, or thermophilic organisms. The other seven protein sequences were obtained using ancestral sequence reconstruction and can be dated back over 4 billion years. We found that all studied proteins fold with very similar rates but unfold with rates that differ up to three orders of magnitude. The unfolding rates correlate well with the thermodynamic stability of the proteins. Moreover, proteins that unfold slower are more resistant to proteolysis. These results provide direct experimental support to the principle of minimal frustration hypothesis.

  16. Electrodynamic pressure modulation of protein stability in cosolvents.

    PubMed

    Damodaran, Srinivasan

    2013-11-19

    Cosolvents affect structural stability of proteins in aqueous solutions. A clear understanding of the mechanism by which cosolvents impact protein stability is critical to understanding protein folding in a biological milieu. In this study, we investigated the Lifshitz-van der Waals dispersion interaction of seven different solutes with nine globular proteins and report that in an aqueous medium the structure-stabilizing solutes exert a positive electrodynamic pressure, whereas the structure-destabilizing solutes exert a negative electrodynamic pressure on the proteins. The net increase in the thermal denaturation temperature (ΔTd) of a protein in 1 M solution of various solutes was linearly related to the electrodynamic pressure (PvdW) between the solutes and the protein. The slope of the PvdW versus ΔTd plots was protein-dependent. However, we find a positive linear relationship (r(2) = 0.79) between the slope (i.e., d(ΔTd)/dPvdW) and the adiabatic compressibility (βs) of the proteins. Together, these results clearly indicate that the Lifshitz's dispersion forces are inextricably involved in solute-induced stabilization/destabilization of globular proteins. The positive and/or negative electrodynamic pressure generated by the solute-protein interaction across the water medium seems to be the fundamental mechanism by which solutes affect protein stability. This is at variance with the existing preferential hydration concept. The implication of these results is significant in the sense that, in addition to the hydrophobic effect that drives protein folding, the electrodynamic forces between the proteins and solutes in the biological milieu also might play a role in the folding process as well as in the stability of the folded state.

  17. Topography of funneled landscapes determines the thermodynamics and kinetics of protein folding

    PubMed Central

    Wang, Jin; Oliveira, Ronaldo J.; Chu, Xiakun; Whitford, Paul C.; Chahine, Jorge; Han, Wei; Wang, Erkang; Onuchic, José N.; Leite, Vitor B.P.

    2012-01-01

    The energy landscape approach has played a fundamental role in advancing our understanding of protein folding. Here, we quantify protein folding energy landscapes by exploring the underlying density of states. We identify three quantities essential for characterizing landscape topography: the stabilizing energy gap between the native and nonnative ensembles δE, the energetic roughness ΔE, and the scale of landscape measured by the entropy S. We show that the dimensionless ratio between the gap, roughness, and entropy of the system accurately predicts the thermodynamics, as well as the kinetics of folding. Large Λ implies that the energy gap (or landscape slope towards the native state) is dominant, leading to more funneled landscapes. We investigate the role of topological and energetic roughness for proteins of different sizes and for proteins of the same size, but with different structural topologies. The landscape topography ratio Λ is shown to be monotonically correlated with the thermodynamic stability against trapping, as characterized by the ratio of folding temperature versus trapping temperature. Furthermore, Λ also monotonically correlates with the folding kinetic rates. These results provide the quantitative bridge between the landscape topography and experimental folding measurements. PMID:23019359

  18. Molecular dynamics studies of protein folding and aggregation

    NASA Astrophysics Data System (ADS)

    Ding, Feng

    that globular proteins under a denaturing environment partially unfold and aggregate by forming stabilizing hydrogen bonds between the backbones of the partial folded substructures. Proteins or peptides rich in alpha-helices also aggregate into beta-rich amyloid fibrils. Upon aggregation, the protein or peptide undergoes a conformational transition from alpha-helices to beta-sheets. The transition of alpha-helix to beta-hairpin (two-stranded beta-sheet) is studied in an all-heavy-atom discrete molecular dynamics model of a polyalanine chain. An entropical driving scenario for the alpha-helix to beta-hairpin transition is discovered.

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

    PubMed

    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.

  20. Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

    PubMed Central

    Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

    2014-01-01

    Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

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

    PubMed

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

    2014-10-01

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

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

    NASA Astrophysics Data System (ADS)

    Fitter, Jörg

    2003-08-01

    In a quasielastic neutron scattering experiment, the picosecond dynamics of α-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 2O-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 Å; unfolded state, 1.8 Å). 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.

  3. Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

    PubMed

    Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

    2018-05-03

    Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

  4. Principles of protein folding--a perspective from simple exact models.

    PubMed Central

    Dill, K. A.; Bromberg, S.; Yue, K.; Fiebig, K. M.; Yee, D. P.; Thomas, P. D.; Chan, H. S.

    1995-01-01

    General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse. PMID:7613459

  5. Energy landscape of knotted protein folding

    PubMed Central

    Sułkowska, Joanna I.; Noel, Jeffrey K.; Onuchic, Jose N.

    2012-01-01

    Recent experiments have conclusively shown that proteins are able to fold from an unknotted, denatured polypeptide to the knotted, native state without the aid of chaperones. These experiments are consistent with a growing body of theoretical work showing that a funneled, minimally frustrated energy landscape is sufficient to fold small proteins with complex topologies. Here, we present a theoretical investigation of the folding of a knotted protein, 2ouf, engineered in the laboratory by a domain fusion that mimics an evolutionary pathway for knotted proteins. Unlike a previously studied knotted protein of similar length, we see reversible folding/knotting and a surprising lack of deep topological traps with a coarse-grained structure-based model. Our main interest is to investigate how evolution might further select the geometry and stiffness of the threading region of the newly fused protein. We compare the folding of the wild-type protein to several mutants. Similarly to the wild-type protein, all mutants show robust and reversible folding, and knotting coincides with the transition state ensemble. As observed experimentally, our simulations show that the knotted protein folds about ten times slower than an unknotted construct with an identical contact map. Simulated folding kinetics reflect the experimentally observed rollover in the folding limbs of chevron plots. Successful folding of the knotted protein is restricted to a narrow range of temperature as compared to the unknotted protein and fits of the kinetic folding data below folding temperature suggest slow, nondiffusive dynamics for the knotted protein. PMID:22891304

  6. Inversion of the Balance between Hydrophobic and Hydrogen Bonding Interactions in Protein Folding and Aggregation

    PubMed Central

    Fitzpatrick, Anthony W.; Knowles, Tuomas P. J.; Waudby, Christopher A.; Vendruscolo, Michele; Dobson, Christopher M.

    2011-01-01

    Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions. PMID:22022239

  7. How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway

    PubMed Central

    Dyson, H. Jane; Wright, Peter E.

    2017-01-01

    Conspectus Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (~2 seconds) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ~ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies

  8. A lattice protein with an amyloidogenic latent state: stability and folding kinetics.

    PubMed

    Palyanov, Andrey Yu; Krivov, Sergei V; Karplus, Martin; Chekmarev, Sergei F

    2007-03-15

    We have designed a model lattice protein that has two stable folded states, the lower free energy native state and a latent state of somewhat higher energy. The two states have a sizable part of their structures in common (two "alpha-helices") and differ in the content of "alpha-helices" and "beta-strands" in the rest of their structures; i.e. for the native state, this part is alpha-helical, and for the latent state it is composed of beta-strands. Thus, the lattice protein free energy surface mimics that of amyloidogenic proteins that form well organized fibrils under appropriate conditions. A Go-like potential was used and the folding process was simulated with a Monte Carlo method. To gain insight into the equilibrium free energy surface and the folding kinetics, we have combined standard approaches (reduced free energy surfaces, contact maps, time-dependent populations of the characteristic states, and folding time distributions) with a new approach. The latter is based on a principal coordinate analysis of the entire set of contacts, which makes possible the introduction of unbiased reaction coordinates and the construction of a kinetic network for the folding process. The system is found to have four characteristic basins, namely a semicompact globule, an on-pathway intermediate (the bifurcation basin), and the native and latent states. The bifurcation basin is shallow and consists of the structure common to the native and latent states, with the rest disorganized. On the basis of the simulation results, a simple kinetic model describing the transitions between the characteristic states was developed, and the rate constants for the essential transitions were estimated. During the folding process the system dwells in the bifurcation basin for a relatively short time before it proceeds to the native or latent state. We suggest that such a bifurcation may occur generally for proteins in which native and latent states have a sizable part of their structures in

  9. A strategy for detecting the conservation of folding-nucleus residues in protein superfamilies.

    PubMed

    Michnick, S W; Shakhnovich, E

    1998-01-01

    Nucleation-growth theory predicts that fast-folding peptide sequences fold to their native structure via structures in a transition-state ensemble that share a small number of native contacts (the folding nucleus). Experimental and theoretical studies of proteins suggest that residues participating in folding nuclei are conserved among homologs. We attempted to determine if this is true in proteins with highly diverged sequences but identical folds (superfamilies). We describe a strategy based on comparisons of residue conservation in natural superfamily sequences with simulated sequences (generated with a Monte-Carlo sequence design strategy) for the same proteins. The basic assumptions of the strategy were that natural sequences will conserve residues needed for folding and stability plus function, the simulated sequences contain no functional conservation, and nucleus residues make native contacts with each other. Based on these assumptions, we identified seven potential nucleus residues in ubiquitin superfamily members. Non-nucleus conserved residues were also identified; these are proposed to be involved in stabilizing native interactions. We found that all superfamily members conserved the same potential nucleus residue positions, except those for which the structural topology is significantly different. Our results suggest that the conservation of the nucleus of a specific fold can be predicted by comparing designed simulated sequences with natural highly diverged sequences that fold to the same structure. We suggest that such a strategy could be used to help plan protein folding and design experiments, to identify new superfamily members, and to subdivide superfamilies further into classes having a similar folding mechanism.

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

    PubMed

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

    2017-10-01

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

  11. Extant fold-switching proteins are widespread.

    PubMed

    Porter, Lauren L; Looger, Loren L

    2018-06-05

    A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ∼100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ∼100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5-4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health. Copyright © 2018 the Author(s). Published by PNAS.

  12. FROM FOLDING THEORIES TO FOLDING PROTEINS: A Review and Assessment of Simulation Studies of Protein Folding and Unfolding

    NASA Astrophysics Data System (ADS)

    Shea, Joan-Emma; Brooks, Charles L., III

    2001-10-01

    Beginning with simplified lattice and continuum "minimalist" models and progressing to detailed atomic models, simulation studies have augmented and directed development of the modern landscape perspective of protein folding. In this review we discuss aspects of detailed atomic simulation methods applied to studies of protein folding free energy surfaces, using biased-sampling free energy methods and temperature-induced protein unfolding. We review studies from each on systems of particular experimental interest and assess the strengths and weaknesses of each approach in the context of "exact" results for both free energies and kinetics of a minimalist model for a beta-barrel protein. We illustrate in detail how each approach is implemented and discuss analysis methods that have been developed as components of these studies. We describe key insights into the relationship between protein topology and the folding mechanism emerging from folding free energy surface calculations. We further describe the determination of detailed "pathways" and models of folding transition states that have resulted from unfolding studies. Our assessment of the two methods suggests that both can provide, often complementary, details of folding mechanism and thermodynamics, but this success relies on (a) adequate sampling of diverse conformational regions for the biased-sampling free energy approach and (b) many trajectories at multiple temperatures for unfolding studies. Furthermore, we find that temperature-induced unfolding provides representatives of folding trajectories only when the topology and sequence (energy) provide a relatively funneled landscape and "off-pathway" intermediates do not exist.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Peptide folding in the presence of interacting protein crowders

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bille, Anna, E-mail: anna.bille@thep.lu.se; Irbäck, Anders, E-mail: anders@thep.lu.se; Mohanty, Sandipan, E-mail: s.mohanty@fz-juelich.de

    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 largermore » 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.« less

  15. Physics of protein folding

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  17. Visualizing chaperone-assisted protein folding

    DOE PAGES

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...

    2016-05-30

    We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

  18. A stoichiometry driven universal spatial organization of backbones of folded proteins: are there Chargaff's rules for protein folding?

    PubMed

    Mittal, A; Jayaram, B; Shenoy, Sandhya; Bawa, Tejdeep Singh

    2010-10-01

    Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that "preferential interactions" between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.

  19. A minimalist model protein with multiple folding funnels

    PubMed Central

    Locker, C. Rebecca; Hernandez, Rigoberto

    2001-01-01

    Kinetic and structural studies of wild-type proteins such as prions and amyloidogenic proteins provide suggestive evidence that proteins may adopt multiple long-lived states in addition to the native state. All of these states differ structurally because they lie far apart in configuration space, but their stability is not necessarily caused by cooperative (nucleation) effects. In this study, a minimalist model protein is designed to exhibit multiple long-lived states to explore the dynamics of the corresponding wild-type proteins. The minimalist protein is modeled as a 27-monomer sequence confined to a cubic lattice with three different monomer types. An order parameter—the winding index—is introduced to characterize the extent of folding. The winding index has several advantages over other commonly used order parameters like the number of native contacts. It can distinguish between enantiomers, its calculation requires less computational time than the number of native contacts, and reduced-dimensional landscapes can be developed when the native state structure is not known a priori. The results for the designed model protein prove by existence that the rugged energy landscape picture of protein folding can be generalized to include protein “misfolding” into long-lived states. PMID:11470921

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

    PubMed

    Wang, Moye; Hu, Jie; Zhang, Zhuqing

    2016-04-26

    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.

  1. Detecting protein folding by thermal fluctuations of microcantilevers

    PubMed Central

    Aguilar-Sandoval, Felipe; Bellon, Ludovic; Melo, Francisco

    2017-01-01

    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. PMID:29267316

  2. Energy landscape in protein folding and unfolding

    DOE PAGES

    Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; ...

    2016-03-08

    Protein folding represents an open question in science, and the free-energy landscape framework is one way to describe it. In particular, the role played by water in the processes is of special interest. To clarify these issues we study, during folding–unfolding, the temperature evolution of the magnetization for hydrophilic and hydrophobic groups of hydrated lysozyme using NMR spectroscopy. Our findings confirm the validity of the theoretical scenario of a process dominated by different energetic routes, also explaining the water role in the protein configuration stability. Here, we also highlight that the protein native state limit is represented by the watermore » singular temperature that characterizes its compressibility and expansivity and is the origin of the thermodynamical anomalies of its liquid state.« less

  3. Effective Potentials for Folding Proteins

    NASA Astrophysics Data System (ADS)

    Chen, Nan-Yow; Su, Zheng-Yao; Mou, Chung-Yu

    2006-02-01

    A coarse-grained off-lattice model that is not biased in any way to the native state is proposed to fold proteins. To predict the native structure in a reasonable time, the model has included the essential effects of water in an effective potential. Two new ingredients, the dipole-dipole interaction and the local hydrophobic interaction, are introduced and are shown to be as crucial as the hydrogen bonding. The model allows successful folding of the wild-type sequence of protein G and may have provided important hints to the study of protein folding.

  4. Accelerated molecular dynamics simulations of protein folding.

    PubMed

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

    2015-07-30

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

  5. Protein Folding Using a Vortex Fluidic Device.

    PubMed

    Britton, Joshua; Smith, Joshua N; Raston, Colin L; Weiss, Gregory A

    2017-01-01

    Essentially all biochemistry and most molecular biology experiments require recombinant proteins. However, large, hydrophobic proteins typically aggregate into insoluble and misfolded species, and are directed into inclusion bodies. Current techniques to fold proteins recovered from inclusion bodies rely on denaturation followed by dialysis or rapid dilution. Such approaches can be time consuming, wasteful, and inefficient. Here, we describe rapid protein folding using a vortex fluidic device (VFD). This process uses mechanical energy introduced into thin films to rapidly and efficiently fold proteins. With the VFD in continuous flow mode, large volumes of protein solution can be processed per day with 100-fold reductions in both folding times and buffer volumes.

  6. Exploring the Energy Landscapes of Protein Folding Simulations with Bayesian Computation

    PubMed Central

    Burkoff, Nikolas S.; Várnai, Csilla; Wells, Stephen A.; Wild, David L.

    2012-01-01

    Nested sampling is a Bayesian sampling technique developed to explore probability distributions localized in an exponentially small area of the parameter space. The algorithm provides both posterior samples and an estimate of the evidence (marginal likelihood) of the model. The nested sampling algorithm also provides an efficient way to calculate free energies and the expectation value of thermodynamic observables at any temperature, through a simple post processing of the output. Previous applications of the algorithm have yielded large efficiency gains over other sampling techniques, including parallel tempering. In this article, we describe a parallel implementation of the nested sampling algorithm and its application to the problem of protein folding in a Gō-like force field of empirical potentials that were designed to stabilize secondary structure elements in room-temperature simulations. We demonstrate the method by conducting folding simulations on a number of small proteins that are commonly used for testing protein-folding procedures. A topological analysis of the posterior samples is performed to produce energy landscape charts, which give a high-level description of the potential energy surface for the protein folding simulations. These charts provide qualitative insights into both the folding process and the nature of the model and force field used. PMID:22385859

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

    PubMed Central

    Wang, Moye; Hu, Jie; Zhang, Zhuqing

    2016-01-01

    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. PMID:27128902

  8. Cooperativity and modularity in protein folding

    PubMed Central

    Sasai, Masaki; Chikenji, George; Terada, Tomoki P.

    2016-01-01

    A simple statistical mechanical model proposed by Wako and Saitô has explained the aspects of protein folding surprisingly well. This model was systematically applied to multiple proteins by Muñoz and Eaton and has since been referred to as the Wako-Saitô-Muñoz-Eaton (WSME) model. The success of the WSME model in explaining the folding of many proteins has verified the hypothesis that the folding is dominated by native interactions, which makes the energy landscape globally biased toward native conformation. Using the WSME and other related models, Saitô emphasized the importance of the hierarchical pathway in protein folding; folding starts with the creation of contiguous segments having a native-like configuration and proceeds as growth and coalescence of these segments. The Φ-values calculated for barnase with the WSME model suggested that segments contributing to the folding nucleus are similar to the structural modules defined by the pattern of native atomic contacts. The WSME model was extended to explain folding of multi-domain proteins having a complex topology, which opened the way to comprehensively understanding the folding process of multi-domain proteins. The WSME model was also extended to describe allosteric transitions, indicating that the allosteric structural movement does not occur as a deterministic sequential change between two conformations but as a stochastic diffusive motion over the dynamically changing energy landscape. Statistical mechanical viewpoint on folding, as highlighted by the WSME model, has been renovated in the context of modern methods and ideas, and will continue to provide insights on equilibrium and dynamical features of proteins. PMID:28409080

  9. Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

    PubMed Central

    Romero-Romero, M. Luisa; Inglés-Prieto, Alvaro; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2011-01-01

    Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions. PMID:21829584

  10. Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein†

    PubMed Central

    Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

    2013-01-01

    Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in mixed micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22. PMID:23639031

  11. Protein Folding and Self-Organized Criticality

    NASA Astrophysics Data System (ADS)

    Bajracharya, Arun; Murray, Joelle

    Proteins are known to fold into tertiary structures that determine their functionality in living organisms. However, the complex dynamics of protein folding and the way they consistently fold into the same structures is not fully understood. Self-organized criticality (SOC) has provided a framework for understanding complex systems in various systems (earthquakes, forest fires, financial markets, and epidemics) through scale invariance and the associated power law behavior. In this research, we use a simple hydrophobic-polar lattice-bound computational model to investigate self-organized criticality as a possible mechanism for generating complexity in protein folding.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Simplified Protein Models: Predicting Folding Pathways and Structure Using Amino Acid Sequences

    NASA Astrophysics Data System (ADS)

    Adhikari, Aashish N.; Freed, Karl F.; Sosnick, Tobin R.

    2013-07-01

    We demonstrate the ability of simultaneously determining a protein’s folding pathway and structure using a properly formulated model without prior knowledge of the native structure. Our model employs a natural coordinate system for describing proteins and a search strategy inspired by the observation that real proteins fold in a sequential fashion by incrementally stabilizing nativelike substructures or “foldons.” Comparable folding pathways and structures are obtained for the twelve proteins recently studied using atomistic molecular dynamics simulations [K. Lindorff-Larsen, S. Piana, R. O. Dror, D. E. Shaw, Science 334, 517 (2011)], with our calculations running several orders of magnitude faster. We find that nativelike propensities in the unfolded state do not necessarily determine the order of structure formation, a departure from a major conclusion of the molecular dynamics study. Instead, our results support a more expansive view wherein intrinsic local structural propensities may be enhanced or overridden in the folding process by environmental context. The success of our search strategy validates it as an expedient mechanism for folding both in silico and in vivo.

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

    PubMed

    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

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

    PubMed

    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.

  16. In vivo protein stabilization based on fragment complementation and a split GFP system.

    PubMed

    Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

    2010-11-16

    Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.

  17. Improving Protein Fold Recognition by Deep Learning Networks

    NASA Astrophysics Data System (ADS)

    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.

  18. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

    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.

  19. Three key residues form a critical contact network in a protein folding transition state

    NASA Astrophysics Data System (ADS)

    Vendruscolo, Michele; Paci, Emanuele; Dobson, Christopher M.; Karplus, Martin

    2001-02-01

    Determining how a protein folds is a central problem in structural biology. The rate of folding of many proteins is determined by the transition state, so that a knowledge of its structure is essential for understanding the protein folding reaction. Here we use mutation measurements-which determine the role of individual residues in stabilizing the transition state-as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures that make up the transition state. We apply this approach to the experimental data for the 98-residue protein acylphosphatase, and obtain a transition-state ensemble with the native-state topology and an average root-mean-square deviation of 6Å from the native structure. Although about 20 residues with small positional fluctuations form the structural core of this transition state, the native-like contact network of only three of these residues is sufficient to determine the overall fold of the protein. This result reveals how a nucleation mechanism involving a small number of key residues can lead to folding of a polypeptide chain to its unique native-state structure.

  20. Exploring the energy landscapes of protein folding simulations with Bayesian computation.

    PubMed

    Burkoff, Nikolas S; Várnai, Csilla; Wells, Stephen A; Wild, David L

    2012-02-22

    Nested sampling is a Bayesian sampling technique developed to explore probability distributions localized in an exponentially small area of the parameter space. The algorithm provides both posterior samples and an estimate of the evidence (marginal likelihood) of the model. The nested sampling algorithm also provides an efficient way to calculate free energies and the expectation value of thermodynamic observables at any temperature, through a simple post processing of the output. Previous applications of the algorithm have yielded large efficiency gains over other sampling techniques, including parallel tempering. In this article, we describe a parallel implementation of the nested sampling algorithm and its application to the problem of protein folding in a Gō-like force field of empirical potentials that were designed to stabilize secondary structure elements in room-temperature simulations. We demonstrate the method by conducting folding simulations on a number of small proteins that are commonly used for testing protein-folding procedures. A topological analysis of the posterior samples is performed to produce energy landscape charts, which give a high-level description of the potential energy surface for the protein folding simulations. These charts provide qualitative insights into both the folding process and the nature of the model and force field used. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Picosecond to nanosecond dynamics provide a source of conformational entropy for protein folding.

    PubMed

    Stadler, Andreas M; Demmel, Franz; Ollivier, Jacques; Seydel, Tilo

    2016-08-03

    Myoglobin can be trapped in fully folded structures, partially folded molten globules, and unfolded states under stable equilibrium conditions. Here, we report an experimental study on the conformational dynamics of different folded conformational states of apo- and holomyoglobin in solution. Global protein diffusion and internal molecular motions were probed by neutron time-of-flight and neutron backscattering spectroscopy on the picosecond and nanosecond time scales. Global protein diffusion was found to depend on the α-helical content of the protein suggesting that charges on the macromolecule increase the short-time diffusion of protein. With regard to the molten globules, a gel-like phase due to protein entanglement and interactions with neighbouring macromolecules was visible due to a reduction of the global diffusion coefficients on the nanosecond time scale. Diffusion coefficients, residence and relaxation times of internal protein dynamics and root mean square displacements of localised internal motions were determined for the investigated structural states. The difference in conformational entropy ΔSconf of the protein between the unfolded and the partially or fully folded conformations was extracted from the measured root mean square displacements. Using thermodynamic parameters from the literature and the experimentally determined ΔSconf values we could identify the entropic contribution of the hydration shell ΔShydr of the different folded states. Our results point out the relevance of conformational entropy of the protein and the hydration shell for stability and folding of myoglobin.

  2. Prediction of protein mutant stability using classification and regression tool.

    PubMed

    Huang, Liang-Tsung; Saraboji, K; Ho, Shinn-Ying; Hwang, Shiow-Fen; Ponnuswamy, M N; Gromiha, M Michael

    2007-02-01

    Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.

  3. Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

    PubMed Central

    Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

    2017-01-01

    Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be

  4. N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

    PubMed

    Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

    2009-03-23

    N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

  5. Solvent Effects on Protein Folding/Unfolding

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    PubMed

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

    2017-05-09

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

  8. Mapping the energy landscape for second-stage folding of a single membrane protein

    PubMed Central

    Min, Duyoung; Jefferson, Robert E; Bowie, James U; Yoon, Tae-Young

    2016-01-01

    Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ~3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. PMID:26479439

  9. Modulation of protein stability and aggregation properties by surface charge engineering.

    PubMed

    Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

    2013-09-01

    An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

  10. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins

    PubMed Central

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2017-01-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. PMID:23035660

  11. Chevron Behavior and Isostable Enthalpic Barriers in Protein Folding: Successes and Limitations of Simple Gō-like Modeling

    PubMed Central

    Kaya, Hüseyin; Liu, Zhirong; Chan, Hue Sun

    2005-01-01

    It has been demonstrated that a “near-Levinthal” cooperative mechanism, whereby the common Gō interaction scheme is augmented by an extra favorability for the native state as a whole, can lead to apparent two-state folding/unfolding kinetics over a broad range of native stabilities in lattice models of proteins. Here such a mechanism is shown to be generalizable to a simplified continuum (off-lattice) Langevin dynamics model with a Cα protein chain representation, with the resulting chevron plots exhibiting an extended quasilinear regime reminiscent of that of apparent two-state real proteins. Similarly high degrees of cooperativity are possible in Gō-like continuum models with rudimentary pairwise desolvation barriers as well. In these models, cooperativity increases with increasing desolvation barrier height, suggesting strongly that two-state-like folding/unfolding kinetics would be achievable when the pairwise desolvation barrier becomes sufficiently high. Besides cooperativity, another generic folding property of interest that has emerged from published experiments on several apparent two-state proteins is that their folding relaxation under constant native stability (isostability) conditions is essentially Arrhenius, entailing high intrinsic enthalpic folding barriers of ∼17–30 kcal/mol. Based on a new analysis of published data on barnase, here we propose that a similar property should also apply to a certain class of non-two-state proteins that fold with chevron rollovers. However, several continuum Gō-like constructs considered here fail to predict any significant intrinsic enthalpic folding barrier under isostability conditions; thus the physical origin of such barriers in real proteins remains to be elucidated. PMID:15863486

  12. Mining sequential patterns for protein fold recognition.

    PubMed

    Exarchos, Themis P; Papaloukas, Costas; Lampros, Christos; Fotiadis, Dimitrios I

    2008-02-01

    Protein data contain discriminative patterns that can be used in many beneficial applications if they are defined correctly. In this work sequential pattern mining (SPM) is utilized for sequence-based fold recognition. Protein classification in terms of fold recognition plays an important role in computational protein analysis, since it can contribute to the determination of the function of a protein whose structure is unknown. Specifically, one of the most efficient SPM algorithms, cSPADE, is employed for the analysis of protein sequence. A classifier uses the extracted sequential patterns to classify proteins in the appropriate fold category. For training and evaluating the proposed method we used the protein sequences from the Protein Data Bank and the annotation of the SCOP database. The method exhibited an overall accuracy of 25% in a classification problem with 36 candidate categories. The classification performance reaches up to 56% when the five most probable protein folds are considered.

  13. Different Members of a Simple Three-Helix Bundle Protein Family Have Very Different Folding Rate Constants and Fold by Different Mechanisms

    PubMed Central

    Wensley, Beth G.; Gärtner, Martina; Choo, Wan Xian; Batey, Sarah; Clarke, Jane

    2009-01-01

    The 15th, 16th, and 17th repeats of chicken brain α-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected. The rate-limiting transition state for R15 folding is investigated using protein engineering methods (Φ-value analysis) and compared with previously completed analyses of R16 and R17. Characterisation of many mutants suggests that all three proteins have similar complexity in the folding landscape. The early rate-limiting transition states of the three domains are similar in terms of overall structure, but there are significant differences in the patterns of Φ-values. R15 apparently folds via a nucleation–condensation mechanism, which involves concomitant folding and packing of the A- and C-helices, establishing the correct topology. R16 and R17 fold via a more framework-like mechanism, which may impede the search to find the correct packing of the helices, providing a possible explanation for the fast folding of R15. PMID:19445951

  14. Frustration in Condensed Matter and Protein Folding

    NASA Astrophysics Data System (ADS)

    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.

  15. Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

    PubMed Central

    Crespo, Maria D.; Rubini, Marina

    2011-01-01

    Background Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cγ-exo or a Cγ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cγ-exo puckering. Methodology/Principal Findings While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the Cγ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol−1 in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub. Conclusions/Significance The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein. PMID:21625626

  16. Progress towards mapping the universe of protein folds

    PubMed Central

    Grant, Alastair; Lee, David; Orengo, Christine

    2004-01-01

    Although the precise aims differ between the various international structural genomics initiatives currently aiming to illuminate the universe of protein folds, many selectively target protein families for which the fold is unknown. How well can the current set of known protein families and folds be used to estimate the total number of folds in nature, and will structural genomics initiatives yield representatives for all the major protein families within a reasonable time scale? PMID:15128436

  17. Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

    PubMed Central

    Zhukov, I.; Jaroszewski, L.; Bierzyński, A.

    2000-01-01

    Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process. PMID:10716179

  18. De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints

    PubMed Central

    Smock, Robert G.; Yadid, Itamar; Dym, Orly; Clarke, Jane; Tawfik, Dan S.

    2016-01-01

    Summary Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence. PMID:26806127

  19. Protein folding on the ribosome studied using NMR spectroscopy

    PubMed Central

    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

  20. Principal component analysis for protein folding dynamics.

    PubMed

    Maisuradze, Gia G; Liwo, Adam; Scheraga, Harold A

    2009-01-09

    Protein folding is considered here by studying the dynamics of the folding of the triple beta-strand WW domain from the Formin-binding protein 28. Starting from the unfolded state and ending either in the native or nonnative conformational states, trajectories are generated with the coarse-grained united residue (UNRES) force field. The effectiveness of principal components analysis (PCA), an already established mathematical technique for finding global, correlated motions in atomic simulations of proteins, is evaluated here for coarse-grained trajectories. The problems related to PCA and their solutions are discussed. The folding and nonfolding of proteins are examined with free-energy landscapes. Detailed analyses of many folding and nonfolding trajectories at different temperatures show that PCA is very efficient for characterizing the general folding and nonfolding features of proteins. It is shown that the first principal component captures and describes in detail the dynamics of a system. Anomalous diffusion in the folding/nonfolding dynamics is examined by the mean-square displacement (MSD) and the fractional diffusion and fractional kinetic equations. The collisionless (or ballistic) behavior of a polypeptide undergoing Brownian motion along the first few principal components is accounted for.

  1. On the Role of Entropy in the Protein Folding Process

    NASA Astrophysics Data System (ADS)

    Hoppe, Travis

    2011-12-01

    A protein's ultimate function and activity is determined by the unique three-dimensional structure taken by the folding process. Protein malfunction due to misfolding is the culprit of many clinical disorders, such as abnormal protein aggregations. This leads to neurodegenerative disorders like Huntington's and Alzheimer's disease. We focus on a subset of the folding problem, exploring the role and effects of entropy on the process of protein folding. Four major concepts and models are developed and each pertains to a specific aspect of the folding process: entropic forces, conformational states under crowding, aggregation, and macrostate kinetics from microstate trajectories. The exclusive focus on entropy is well-suited for crowding studies, as many interactions are nonspecific. We show how a stabilizing entropic force can arise purely from the motion of crowders in solution. In addition we are able to make a a quantitative prediction of the crowding effect with an implicit crowding approximation using an aspherical scaled-particle theory. In order to investigate the effects of aggregation, we derive a new operator expansion method to solve the Ising/Potts model with external fields over an arbitrary graph. Here the external fields are representative of the entropic forces. We show that this method reduces the problem of calculating the partition function to the solution of recursion relations. Many of the methods employed are coarse-grained approximations. As such, it is useful to have a viable method for extracting macrostate information from time series data. We develop a method to cluster the microstates into physically meaningful macrostates by grouping similar relaxation times from a transition matrix. Overall, the studied topics allow us to understand deeper the complicated process involving proteins.

  2. Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

    PubMed Central

    Gruber, Tobias; Balbach, Jochen

    2015-01-01

    The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. PMID:26368922

  3. Solvent viscosity and friction in protein folding dynamics.

    PubMed

    Hagen, Stephen J

    2010-08-01

    The famous Kramers rate theory for diffusion-controlled reactions has been extended in numerous ways and successfully applied to many types of reactions. Its application to protein folding reactions has been of particular interest in recent years, as many researchers have performed experiments and simulations to test whether folding reactions are diffusion-controlled, whether the solvent is the source of the reaction friction, and whether the friction-dependence of folding rates generally can provide insight into folding dynamics. These experiments involve many practical difficulties, however. They have also produced some unexpected results. Here we briefly review the Kramers theory for reactions in the presence of strong friction and summarize some of the subtle problems that arise in the application of the theory to protein folding. We discuss how the results of these experiments ultimately point to a significant role for internal friction in protein folding dynamics. Studies of friction in protein folding, far from revealing any weakness in Kramers theory, may actually lead to new approaches for probing diffusional dynamics and energy landscapes in protein folding.

  4. Method of generating ploynucleotides encoding enhanced folding variants

    DOEpatents

    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.

  5. In vitro folding of inclusion body proteins.

    PubMed

    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.

  6. Heterochiral Knottin Protein: Folding and Solution Structure.

    PubMed

    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.

  7. Interstitial protein alterations in rabbit vocal fold with scar.

    PubMed

    Thibeault, Susan L; Bless, Diane M; Gray, Steven D

    2003-09-01

    Fibrous and interstitial proteins compose the extracellular matrix of the vocal fold lamina propria and account for its biomechanic properties. Vocal fold scarring is characterized by altered biomechanical properties, which create dysphonia. Although alterations of the fibrous proteins have been confirmed in the rabbit vocal fold scar, interstitial proteins, which are known to be important in wound repair, have not been investigated to date. Using a rabbit model, interstitial proteins decorin, fibromodulin, and fibronectin were examined immunohistologically, two months postinduction of vocal fold scar by means of forcep biopsy. Significantly decreased decorin and fibromodulin with significantly increased fibronectin characterized scarred vocal fold tissue. The implications of altered interstitial proteins levels and their affect on the fibrous proteins will be discussed in relation to increased vocal fold stiffness and viscosity, which characterizes vocal fold scar.

  8. Stability and the Evolvability of Function in a Model Protein

    PubMed Central

    Bloom, Jesse D.; Wilke, Claus O.; Arnold, Frances H.; Adami, Christoph

    2004-01-01

    Functional proteins must fold with some minimal stability to a structure that can perform a biochemical task. Here we use a simple model to investigate the relationship between the stability requirement and the capacity of a protein to evolve the function of binding to a ligand. Although our model contains no built-in tradeoff between stability and function, proteins evolved function more efficiently when the stability requirement was relaxed. Proteins with both high stability and high function evolved more efficiently when the stability requirement was gradually increased than when there was constant selection for high stability. These results show that in our model, the evolution of function is enhanced by allowing proteins to explore sequences corresponding to marginally stable structures, and that it is easier to improve stability while maintaining high function than to improve function while maintaining high stability. Our model also demonstrates that even in the absence of a fundamental biophysical tradeoff between stability and function, the speed with which function can evolve is limited by the stability requirement imposed on the protein. PMID:15111394

  9. Cold denaturation as a tool to measure protein stability

    PubMed Central

    Sanfelice, Domenico; Temussi, Piero Andrea

    2016-01-01

    Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves. PMID:26026885

  10. Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins

    PubMed Central

    Liu, Zhenxing; Reddy, Govardhan; O’Brien, Edward P.; Thirumalai, D.

    2011-01-01

    Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained Cα-side chain model for polypeptide chains to simulate the folding of src SH3 domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln kobs (kobs is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (mf is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations. PMID:21512127

  11. Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins.

    PubMed

    Liu, Zhenxing; Reddy, Govardhan; O'Brien, Edward P; Thirumalai, D

    2011-05-10

    Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained C(α)-side chain model for polypeptide chains to simulate the folding of src SH(3) domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln k(obs) (k(obs) is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (m(f) is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations.

  12. Retarded protein folding of deficient human α1-antitrypsin D256V and L41P variants

    PubMed Central

    Jung, Chan-Hun; Na, Yu-Ran; Im, Hana

    2004-01-01

    α1-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human α1-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant α1-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient α1-antitrypsin variants. Unlike most α1-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing α1-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional. PMID:14767073

  13. Evolution of a protein folding nucleus.

    PubMed

    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.

  14. Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

    DOE PAGES

    Shi, Jade; Nobrega, R. Paul; Schwantes, Christian; ...

    2017-03-08

    The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. We report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structuremore » of the excited state ensemble. The resulting prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. We then predict incisive single molecule FRET experiments, using these results, as a means of model validation. Our study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.« less

  15. Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

    NASA Astrophysics Data System (ADS)

    Shi, Jade; Nobrega, R. Paul; Schwantes, Christian; Kathuria, Sagar V.; Bilsel, Osman; Matthews, C. Robert; Lane, T. J.; Pande, Vijay S.

    2017-03-01

    The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

  16. Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP.

    PubMed

    Iyer, Bharat Ramasubramanian; Mahalakshmi, Radhakrishnan

    2016-09-06

    The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gram-negative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. In addition, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix to folding and stability and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequence-dependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the W78F substitution in S. typhimurium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to that of E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

  17. Role of Hydrophobic Clusters and Long-Range Contact Networks in the Folding of (α/β)8 Barrel Proteins

    PubMed Central

    Selvaraj, S.; Gromiha, M. Michael

    2003-01-01

    Analysis on the three dimensional structures of (α/β)8 barrel proteins provides ample light to understand the factors that are responsible for directing and maintaining their common fold. In this work, the hydrophobically enriched clusters are identified in 92% of the considered (α/β)8 barrel proteins. The residue segments with hydrophobic clusters have high thermal stability. Further, these clusters are formed and stabilized through long-range interactions. Specifically, a network of long-range contacts connects adjacent β-strands of the (α/β)8 barrel domain and the hydrophobic clusters. The implications of hydrophobic clusters and long-range networks in providing a feasible common mechanism for the folding of (α/β)8 barrel proteins are proposed. PMID:12609894

  18. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  19. Energetics of side-chain partitioning of β-signal residues in unassisted folding of a transmembrane β-barrel protein

    PubMed Central

    Iyer, Bharat Ramasubramanian; Zadafiya, Punit; Vetal, Pallavi Vijay

    2017-01-01

    The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain. PMID:28592485

  20. Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

    PubMed

    Buchner, Ginka S; Murphy, Ronan D; Buchete, Nicolae-Viorel; Kubelka, Jan

    2011-08-01

    The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at

  1. A First-Principles Model of Early Evolution: Emergence of Gene Families, Species, and Preferred Protein Folds

    PubMed Central

    Zeldovich, Konstantin B; Chen, Peiqiu; Shakhnovich, Boris E; Shakhnovich, Eugene I

    2007-01-01

    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. PMID:17630830

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

    PubMed

    Zeldovich, Konstantin B; Chen, Peiqiu; Shakhnovich, Boris E; Shakhnovich, Eugene I

    2007-07-01

    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.

  3. Functional modulation of a protein folding landscape via side-chain distortion

    PubMed Central

    Kelch, Brian A.; Salimi, Neema L.; Agard, David A.

    2012-01-01

    Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions. PMID:22635267

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

    PubMed

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

    2016-01-01

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

  5. Solitons and protein folding: An In Silico experiment

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    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.

  6. The Safety Dance: Biophysics of Membrane Protein Folding and Misfolding in a Cellular Context

    PubMed Central

    Schlebach, Jonathan P.; Sanders, Charles R.

    2015-01-01

    Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine. PMID:25420508

  7. Small protein domains fold inside the ribosome exit tunnel.

    PubMed

    Marino, Jacopo; von Heijne, Gunnar; Beckmann, Roland

    2016-03-01

    Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel. © 2016 Federation of European Biochemical Societies.

  8. The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

    PubMed Central

    Komives, Elizabeth A.; Wolynes, Peter G.

    2008-01-01

    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 (ΔGwater) 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. PMID:18483553

  9. Protein folding on Biosensor tips: Folding of Maltodextrin glucosidase monitored by its interactions with GroEL

    PubMed Central

    Pastor, Ashutosh; Singh, Amit K.; Fisher, Mark T.; Chaudhuri, Tapan K.

    2016-01-01

    Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution. PMID:27367928

  10. Frustration in Condensed Matter and Protein Folding

    NASA Astrophysics Data System (ADS)

    Lorelli, S.; Cabot, A.; Sundarprasad, N.; Boekema, C.

    Using computer modeling we study frustration in condensed matter and protein folding. Frustration is due to random and/or competing interactions. One definition of frustration is the sum of squares of the differences between actual and expected distances between characters. If this sum is non-zero, then the system is said to have frustration. A simulation tracks the movement of characters to lower their frustration. Our research is conducted on frustration as a function of temperature using a logarithmic scale. At absolute zero, the relaxation for frustration is a power function for randomly assigned patterns or an exponential function for regular patterns like Thomson figures. These findings have implications for protein folding; we attempt to apply our frustration modeling to protein folding and dynamics. We use coding in Python to simulate different ways a protein can fold. An algorithm is being developed to find the lowest frustration (and thus energy) states possible. Research supported by SJSU & AFC.

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

    PubMed Central

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

    2007-01-01

    Background 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. Results 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 . 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 significantly improves

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

    PubMed Central

    Barrick, Doug

    2011-01-01

    Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

  14. Metamorphic Proteins: Emergence of Dual Protein Folds from One Primary Sequence.

    PubMed

    Lella, Muralikrishna; Mahalakshmi, Radhakrishnan

    2017-06-20

    Every amino acid exhibits a different propensity for distinct structural conformations. Hence, decoding how the primary amino acid sequence undergoes the transition to a defined secondary structure and its final three-dimensional fold is presently considered predictable with reasonable certainty. However, protein sequences that defy the first principles of secondary structure prediction (they attain two different folds) have recently been discovered. Such proteins, aptly named metamorphic proteins, decrease the conformational constraint by increasing flexibility in the secondary structure and thereby result in efficient functionality. In this review, we discuss the major factors driving the conformational switch related both to protein sequence and to structure using illustrative examples. We discuss the concept of an evolutionary transition in sequence and structure, the functional impact of the tertiary fold, and the pressure of intrinsic and external factors that give rise to metamorphic proteins. We mainly focus on the major components of protein architecture, namely, the α-helix and β-sheet segments, which are involved in conformational switching within the same or highly similar sequences. These chameleonic sequences are widespread in both cytosolic and membrane proteins, and these folds are equally important for protein structure and function. We discuss the implications of metamorphic proteins and chameleonic peptide sequences in de novo peptide design.

  15. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

    PubMed Central

    Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

    2016-01-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally—a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process. PMID:26887592

  16. SeqRate: sequence-based protein folding type classification and rates prediction

    PubMed Central

    2010-01-01

    Background Protein folding rate is an important property of a protein. Predicting protein folding rate is useful for understanding protein folding process and guiding protein design. Most previous methods of predicting protein folding rate require the tertiary structure of a protein as an input. And most methods do not distinguish the different kinetic nature (two-state folding or multi-state folding) of the proteins. Here we developed a method, SeqRate, to predict both protein folding kinetic type (two-state versus multi-state) and real-value folding rate using sequence length, amino acid composition, contact order, contact number, and secondary structure information predicted from only protein sequence with support vector machines. Results We systematically studied the contributions of individual features to folding rate prediction. On a standard benchmark dataset, the accuracy of folding kinetic type classification is 80%. The Pearson correlation coefficient and the mean absolute difference between predicted and experimental folding rates (sec-1) in the base-10 logarithmic scale are 0.81 and 0.79 for two-state protein folders, and 0.80 and 0.68 for three-state protein folders. SeqRate is the first sequence-based method for protein folding type classification and its accuracy of fold rate prediction is improved over previous sequence-based methods. Its performance can be further enhanced with additional information, such as structure-based geometric contacts, as inputs. Conclusions Both the web server and software of predicting folding rate are publicly available at http://casp.rnet.missouri.edu/fold_rate/index.html. PMID:20438647

  17. Improvement on a simplified model for protein folding simulation.

    PubMed

    Zhang, Ming; Chen, Changjun; He, Yi; Xiao, Yi

    2005-11-01

    Improvements were made on a simplified protein model--the Ramachandran model-to achieve better computer simulation of protein folding. To check the validity of such improvements, we chose the ultrafast folding protein Engrailed Homeodomain as an example and explored several aspects of its folding. The engrailed homeodomain is a mainly alpha-helical protein of 61 residues from Drosophila melanogaster. We found that the simplified model of Engrailed Homeodomain can fold into a global minimum state with a tertiary structure in good agreement with its native structure.

  18. Experimental investigation of protein folding and misfolding.

    PubMed

    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.

  19. Outer Membrane Protein Folding and Topology from a Computational Transfer Free Energy Scale.

    PubMed

    Lin, Meishan; Gessmann, Dennis; Naveed, Hammad; Liang, Jie

    2016-03-02

    Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane β-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic core, and its NC-IN topology is determined by the differential stabilities of OMPs in the asymmetrical outer membrane. The folding free energy is further reduced by lipid A and assisted by general depth-dependent cooperativities that exist between polar and ionizable residues. Moreover, context-dependency of transfer free energies at specific positions in OmpLA predict regions important for protein function as well as structural anomalies. Our computational approach is fast, efficient and applicable to any OMP.

  20. Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

    PubMed Central

    Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

    2011-01-01

    To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

  1. Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer.

    PubMed

    Guinn, Emily J; Pegram, Laurel M; Capp, Michael W; Pollock, Michelle N; Record, M Thomas

    2011-10-11

    To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K(p) for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K(p) values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH--amide O and amide NH--amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K(p) values.

  2. Dodging the crisis of folding proteins with knots

    NASA Astrophysics Data System (ADS)

    Sulkowska, Joanna

    2009-03-01

    Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, which involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in around 1-2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies.

  3. Design and structure of an equilibrium protein folding intermediate: a hint into dynamical regions of proteins.

    PubMed

    Ayuso-Tejedor, Sara; Angarica, Vladimir Espinosa; Bueno, Marta; Campos, Luis A; Abián, Olga; Bernadó, Pau; Sancho, Javier; Jiménez, M Angeles

    2010-07-23

    Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  4. Replica exchange molecular dynamics simulation of structure variation from α/4β-fold to 3α-fold protein.

    PubMed

    Lazim, Raudah; Mei, Ye; Zhang, Dawei

    2012-03-01

    Replica exchange molecular dynamics (REMD) simulation provides an efficient conformational sampling tool for the study of protein folding. In this study, we explore the mechanism directing the structure variation from α/4β-fold protein to 3α-fold protein after mutation by conducting REMD simulation on 42 replicas with temperatures ranging from 270 K to 710 K. The simulation began from a protein possessing the primary structure of GA88 but the tertiary structure of GB88, two G proteins with "high sequence identity." Albeit the large Cα-root mean square deviation (RMSD) of the folded protein (4.34 Å at 270 K and 4.75 Å at 304 K), a variation in tertiary structure was observed. Together with the analysis of secondary structure assignment, cluster analysis and principal component, it provides insights to the folding and unfolding pathway of 3α-fold protein and α/4β-fold protein respectively paving the way toward the understanding of the ongoings during conformational variation.

  5. Conformational stability as a design target to control protein aggregation.

    PubMed

    Costanzo, Joseph A; O'Brien, Christopher J; Tiller, Kathryn; Tamargo, Erin; Robinson, Anne Skaja; Roberts, Christopher J; Fernandez, Erik J

    2014-05-01

    Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

  6. Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO).

    PubMed

    Ma, Jianqiang; Pazos, Ileana M; Gai, Feng

    2014-06-10

    Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.

  7. Navigating ligand protein binding free energy landscapes: universality and diversity of protein folding and molecular recognition mechanisms

    NASA Astrophysics Data System (ADS)

    Verkhivker, Gennady M.; Rejto, Paul A.; Bouzida, Djamal; Arthurs, Sandra; Colson, Anthony B.; Freer, Stephan T.; Gehlhaar, Daniel K.; Larson, Veda; Luty, Brock A.; Marrone, Tami; Rose, Peter W.

    2001-03-01

    Thermodynamic and kinetic aspects of ligand-protein binding are studied for the methotrexate-dihydrofolate reductase system from the binding free energy profile constructed as a function of the order parameter. Thermodynamic stability of the native complex and a cooperative transition to the unique native structure suggest the nucleation kinetic mechanism at the equilibrium transition temperature. Structural properties of the transition state ensemble and the ensemble of nucleation conformations are determined by kinetic simulations of the transmission coefficient and ligand-protein association pathways. Structural analysis of the transition states and the nucleation conformations reconciles different views on the nucleation mechanism in protein folding.

  8. Polymer Uncrossing and Knotting in Protein Folding, and Their Role in Minimal Folding Pathways

    PubMed Central

    Mohazab, Ali R.; Plotkin, Steven S.

    2013-01-01

    We introduce a method for calculating the extent to which chain non-crossing is important in the most efficient, optimal trajectories or pathways for a protein to fold. This involves recording all unphysical crossing events of a ghost chain, and calculating the minimal uncrossing cost that would have been required to avoid such events. A depth-first tree search algorithm is applied to find minimal transformations to fold , , , and knotted proteins. In all cases, the extra uncrossing/non-crossing distance is a small fraction of the total distance travelled by a ghost chain. Different structural classes may be distinguished by the amount of extra uncrossing distance, and the effectiveness of such discrimination is compared with other order parameters. It was seen that non-crossing distance over chain length provided the best discrimination between structural and kinetic classes. The scaling of non-crossing distance with chain length implies an inevitable crossover to entanglement-dominated folding mechanisms for sufficiently long chains. We further quantify the minimal folding pathways by collecting the sequence of uncrossing moves, which generally involve leg, loop, and elbow-like uncrossing moves, and rendering the collection of these moves over the unfolded ensemble as a multiple-transformation “alignment”. The consensus minimal pathway is constructed and shown schematically for representative cases of an , , and knotted protein. An overlap parameter is defined between pathways; we find that proteins have minimal overlap indicating diverse folding pathways, knotted proteins are highly constrained to follow a dominant pathway, and proteins are somewhere in between. Thus we have shown how topological chain constraints can induce dominant pathway mechanisms in protein folding. PMID:23365638

  9. GroEL stimulates protein folding through forced unfolding

    PubMed Central

    Lin, Zong; Madan, Damian; Rye, Hays S

    2013-01-01

    Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding. PMID:18311152

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

    PubMed

    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.

  11. Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

    PubMed Central

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

    2017-01-01

    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. PMID:28218913

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

    PubMed

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

    2013-10-01

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

  13. Semiempirical prediction of protein folds

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel; Colubri, Andrés; Appignanesi, Gustavo

    2001-08-01

    We introduce a semiempirical approach to predict ab initio expeditious pathways and native backbone geometries of proteins that fold under in vitro renaturation conditions. The algorithm is engineered to incorporate a discrete codification of local steric hindrances that constrain the movements of the peptide backbone throughout the folding process. Thus, the torsional state of the chain is assumed to be conditioned by the fact that hopping from one basin of attraction to another in the Ramachandran map (local potential energy surface) of each residue is energetically more costly than the search for a specific (Φ, Ψ) torsional state within a single basin. A combinatorial procedure is introduced to evaluate coarsely defined torsional states of the chain defined ``modulo basins'' and translate them into meaningful patterns of long range interactions. Thus, an algorithm for structure prediction is designed based on the fact that local contributions to the potential energy may be subsumed into time-evolving conformational constraints defining sets of restricted backbone geometries whereupon the patterns of nonbonded interactions are constructed. The predictive power of the algorithm is assessed by (a) computing ab initio folding pathways for mammalian ubiquitin that ultimately yield a stable structural pattern reproducing all of its native features, (b) determining the nucleating event that triggers the hydrophobic collapse of the chain, and (c) comparing coarse predictions of the stable folds of moderately large proteins (N~100) with structural information extracted from the protein data bank.

  14. A specific transition state for S-peptide combining with folded S-protein and then refolding

    PubMed Central

    Goldberg, Jonathan M.; Baldwin, Robert L.

    1999-01-01

    We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the α-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the α-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of −0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state. PMID:10051587

  15. Designed protein reveals structural determinants of extreme kinetic stability

    PubMed Central

    Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

    2015-01-01

    The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

  16. From laws of inference to protein folding dynamics.

    PubMed

    Tseng, Chih-Yuan; Yu, Chun-Ping; Lee, H C

    2010-08-01

    Protein folding dynamics is one of major issues constantly investigated in the study of protein functions. The molecular dynamic (MD) simulation with the replica exchange method (REM) is a common theoretical approach considered. Yet a trade-off in applying the REM is that the dynamics toward the native configuration in the simulations seems lost. In this work, we show that given REM-MD simulation results, protein folding dynamics can be directly derived from laws of inference. The applicability of the resulting approach, the entropic folding dynamics, is illustrated by investigating a well-studied Trp-cage peptide. Our results are qualitatively comparable with those from other studies. The current studies suggest that the incorporation of laws of inference and physics brings in a comprehensive perspective on exploring the protein folding dynamics.

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

    PubMed Central

    2017-01-01

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

  18. Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

    PubMed

    Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

    2017-09-01

    Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Coarse-grained sequences for protein folding and design

    PubMed Central

    Brown, Scott; Fawzi, Nicolas J.; Head-Gordon, Teresa

    2003-01-01

    We present the results of sequence design on our off-lattice minimalist model in which no specification of native-state tertiary contacts is needed. We start with a sequence that adopts a target topology and build on it through sequence mutation to produce new sequences that comprise distinct members within a target fold class. In this work, we use the α/β ubiquitin fold class and design two new sequences that, when characterized through folding simulations, reproduce the differences in folding mechanism seen experimentally for proteins L and G. The primary implication of this work is that patterning of hydrophobic and hydrophilic residues is the physical origin for the success of relative contact-order descriptions of folding, and that these physics-based potentials provide a predictive connection between free energy landscapes and amino acid sequence (the original protein folding problem). We present results of the sequence mapping from a 20- to the three-letter code for determining a sequence that folds into the WW domain topology to illustrate future extensions to protein design. PMID:12963815

  20. Coarse-grained sequences for protein folding and design.

    PubMed

    Brown, Scott; Fawzi, Nicolas J; Head-Gordon, Teresa

    2003-09-16

    We present the results of sequence design on our off-lattice minimalist model in which no specification of native-state tertiary contacts is needed. We start with a sequence that adopts a target topology and build on it through sequence mutation to produce new sequences that comprise distinct members within a target fold class. In this work, we use the alpha/beta ubiquitin fold class and design two new sequences that, when characterized through folding simulations, reproduce the differences in folding mechanism seen experimentally for proteins L and G. The primary implication of this work is that patterning of hydrophobic and hydrophilic residues is the physical origin for the success of relative contact-order descriptions of folding, and that these physics-based potentials provide a predictive connection between free energy landscapes and amino acid sequence (the original protein folding problem). We present results of the sequence mapping from a 20- to the three-letter code for determining a sequence that folds into the WW domain topology to illustrate future extensions to protein design.

  1. Solvent effect on the folding dynamics and structure of E6-associated protein characterized from ab initio protein folding simulations

    NASA Astrophysics Data System (ADS)

    Xu, Zhijun; Lazim, Raudah; Sun, Tiedong; Mei, Ye; Zhang, Dawei

    2012-04-01

    Solvent effect on protein conformation and folding mechanism of E6-associated protein (E6ap) peptide are investigated using a recently developed charge update scheme termed as adaptive hydrogen bond-specific charge (AHBC). On the basis of the close agreement between the calculated helix contents from AHBC simulations and experimental results, we observed based on the presented simulations that the two ends of the peptide may simultaneously take part in the formation of the helical structure at the early stage of folding and finally merge to form a helix with lowest backbone RMSD of about 0.9 Å in 40% 2,2,2-trifluoroethanol solution. However, in pure water, the folding may start at the center of the peptide sequence instead of at the two opposite ends. The analysis of the free energy landscape indicates that the solvent may determine the folding clusters of E6ap, which subsequently leads to the different final folded structure. The current study demonstrates new insight to the role of solvent in the determination of protein structure and folding dynamics.

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

    PubMed

    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.

  3. Tailoring in vitro evolution for protein affinity or stability

    PubMed Central

    Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

    2001-01-01

    We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

  4. Characterization of protein-folding pathways by reduced-space modeling.

    PubMed

    Kmiecik, Sebastian; Kolinski, Andrzej

    2007-07-24

    Ab initio simulations of the folding pathways are currently limited to very small proteins. For larger proteins, some approximations or simplifications in protein models need to be introduced. Protein folding and unfolding are among the basic processes in the cell and are very difficult to characterize in detail by experiment or simulation. Chymotrypsin inhibitor 2 (CI2) and barnase are probably the best characterized experimentally in this respect. For these model systems, initial folding stages were simulated by using CA-CB-side chain (CABS), a reduced-space protein-modeling tool. CABS employs knowledge-based potentials that proved to be very successful in protein structure prediction. With the use of isothermal Monte Carlo (MC) dynamics, initiation sites with a residual structure and weak tertiary interactions were identified. Such structures are essential for the initiation of the folding process through a sequential reduction of the protein conformational space, overcoming the Levinthal paradox in this manner. Furthermore, nucleation sites that initiate a tertiary interactions network were located. The MC simulations correspond perfectly to the results of experimental and theoretical research and bring insights into CI2 folding mechanism: unambiguous sequence of folding events was reported as well as cooperative substructures compatible with those obtained in recent molecular dynamics unfolding studies. The correspondence between the simulation and experiment shows that knowledge-based potentials are not only useful in protein structure predictions but are also capable of reproducing the folding pathways. Thus, the results of this work significantly extend the applicability range of reduced models in the theoretical study of proteins.

  5. Disulfide bonds in ER protein folding and homeostasis

    PubMed Central

    Feige, Matthias J.; Hendershot, Linda M.

    2010-01-01

    Proteins that are expressed outside the cell must be synthesized, folded and assembled in a way that ensures they can function in their designate location. Accordingly these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to their formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery. PMID:21144725

  6. Transiently disordered tails accelerate folding of globular proteins.

    PubMed

    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.

  7. Protein vivisection reveals elusive intermediates in folding

    PubMed Central

    Zheng, Zhongzhou; Sosnick, Tobin R.

    2010-01-01

    Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin, reversibly trapping a folding intermediate in which the β5 strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high energy states. PMID:20144618

  8. Simulating protein folding initiation sites using an alpha-carbon-only knowledge-based force field

    PubMed Central

    Buck, Patrick M.; Bystroff, Christopher

    2015-01-01

    Protein folding is a hierarchical process where structure forms locally first, then globally. Some short sequence segments initiate folding through strong structural preferences that are independent of their three-dimensional context in proteins. We have constructed a knowledge-based force field in which the energy functions are conditional on local sequence patterns, as expressed in the hidden Markov model for local structure (HMMSTR). Carbon-alpha force field (CALF) builds sequence specific statistical potentials based on database frequencies for α-carbon virtual bond opening and dihedral angles, pairwise contacts and hydrogen bond donor-acceptor pairs, and simulates folding via Brownian dynamics. We introduce hydrogen bond donor and acceptor potentials as α-carbon probability fields that are conditional on the predicted local sequence. Constant temperature simulations were carried out using 27 peptides selected as putative folding initiation sites, each 12 residues in length, representing several different local structure motifs. Each 0.6 μs trajectory was clustered based on structure. Simulation convergence or representativeness was assessed by subdividing trajectories and comparing clusters. For 21 of the 27 sequences, the largest cluster made up more than half of the total trajectory. Of these 21 sequences, 14 had cluster centers that were at most 2.6 Å root mean square deviation (RMSD) from their native structure in the corresponding full-length protein. To assess the adequacy of the energy function on nonlocal interactions, 11 full length native structures were relaxed using Brownian dynamics simulations. Equilibrated structures deviated from their native states but retained their overall topology and compactness. A simple potential that folds proteins locally and stabilizes proteins globally may enable a more realistic understanding of hierarchical folding pathways. PMID:19137613

  9. Automated selection of stabilizing mutations in designed and natural proteins.

    PubMed

    Borgo, Benjamin; Havranek, James J

    2012-01-31

    The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins.

  10. Automated selection of stabilizing mutations in designed and natural proteins

    PubMed Central

    Borgo, Benjamin; Havranek, James J.

    2012-01-01

    The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins. PMID:22307603

  11. Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

    PubMed

    Vuillard, L; Rabilloud, T; Goldberg, M E

    1998-08-15

    Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

  12. Machinery of protein folding and unfolding.

    PubMed

    Zhang, Xiaodong; Beuron, Fabienne; Freemont, Paul S

    2002-04-01

    During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding. It has emerged that dual functionality in terms of folding and unfolding might exist for some systems. The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.

  13. Mechanisms of protein-folding diseases at a glance.

    PubMed

    Valastyan, Julie S; Lindquist, Susan

    2014-01-01

    For a protein to function appropriately, it must first achieve its proper conformation and location within the crowded environment inside the cell. Multiple chaperone systems are required to fold proteins correctly. In addition, degradation pathways participate by destroying improperly folded proteins. The intricacy of this multisystem process provides many opportunities for error. Furthermore, mutations cause misfolded, nonfunctional forms of proteins to accumulate. As a result, many pathological conditions are fundamentally rooted in the protein-folding problem that all cells must solve to maintain their function and integrity. Here, to illustrate the breadth of this phenomenon, we describe five examples of protein-misfolding events that can lead to disease: improper degradation, mislocalization, dominant-negative mutations, structural alterations that establish novel toxic functions, and amyloid accumulation. In each case, we will highlight current therapeutic options for battling such diseases.

  14. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

    NASA Astrophysics Data System (ADS)

    Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

    2016-11-01

    The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

  15. Thermodynamics of coupled protein adsorption and stability using hybrid Monte Carlo simulations.

    PubMed

    Zhong, Ellen D; Shirts, Michael R

    2014-05-06

    A better understanding of changes in protein stability upon adsorption can improve the design of protein separation processes. In this study, we examine the coupling of the folding and the adsorption of a model protein, the B1 domain of streptococcal protein G, as a function of surface attraction using a hybrid Monte Carlo (HMC) approach with temperature replica exchange and umbrella sampling. In our HMC implementation, we are able to use a molecular dynamics (MD) time step that is an order of magnitude larger than in a traditional MD simulation protocol and observe a factor of 2 enhancement in the folding and unfolding rate. To demonstrate the convergence of our systems, we measure the travel of our order parameter the fraction of native contacts between folded and unfolded states throughout the length of our simulations. Thermodynamic quantities are extracted with minimum statistical variance using multistate reweighting between simulations at different temperatures and harmonic distance restraints from the surface. The resultant free energies, enthalpies, and entropies of the coupled unfolding and absorption processes are in qualitative agreement with previous experimental and computational observations, including entropic stabilization of the adsorbed, folded state relative to the bulk on surfaces with low attraction.

  16. Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape.

    PubMed

    Adamcik, Jozef; Mezzenga, Raffaele

    2018-02-15

    Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on- or off-pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon↛crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Evolution, Energy Landscapes and the Paradoxes of Protein Folding

    PubMed Central

    Wolynes, Peter G.

    2014-01-01

    Protein folding has been viewed as a difficult problem of molecular self-organization. The search problem involved in folding however has been simplified through the evolution of folding energy landscapes that are funneled. The funnel hypothesis can be quantified using energy landscape theory based on the minimal frustration principle. Strong quantitative predictions that follow from energy landscape theory have been widely confirmed both through laboratory folding experiments and from detailed simulations. Energy landscape ideas also have allowed successful protein structure prediction algorithms to be developed. The selection constraint of having funneled folding landscapes has left its imprint on the sequences of existing protein structural families. Quantitative analysis of co-evolution patterns allows us to infer the statistical characteristics of the folding landscape. These turn out to be consistent with what has been obtained from laboratory physicochemical folding experiments signalling a beautiful confluence of genomics and chemical physics. PMID:25530262

  18. PconsFold: improved contact predictions improve protein models.

    PubMed

    Michel, Mirco; Hayat, Sikander; Skwark, Marcin J; Sander, Chris; Marks, Debora S; Elofsson, Arne

    2014-09-01

    Recently it has been shown that the quality of protein contact prediction from evolutionary information can be improved significantly if direct and indirect information is separated. Given sufficiently large protein families, the contact predictions contain sufficient information to predict the structure of many protein families. However, since the first studies contact prediction methods have improved. Here, we ask how much the final models are improved if improved contact predictions are used. In a small benchmark of 15 proteins, we show that the TM-scores of top-ranked models are improved by on average 33% using PconsFold compared with the original version of EVfold. In a larger benchmark, we find that the quality is improved with 15-30% when using PconsC in comparison with earlier contact prediction methods. Further, using Rosetta instead of CNS does not significantly improve global model accuracy, but the chemistry of models generated with Rosetta is improved. PconsFold is a fully automated pipeline for ab initio protein structure prediction based on evolutionary information. PconsFold is based on PconsC contact prediction and uses the Rosetta folding protocol. Due to its modularity, the contact prediction tool can be easily exchanged. The source code of PconsFold is available on GitHub at https://www.github.com/ElofssonLab/pcons-fold under the MIT license. PconsC is available from http://c.pcons.net/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  19. Evolutionary Dynamics on Protein Bi-stability Landscapes can Potentially Resolve Adaptive Conflicts

    PubMed Central

    Sikosek, Tobias; Bornberg-Bauer, Erich; Chan, Hue Sun

    2012-01-01

    Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bi-stable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009, 106:21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed. PMID:23028272

  20. Importance of α–helix N–capping motif in stabilization of ββα fold

    PubMed Central

    Koscielska-Kasprzak, Katarzyna; Cierpicki, Tomasz; Otlewski, Jacek

    2003-01-01

    FSD-1 (full sequence design 1) is a protein folded in a ββα motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S13RSDH17) into FSD-1 sequence in order to obtain a small DNA-binding module devoid of cross-link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N-capping motif of the α-helix, we analyzed conformational and stability properties of eight FSD-1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17→His substitution at the N3 in S13NEKE17 variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal • mole−1 destabilization) by disruption of N-capping interactions, and had the most significant effect among single mutants studied here. The Ncap Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal • mole−1 destabilization) by disruption of a helix-capping hydrogen bond and destabilization of a helix macrodipole. The N1 Glu 15→Ser mutation also produced a considerable effect (1.0 kcal • mole−1 destabilization), again emphasizing the significance of electrostatic interactions in α-helix stabilization. PMID:12761399

  1. Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    PubMed Central

    Warepam, Marina; Sharma, Gurumayum Suraj; Dar, Tanveer Ali; Khan, Md. Khurshid Alam; Singh, Laishram Rajendrakumar

    2014-01-01

    Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K m and k cat), thermodynamic stability (T m and ΔH m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes. PMID:25313668

  2. Absolute comparison of simulated and experimental protein-folding dynamics

    NASA Astrophysics Data System (ADS)

    Snow, Christopher D.; Nguyen, Houbi; Pande, Vijay S.; Gruebele, Martin

    2002-11-01

    Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as α-helices and β-hairpins can form in 0.1-10µs (ref. 1), whereas small proteins have been shown to fold completely in tens of microseconds. The longest folding simulation to date is a single 1-µs simulation of the villin headpiece; however, such single runs may miss many features of the folding process as it is a heterogeneous reaction involving an ensemble of transition states. Here, we have used a distributed computing implementation to produce tens of thousands of 5-20-ns trajectories (700µs) to simulate mutants of the designed mini-protein BBA5. The fast relaxation dynamics these predict were compared with the results of laser temperature-jump experiments. Our computational predictions are in excellent agreement with the experimentally determined mean folding times and equilibrium constants. The rapid folding of BBA5 is due to the swift formation of secondary structure. The convergence of experimentally and computationally accessible timescales will allow the comparison of absolute quantities characterizing in vitro and in silico (computed) protein folding.

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

    PubMed

    Getov, Ivan; Petukh, Marharyta; Alexov, Emil

    2016-04-07

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

  4. Assessment of local friction in protein folding dynamics using a helix cross-linker.

    PubMed

    Markiewicz, Beatrice N; Jo, Hyunil; Culik, Robert M; DeGrado, William F; Gai, Feng

    2013-11-27

    Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4-1.0k(B)T.

  5. Predicting stability of alpha-helical, orthogonal-bundle proteins on surfaces

    NASA Astrophysics Data System (ADS)

    Wei, Shuai; Knotts, Thomas A.

    2010-09-01

    The interaction of proteins with surfaces is a key phenomenon in many applications, but current understanding of the biophysics involved is lacking. At present, rational design of such emerging technologies is difficult as no methods or theories exist that correctly predict how surfaces influence protein behavior. Using molecular simulation and a coarse-grain model, this study illustrates for the first time that stability of proteins on surfaces can be correlated with tertiary structural elements for alpha-helical, orthogonal-bundle proteins. Results show that several factors contribute to stability on surfaces including the nature of the loop region where the tether is placed and the ability of the protein to freely rotate on the surface. A thermodynamic analysis demonstrates that surfaces stabilize proteins entropically and that any destabilization is an enthalpic effect. Moreover, the entropic effects are concentrated on the unfolded state of the protein while the ethalpic effects are focused on the folded state.

  6. Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

    PubMed Central

    Calosci, Nicoletta; Chi, Celestine N.; Richter, Barbara; Camilloni, Carlo; Engström, Åke; Eklund, Lars; Travaglini-Allocatelli, Carlo; Gianni, Stefano; Vendruscolo, Michele; Jemth, Per

    2008-01-01

    The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous three-state proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of Φ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state. PMID:19033470

  7. Examining a Thermodynamic Order Parameter of Protein Folding.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

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

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

    PubMed Central

    Muñoz, Victor; Cerminara, Michele

    2016-01-01

    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. PMID:27574021

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

    NASA Astrophysics Data System (ADS)

    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

  12. Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

    PubMed Central

    Kister, Alexander

    2015-01-01

    We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

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

    PubMed

    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.

  14. Exploring the Sequence-based Prediction of Folding Initiation Sites in Proteins.

    PubMed

    Raimondi, Daniele; Orlando, Gabriele; Pancsa, Rita; Khan, Taushif; Vranken, Wim F

    2017-08-18

    Protein folding is a complex process that can lead to disease when it fails. Especially poorly understood are the very early stages of protein folding, which are likely defined by intrinsic local interactions between amino acids close to each other in the protein sequence. We here present EFoldMine, a method that predicts, from the primary amino acid sequence of a protein, which amino acids are likely involved in early folding events. The method is based on early folding data from hydrogen deuterium exchange (HDX) data from NMR pulsed labelling experiments, and uses backbone and sidechain dynamics as well as secondary structure propensities as features. The EFoldMine predictions give insights into the folding process, as illustrated by a qualitative comparison with independent experimental observations. Furthermore, on a quantitative proteome scale, the predicted early folding residues tend to become the residues that interact the most in the folded structure, and they are often residues that display evolutionary covariation. The connection of the EFoldMine predictions with both folding pathway data and the folded protein structure suggests that the initial statistical behavior of the protein chain with respect to local structure formation has a lasting effect on its subsequent states.

  15. Enhancing protein stability with extended disulfide bonds

    DOE PAGES

    Liu, Tao; Wang, Yan; Luo, Xiaozhou; ...

    2016-05-09

    Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. In this paper, we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a librarymore » of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ~9 °C was identified. Finally, this result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.« less

  16. Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

    PubMed Central

    Lim, Shion A.; Hart, Kathryn M.; Marqusee, Susan

    2016-01-01

    Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein’s folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein’s energy landscape is maintained or altered throughout evolution is unclear. To study how a protein’s energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics. PMID:27799545

  17. Mutational analysis of the folding transition state of the C-terminal domain of ribosomal protein L9: a protein with an unusual beta-sheet topology.

    PubMed

    Li, Ying; Gupta, Ruchi; Cho, Jae-Hyun; Raleigh, Daniel P

    2007-01-30

    The C-terminal domain of ribosomal protein L9 (CTL9) is a 92-residue alpha-beta protein which contains an unusual three-stranded mixed parallel and antiparallel beta-sheet. The protein folds in a two-state fashion, and the folding rate is slow. It is thought that the slow folding may be caused by the necessity of forming this unusual beta-sheet architecture in the transition state for folding. This hypothesis makes CTL9 an interesting target for folding studies. The transition state for the folding of CTL9 was characterized by phi-value analysis. The folding of a set of hydrophobic core mutants was analyzed together with a set of truncation mutants. The results revealed a few positions with high phi-values (> or = 0.5), notably, V131, L133, H134, V137, and L141. All of these residues were found in the beta-hairpin region, indicating that the formation of this structure is likely to be the rate-limiting step in the folding of CTL9. One face of the beta-hairpin docks against the N-terminal helix. Analysis of truncation mutants of this helix confirmed its importance in folding. Mutations at other sites in the protein gave small phi-values, despite the fact that some of them had major effects on stability. The analysis indicates that formation of the antiparallel hairpin is critical and its interactions with the first helix are also important. Thus, the slow folding is not a consequence of the need to fully form the unusual three-stranded beta-sheet in the transition state. Analysis of the urea dependence of the folding rates indicates that mutations modulate the unfolded state. The folding of CTL9 is broadly consistent with the nucleation-condensation model of protein folding.

  18. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

    PubMed Central

    Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

    2016-01-01

    The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits. PMID:27808280

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

    NASA Astrophysics Data System (ADS)

    Nilsson, Daniel; Mohanty, Sandipan; Irbäck, Anders

    2018-02-01

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

  20. Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems

    NASA Technical Reports Server (NTRS)

    Weaver, D. L.

    1982-01-01

    Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered.

  1. Methyl Transfer by Substrate Signaling from a Knotted Protein Fold

    PubMed Central

    Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P.; Lahoud, Georges; Ito, Takuhiro; Taylor, Erika A.; Yokoyama, Shigeyuki; Sulkowska, Joanna I.; Hou, Ya-Ming

    2017-01-01

    Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis. PMID:27571175

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

    PubMed

    Sun, Yunxiang; Ming, Dengming

    2014-01-01

    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.

  3. Nanoscale Dewetting Transition in Protein Complex Folding

    PubMed Central

    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

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

    PubMed

    Christensen, Niels J; Kepp, Kasper P

    2013-07-09

    A recent computational protocol that accurately predicts and rationalizes protein multisite mutant stabilities has been extended to handle widely different isoforms of laccases. We apply the protocol to four isoenzymes of Trametes versicolor laccase (TvL) with variable lengths (498-503 residues) and thermostability (Topt ∼ 45-80 °C) and with 67-77% sequence identity. The extended protocol uses (i) statistical averaging, (ii) a molecular-dynamics-validated "compromise" homology model to minimize bias that causes proteins close in sequence to a structural template to be too stable due to having the benefits of the better sampled template (typically from a crystal structure), (iii) correction for hysteresis that favors the input template to overdestabilize, and (iv) a preparative protocol to provide robust input sequences of equal length. The computed ΔΔG values are in good agreement with the major trends in experimental stabilities; that is, the approach may be applicable for fast estimates of the relative stabilities of proteins with as little as 70% identity, something that is currently extremely challenging. The computed stability changes associated with variations are Gaussian-distributed, in good agreement with experimental distributions of stability effects from mutation. The residues causing the differential stability of the four isoforms are consistent with a range of compiled laccase wild type data, suggesting that we may have identified general drivers of laccase stability. Several sites near Cu, notably 79, 241, and 245, or near substrate, mainly 265, are identified that contribute to stability-function trade-offs, of relevance to the search for new proficient and stable variants of these important industrial enzymes.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    PubMed

    Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

    2014-07-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  9. Unraveling protein folding mechanism by analyzing the hierarchy of models with increasing level of detail

    NASA Astrophysics Data System (ADS)

    Hayashi, Tomohiko; Yasuda, Satoshi; Škrbić, Tatjana; Giacometti, Achille; Kinoshita, Masahiro

    2017-09-01

    Taking protein G with 56 residues for a case study, we investigate the mechanism of protein folding. In addition to its native structure possessing α-helix and β-sheet contents of 27% and 39%, respectively, we construct a number of misfolded decoys with a wide variety of α-helix and β-sheet contents. We then consider a hierarchy of 8 different models with increasing level of detail in terms of the number of entropic and energetic physical factors incorporated. The polyatomic structure is always taken into account, but the side chains are removed in half of the models. The solvent is formed by either neutral hard spheres or water molecules. Protein intramolecular hydrogen bonds (H-bonds) and protein-solvent H-bonds (the latter is present only in water) are accounted for or not, depending on the model considered. We then apply a physics-based free-energy function (FEF) corresponding to each model and investigate which structures are most stabilized. This special approach taken on a step-by-step basis enables us to clarify the role of each physical factor in contributing to the structural stability and separately elucidate its effect. Depending on the model employed, significantly different structures such as very compact configurations with no secondary structures and configurations of associated α-helices are optimally stabilized. The native structure can be identified as that with lowest FEF only when the most detailed model is employed. This result is significant for at least the two reasons: The most detailed model considered here is able to capture the fundamental aspects of protein folding notwithstanding its simplicity; and it is shown that the native structure is stabilized by a complex interplay of minimal multiple factors that must be all included in the description. In the absence of even a single of these factors, the protein is likely to be driven towards a different, more stable state.

  10. Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

    PubMed Central

    Chen, Tao; Chan, Hue Sun

    2015-01-01

    The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate. PMID:26016652

  11. How the hydrophobic factor drives protein folding

    PubMed Central

    Baldwin, Robert L.; Rose, George D.

    2016-01-01

    How hydrophobicity (HY) drives protein folding is studied. The 1971 Nozaki–Tanford method of measuring HY is modified to use gases as solutes, not crystals, and this makes the method easy to use. Alkanes are found to be much more hydrophobic than rare gases, and the two different kinds of HY are termed intrinsic (rare gases) and extrinsic (alkanes). The HY values of rare gases are proportional to solvent-accessible surface area (ASA), whereas the HY values of alkanes depend on special hydration shells. Earlier work showed that hydration shells produce the hydration energetics of alkanes. Evidence is given here that the transfer energetics of alkanes to cyclohexane [Wolfenden R, Lewis CA, Jr, Yuan Y, Carter CW, Jr (2015) Proc Natl Acad Sci USA 112(24):7484–7488] measure the release of these shells. Alkane shells are stabilized importantly by van der Waals interactions between alkane carbon and water oxygen atoms. Thus, rare gases cannot form this type of shell. The very short (approximately picoseconds) lifetime of the van der Waals interaction probably explains why NMR efforts to detect alkane hydration shells have failed. The close similarity between the sizes of the opposing energetics for forming or releasing alkane shells confirms the presence of these shells on alkanes and supports Kauzmann's 1959 mechanism of protein folding. A space-filling model is given for the hydration shells on linear alkanes. The model reproduces the n values of Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473] for the number of waters in alkane hydration shells. PMID:27791131

  12. Topological frustration in βα-repeat proteins: sequence diversity modulates the conserved folding mechanisms of α/β/α sandwich proteins

    PubMed Central

    Hills, Ronald D.; Kathuria, Sagar V.; Wallace, Louise A.; Day, Iain J.; Brooks, Charles L.; Matthews, C. Robert

    2010-01-01

    The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of non-local interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα–repeat proteins, which are dominated by local interactions, a combined experimental and simulation analysis was performed on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured sub-millisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine and valine (ILV) side-chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3 and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side-chains in its corresponding hydrophobic cluster. The location of the hydrophobic

  13. Protein collapse is encoded in the folded state architecture.

    PubMed

    Samanta, Himadri S; Zhuravlev, Pavel I; Hinczewski, Michael; Hori, Naoto; Chakrabarti, Shaon; Thirumalai, D

    2017-05-21

    Folded states of single domain globular proteins are compact with high packing density. The radius of gyration, R g , of both the folded and unfolded states increase as N ν where N is the number of amino acids in the protein. The values of the Flory exponent ν are, respectively, ≈⅓ and ≈0.6 in the folded and unfolded states, coinciding with those for homopolymers. However, the extent of compaction of the unfolded state of a protein under low denaturant concentration (collapsibility), conditions favoring the formation of the folded state, is unknown. We develop a theory that uses the contact map of proteins as input to quantitatively assess collapsibility of proteins. Although collapsibility is universal, the propensity to be compact depends on the protein architecture. Application of the theory to over two thousand proteins shows that collapsibility depends not only on N but also on the contact map reflecting the native structure. A major prediction of the theory is that β-sheet proteins are far more collapsible than structures dominated by α-helices. The theory and the accompanying simulations, validating the theoretical predictions, provide insights into the differing conclusions reached using different experimental probes assessing the extent of compaction of proteins. By calculating the criterion for collapsibility as a function of protein length we provide quantitative insights into the reasons why single domain proteins are small and the physical reasons for the origin of multi-domain proteins. Collapsibility of non-coding RNA molecules is similar β-sheet proteins structures adding support to "Compactness Selection Hypothesis".

  14. Statistical Mechanical Foundation for the Two-State Transition in Protein Folding of Small Globular Proteins

    NASA Astrophysics Data System (ADS)

    Iguchi, Kazumoto

    We discuss the statistical mechanical foundation for the two-state transition in the protein folding of small globular proteins. In the standard arguments of protein folding, the statistical search for the ground state is carried out from astronomically many conformations in the configuration space. This leads us to the famous Levinthal's paradox. To resolve the paradox, Gō first postulated that the two-state transition - all-or-none type transition - is very crucial for the protein folding of small globular proteins and used the Gō's lattice model to show the two-state transition nature. Recently, there have been accumulated many experimental results that support the two-state transition for small globular proteins. Stimulated by such recent experiments, Zwanzig has introduced a minimal statistical mechanical model that exhibits the two-state transition. Also, Finkelstein and coworkers have discussed the solution of the paradox by considering the sequential folding of a small globular protein. On the other hand, recently Iguchi have introduced a toy model of protein folding using the Rubik's magic snake model, in which all folded structures are exactly known and mathematically represented in terms of the four types of conformations: cis-, trans-, left and right gauche-configurations between the unit polyhedrons. In this paper, we study the relationship between the Gō's two-state transition, the Zwanzig's statistical mechanics model and the Finkelsteinapos;s sequential folding model by applying them to the Rubik's magic snake models. We show that the foundation of the Gō's two-state transition model relies on the search within the equienergy surface that is labeled by the contact order of the hydrophobic condensation. This idea reproduces the Zwanzig's statistical model as a special case, realizes the Finkelstein's sequential folding model and fits together to understand the nature of the two-state transition of a small globular protein by calculating the

  15. Identification of the protein folding transition state from molecular dynamics trajectories

    NASA Astrophysics Data System (ADS)

    Muff, S.; Caflisch, A.

    2009-03-01

    The rate of protein folding is governed by the transition state so that a detailed characterization of its structure is essential for understanding the folding process. In vitro experiments have provided a coarse-grained description of the folding transition state ensemble (TSE) of small proteins. Atomistic details could be obtained by molecular dynamics (MD) simulations but it is not straightforward to extract the TSE directly from the MD trajectories, even for small peptides. Here, the structures in the TSE are isolated by the cut-based free-energy profile (cFEP) using the network whose nodes and links are configurations sampled by MD and direct transitions among them, respectively. The cFEP is a barrier-preserving projection that does not require arbitrarily chosen progress variables. First, a simple two-dimensional free-energy surface is used to illustrate the successful determination of the TSE by the cFEP approach and to explain the difficulty in defining boundary conditions of the Markov state model for an entropically stabilized free-energy minimum. The cFEP is then used to extract the TSE of a β-sheet peptide with a complex free-energy surface containing multiple basins and an entropic region. In contrast, Markov state models with boundary conditions defined by projected variables and conventional histogram-based free-energy profiles are not able to identify the TSE of the β-sheet peptide.

  16. On the role of conformational geometry in protein folding

    NASA Astrophysics Data System (ADS)

    Du, Rose; Pande, Vijay S.; Grosberg, Alexander Yu.; Tanaka, Toyoichi; Shakhnovich, Eugene

    1999-12-01

    Using a lattice model of protein folding, we find that once certain native contacts have been formed, folding to the native state is inevitable, even if the only energetic bias in the system is nonspecific, homopolymeric attraction to a collapsed state. These conformations can be quite geometrically unrelated to the native state (with as low as only 53% of the native contacts formed). We demonstrate these results by examining the Monte Carlo kinetics of both heteropolymers under Go interactions and homopolymers, with the folding of both types of polymers to the native state of the heteropolymer. Although we only consider a 48-mer lattice model, our findings shed light on the effects of geometrical restrictions, including those of chain connectivity and steric excluded volume, on protein folding. These effects play a complementary role to that of the rugged energy landscape. In addition, the results of this work can aid in the interpretation of experiments and computer simulations of protein folding performed at elevated temperatures.

  17. The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

    PubMed Central

    King, Steven C

    2004-01-01

    structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity). PMID:15548327

  18. Ab initio folding of proteins using all-atom discrete molecular dynamics

    PubMed Central

    Ding, Feng; Tsao, Douglas; Nie, Huifen; Dokholyan, Nikolay V.

    2008-01-01

    Summary Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. Using the replica exchange method, we perform folding simulations of six small proteins (20–60 residues) with distinct native structures. In all cases, native or near-native states are reached in simulations. For three small proteins, multiple folding transitions are observed and the computationally-characterized thermodynamics are in quantitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes, and applied to protein engineering and design of protein-protein interactions. PMID:18611374

  19. Protein fold recognition using geometric kernel data fusion.

    PubMed

    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.

  20. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    PubMed

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. A Simple and Effective Protein Folding Activity Suitable for Large Lectures

    ERIC Educational Resources Information Center

    White, Brian

    2006-01-01

    This article describes a simple and inexpensive hands-on simulation of protein folding suitable for use in large lecture classes. This activity uses a minimum of parts, tools, and skill to simulate some of the fundamental principles of protein folding. The major concepts targeted are that proteins begin as linear polypeptides and fold to…

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

    NASA Astrophysics Data System (ADS)

    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].

  3. The topomer-sampling model of protein folding

    PubMed Central

    Debe, Derek A.; Carlson, Matt J.; Goddard, William A.

    1999-01-01

    Clearly, a protein cannot sample all of its conformations (e.g., ≈3100 ≈ 1048 for a 100 residue protein) on an in vivo folding timescale (<1 s). To investigate how the conformational dynamics of a protein can accommodate subsecond folding time scales, we introduce the concept of the native topomer, which is the set of all structures similar to the native structure (obtainable from the native structure through local backbone coordinate transformations that do not disrupt the covalent bonding of the peptide backbone). We have developed a computational procedure for estimating the number of distinct topomers required to span all conformations (compact and semicompact) for a polypeptide of a given length. For 100 residues, we find ≈3 × 107 distinct topomers. Based on the distance calculated between different topomers, we estimate that a 100-residue polypeptide diffusively samples one topomer every ≈3 ns. Hence, a 100-residue protein can find its native topomer by random sampling in just ≈100 ms. These results suggest that subsecond folding of modest-sized, single-domain proteins can be accomplished by a two-stage process of (i) topomer diffusion: random, diffusive sampling of the 3 × 107 distinct topomers to find the native topomer (≈0.1 s), followed by (ii) intratopomer ordering: nonrandom, local conformational rearrangements within the native topomer to settle into the precise native state. PMID:10077555

  4. Learning To Fold Proteins Using Energy Landscape Theory

    PubMed Central

    Schafer, N.P.; Kim, B.L.; Zheng, W.; Wolynes, P.G.

    2014-01-01

    This review is a tutorial for scientists interested in the problem of protein structure prediction, particularly those interested in using coarse-grained molecular dynamics models that are optimized using lessons learned from the energy landscape theory of protein folding. We also present a review of the results of the AMH/AMC/AMW/AWSEM family of coarse-grained molecular dynamics protein folding models to illustrate the points covered in the first part of the article. Accurate coarse-grained structure prediction models can be used to investigate a wide range of conceptual and mechanistic issues outside of protein structure prediction; specifically, the paper concludes by reviewing how AWSEM has in recent years been able to elucidate questions related to the unusual kinetic behavior of artificially designed proteins, multidomain protein misfolding, and the initial stages of protein aggregation. PMID:25308991

  5. High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

    PubMed

    Fossat, Martin J; Dao, Thuy P; Jenkins, Kelly; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A; Garcia, Angel E; Barrick, Doug; Roumestand, Christian; Royer, Catherine A

    2016-12-06

    A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1 H- 15 N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

    PubMed

    Robinson, Philip J; Pringle, Marie Anne; Woolhead, Cheryl A; Bulleid, Neil J

    2017-04-28

    The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

    PubMed

    Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

    2016-05-18

    Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

  8. Hidden complexity of free energy surfaces for peptide (protein) folding.

    PubMed

    Krivov, Sergei V; Karplus, Martin

    2004-10-12

    An understanding of the thermodynamics and kinetics of protein folding requires a knowledge of the free energy surface governing the motion of the polypeptide chain. Because of the many degrees of freedom involved, surfaces projected on only one or two progress variables are generally used in descriptions of the folding reaction. Such projections result in relatively smooth surfaces, but they could mask the complexity of the unprojected surface. Here we introduce an approach to determine the actual (unprojected) free energy surface and apply it to the second beta-hairpin of protein G, which has been used as a model system for protein folding. The surface is represented by a disconnectivity graph calculated from a long equilibrium folding-unfolding trajectory. The denatured state is found to have multiple low free energy basins. Nevertheless, the peptide shows exponential kinetics in folding to the native basin. Projected surfaces obtained from the present analysis have a simple form in agreement with other studies of the beta-hairpin. The hidden complexity found for the beta-hairpin surface suggests that the standard funnel picture of protein folding should be revisited.

  9. Frustration Sculpts the Early Stages of Protein Folding.

    PubMed

    Di Silvio, Eva; Brunori, Maurizio; Gianni, Stefano

    2015-09-07

    The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site-directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy-landscape theory. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Folding energy landscape and network dynamics of small globular proteins

    PubMed Central

    Hori, Naoto; Chikenji, George; Berry, R. Stephen; Takada, Shoji

    2009-01-01

    The folding energy landscape of proteins has been suggested to be funnel-like with some degree of ruggedness on the slope. How complex the landscape, however, is still rather unclear. Many experiments for globular proteins suggested relative simplicity, whereas molecular simulations of shorter peptides implied more complexity. Here, by using complete conformational sampling of 2 globular proteins, protein G and src SH3 domain and 2 related random peptides, we investigated their energy landscapes, topological properties of folding networks, and folding dynamics. The projected energy surfaces of globular proteins were funneled in the vicinity of the native but also have other quite deep, accessible minima, whereas the randomized peptides have many local basins, including some leading to seriously misfolded forms. Dynamics in the denatured part of the network exhibited basin-hopping itinerancy among many conformations, whereas the protein reached relatively well-defined final stages that led to their native states. We also found that the folding network has the hierarchic nature characterized by the scale-free and the small-world properties. PMID:19114654

  11. Folding energy landscape and network dynamics of small globular proteins.

    PubMed

    Hori, Naoto; Chikenji, George; Berry, R Stephen; Takada, Shoji

    2009-01-06

    The folding energy landscape of proteins has been suggested to be funnel-like with some degree of ruggedness on the slope. How complex the landscape, however, is still rather unclear. Many experiments for globular proteins suggested relative simplicity, whereas molecular simulations of shorter peptides implied more complexity. Here, by using complete conformational sampling of 2 globular proteins, protein G and src SH3 domain and 2 related random peptides, we investigated their energy landscapes, topological properties of folding networks, and folding dynamics. The projected energy surfaces of globular proteins were funneled in the vicinity of the native but also have other quite deep, accessible minima, whereas the randomized peptides have many local basins, including some leading to seriously misfolded forms. Dynamics in the denatured part of the network exhibited basin-hopping itinerancy among many conformations, whereas the protein reached relatively well-defined final stages that led to their native states. We also found that the folding network has the hierarchic nature characterized by the scale-free and the small-world properties.

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

    PubMed

    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.

  13. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    PubMed Central

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  14. Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

    PubMed Central

    Zhang, Xinming; Rebane, Aleksander A.; Ma, Lu; Li, Feng; Jiao, Junyi; Qu, Hong; Pincet, Frederic; Rothman, James E.

    2016-01-01

    Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission. PMID:27911771

  15. Optimizing physical energy functions for protein folding.

    PubMed

    Fujitsuka, Yoshimi; Takada, Shoji; Luthey-Schulten, Zaida A; Wolynes, Peter G

    2004-01-01

    We optimize a physical energy function for proteins with the use of the available structural database and perform three benchmark tests of the performance: (1) recognition of native structures in the background of predefined decoy sets of Levitt, (2) de novo structure prediction using fragment assembly sampling, and (3) molecular dynamics simulations. The energy parameter optimization is based on the energy landscape theory and uses a Monte Carlo search to find a set of parameters that seeks the largest ratio deltaE(s)/DeltaE for all proteins in a training set simultaneously. Here, deltaE(s) is the stability gap between the native and the average in the denatured states and DeltaE is the energy fluctuation among these states. Some of the energy parameters optimized are found to show significant correlation with experimentally observed quantities: (1) In the recognition test, the optimized function assigns the lowest energy to either the native or a near-native structure among many decoy structures for all the proteins studied. (2) Structure prediction with the fragment assembly sampling gives structure models with root mean square deviation less than 6 A in one of the top five cluster centers for five of six proteins studied. (3) Structure prediction using molecular dynamics simulation gives poorer performance, implying the importance of having a more precise description of local structures. The physical energy function solely inferred from a structural database neither utilizes sequence information from the family of the target nor the outcome of the secondary structure prediction but can produce the correct native fold for many small proteins. Copyright 2003 Wiley-Liss, Inc.

  16. Aromatic Cluster Sensor of Protein Folding: Near-UV Electronic Circular Dichroism Bands Assigned to Fold Compactness.

    PubMed

    Farkas, Viktor; Jákli, Imre; Tóth, Gábor K; Perczel, András

    2016-09-19

    Both far- and near-UV electronic circular dichroism (ECD) spectra have bands sensitive to thermal unfolding of Trp and Tyr residues containing proteins. Beside spectral changes at 222 nm reporting secondary structural variations (far-UV range), L b bands (near-UV range) are applicable as 3D-fold sensors of protein's core structure. In this study we show that both L b (Tyr) and L b (Trp) ECD bands could be used as sensors of fold compactness. ECD is a relative method and thus requires NMR referencing and cross-validation, also provided here. The ensemble of 204 ECD spectra of Trp-cage miniproteins is analysed as a training set for "calibrating" Trp↔Tyr folded systems of known NMR structure. While in the far-UV ECD spectra changes are linear as a function of the temperature, near-UV ECD data indicate a non-linear and thus, cooperative unfolding mechanism of these proteins. Ensemble of ECD spectra deconvoluted gives both conformational weights and insight to a protein folding↔unfolding mechanism. We found that the L b 293 band is reporting on the 3D-structure compactness. In addition, the pure near-UV ECD spectrum of the unfolded state is described here for the first time. Thus, ECD folding information now validated can be applied with confidence in a large thermal window (5≤T≤85 °C) compared to NMR for studying the unfolding of Trp↔Tyr residue pairs. In conclusion, folding propensities of important proteins (RNA polymerase II, ubiquitin protein ligase, tryptase-inhibitor etc.) can now be analysed with higher confidence. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    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.

  18. Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model.

    PubMed

    Wako, Hiroshi; Abe, Haruo

    2016-01-01

    The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding.

  19. Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model

    PubMed Central

    Wako, Hiroshi; Abe, Haruo

    2016-01-01

    The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding. PMID:28409079

  20. Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

    PubMed

    Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

    2016-06-07

    Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  2. Molecular origins of internal friction effects on protein-folding rates.

    PubMed

    de Sancho, David; Sirur, Anshul; Best, Robert B

    2014-07-02

    Recent experiments on protein-folding dynamics have revealed strong evidence for internal friction effects. That is, observed relaxation times are not simply proportional to the solvent viscosity as might be expected if the solvent were the only source of friction. However, a molecular interpretation of this remarkable phenomenon is currently lacking. Here, we use all-atom simulations of peptide and protein folding in explicit solvent, to probe the origin of the unusual viscosity dependence. We find that an important contribution to this effect, explaining the viscosity dependence of helix formation and the folding of a helix-containing protein, is the insensitivity of torsion angle isomerization to solvent friction. The influence of this landscape roughness can, in turn, be quantitatively explained by a rate theory including memory friction. This insensitivity of local barrier crossing to solvent friction is expected to contribute to the viscosity dependence of folding rates in larger proteins.

  3. Universality and diversity of folding mechanics for three-helix bundle proteins.

    PubMed

    Yang, Jae Shick; Wallin, Stefan; Shakhnovich, Eugene I

    2008-01-22

    In this study we evaluate, at full atomic detail, the folding processes of two small helical proteins, the B domain of protein A and the Villin headpiece. Folding kinetics are studied by performing a large number of ab initio Monte Carlo folding simulations using a single transferable all-atom potential. Using these trajectories, we examine the relaxation behavior, secondary structure formation, and transition-state ensembles (TSEs) of the two proteins and compare our results with experimental data and previous computational studies. To obtain a detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Moreover, rigorous p(fold) analysis is used to obtain representative samples of the TSEs and a good quantitative agreement between experimental and simulated Phi values is obtained for protein A. Phi values for Villin also are obtained and left as predictions to be tested by future experiments. Our analysis shows that the two-helix hairpin is a common partially stable structural motif that gets formed before entering the TSE in the studied proteins. These results together with our earlier study of Engrailed Homeodomain and recent experimental studies provide a comprehensive, atomic-level picture of folding mechanics of three-helix bundle proteins.

  4. Revealing the global map of protein folding space by large-scale simulations

    NASA Astrophysics Data System (ADS)

    Sinner, Claude; Lutz, Benjamin; Verma, Abhinav; Schug, Alexander

    2015-12-01

    The full characterization of protein folding is a remarkable long-standing challenge both for experiment and simulation. Working towards a complete understanding of this process, one needs to cover the full diversity of existing folds and identify the general principles driving the process. Here, we want to understand and quantify the diversity in folding routes for a large and representative set of protein topologies covering the full range from all alpha helical topologies towards beta barrels guided by the key question: Does the majority of the observed routes contribute to the folding process or only a particular route? We identified a set of two-state folders among non-homologous proteins with a sequence length of 40-120 residues. For each of these proteins, we ran native-structure based simulations both with homogeneous and heterogeneous contact potentials. For each protein, we simulated dozens of folding transitions in continuous uninterrupted simulations and constructed a large database of kinetic parameters. We investigate folding routes by tracking the formation of tertiary structure interfaces and discuss whether a single specific route exists for a topology or if all routes are equiprobable. These results permit us to characterize the complete folding space for small proteins in terms of folding barrier ΔG‡, number of routes, and the route specificity RT.

  5. How Fast is Collapse of Proteins During Folding?

    NASA Astrophysics Data System (ADS)

    Chahine, J.; Onuchic, J. N.; Socci, N. D.

    1998-03-01

    Recent experiments in fast folding proteins are now starting to address the question of how fast is collapse relative to the total folding time. Using minimalist models, we are able to investigate the way in which different scenarios of folding can arise depending on the interplay between the collapse order parameter and the order parameter sensitive to specific tertiary contacts. Most of our earlier studies have focused on the limit that collapse is very fast compared to the total folding time. In this work we focus on the opposite limit, i.e., at the folding temperature, collapse and folding occurs simultaneously. The folding mechanism becomes very different in this limit. Particularly, the non-specific collapse transition, that occurs at temperatures higher than the folding temperature for the fast collapse limit, now occurs between the folding and the glass temperature. We show how this transition can be identified and its consequences for the folding kinetics.

  6. Common fold in helix–hairpin–helix proteins

    PubMed Central

    Shao, Xuguang; Grishin, Nick V.

    2000-01-01

    Helix–hairpin–helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein–protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)2 domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)2 domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each α-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the α-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glyco­s­y­lases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)2 domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)2 functional unit. PMID:10908318

  7. A semi-analytical description of protein folding that incorporates detailed geometrical information

    PubMed Central

    Suzuki, Yoko; Noel, Jeffrey K.; Onuchic, José N.

    2011-01-01

    Much has been done to study the interplay between geometric and energetic effects on the protein folding energy landscape. Numerical techniques such as molecular dynamics simulations are able to maintain a precise geometrical representation of the protein. Analytical approaches, however, often focus on the energetic aspects of folding, including geometrical information only in an average way. Here, we investigate a semi-analytical expression of folding that explicitly includes geometrical effects. We consider a Hamiltonian corresponding to a Gaussian filament with structure-based interactions. The model captures local features of protein folding often averaged over by mean-field theories, for example, loop contact formation and excluded volume. We explore the thermodynamics and folding mechanisms of beta-hairpin and alpha-helical structures as functions of temperature and Q, the fraction of native contacts formed. Excluded volume is shown to be an important component of a protein Hamiltonian, since it both dominates the cooperativity of the folding transition and alters folding mechanisms. Understanding geometrical effects in analytical formulae will help illuminate the consequences of the approximations required for the study of larger proteins. PMID:21721664

  8. Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

    PubMed Central

    Ganguly, Debabani; Zhang, Weihong; Chen, Jianhan

    2013-01-01

    Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for

  9. Molecular Origins of Internal Friction Effects on Protein Folding Rates

    PubMed Central

    Sirur, Anshul

    2014-01-01

    Recent experiments on protein folding dynamics have revealed strong evidence for internal friction effects. That is, observed relaxation times are not simply proportional to the solvent viscosity as might be expected if the solvent were the only source of friction. However, a molecular interpretation of this remarkable phenomenon is currently lacking. Here, we use all-atom simulations of peptide and protein folding in explicit solvent, to probe the origin of the unusual viscosity dependence. We find that an important contribution to this effect, explaining the viscosity dependence of helix formation and the folding of a helix-containing protein, is the insensitivity of torsion angle isomerization to solvent friction. The influence of this landscape roughness can, in turn, be quantitatively explained by a rate theory including memory friction. This insensitivity of local barrier crossing to solvent friction is expected to contribute to the viscosity dependence of folding rates in larger proteins. PMID:24986114

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

    PubMed

    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.

  11. Hydrogen bonds are a primary driving force for de novo protein folding

    DOE PAGES

    Lee, Schuyler; Wang, Chao; Liu, Haolin; ...

    2017-11-10

    The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

  12. Hydrogen bonds are a primary driving force for de novo protein folding

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Schuyler; Wang, Chao; Liu, Haolin

    The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

  13. Acceleration through passive destabilization: protein folding in a weak hydrophobic environment

    NASA Astrophysics Data System (ADS)

    Jewett, Andrew; Baumketner, Andrij; Shea, Joan-Emma

    2004-03-01

    The GroEL chaperonin is a biomolecule which assists the folding of an extremely diverse range of proteins in Eubacteria. Some proteins undergo many rounds of ATP-regulated binding and dissociation from GroEL/ES before folding. It has been proposed that transient stress from ATP-regulated binding and release from GroEL/ES frees frustrated proteins from misfolded conformations. However recent evidence suggests that chaperonin-accelerated protein folding can take place entirely within a mutated GroEL+ES cavity that is unable to open and release the protein. Using molecular dynamics, we demonstrate that static confinement within a weakly hydrophobic (attractive) cavity (similar to the interior of the cavity formed by the GroEL+ES complex) is sufficient to significantly accelerate the folding of a highly frustrated protein-like heteropolymer. Our frustrated molecule benifits kinetically from a static hydrophobic environment that destabilizes misfolded conformations. This may shed light on the mechanisms used by other chaperones which do not depend on ATP.

  14. Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

    PubMed

    Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

    2017-11-01

    The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day -1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. © 2017 Wiley Periodicals, Inc.

  15. Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

    PubMed Central

    Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

    2017-01-01

    The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (~100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day−1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. PMID:28722205

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

    NASA Astrophysics Data System (ADS)

    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.

  17. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    NASA Astrophysics Data System (ADS)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  18. GroEL actively stimulates folding of the endogenous substrate protein PepQ.

    PubMed

    Weaver, Jeremy; Jiang, Mengqiu; Roth, Andrew; Puchalla, Jason; Zhang, Junjie; Rye, Hays S

    2017-06-30

    Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

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

    PubMed

    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.

  20. Computational Insights into the Stability and Folding Pathways of Human Telomeric DNA G-Quadruplexes.

    PubMed

    Luo, Di; Mu, Yuguang

    2016-06-09

    G-quadruplex is a noncanonical yet crucial secondary structure of nucleic acids, which has proven its importance in cell aging, anticancer therapies, gene expression, and genome stability. In this study, the stability and folding dynamics of human telomeric DNA G-quadruplexes were investigated via enhanced sampling techniques. First, temperature-replica exchange MD (REMD) simulations were employed to compare the thermal stabilities among the five established folding topologies. The hybrid-2 type adopted by extended human telomeric sequence is revealed to be the most stable conformation in our simulations. Next, the free energy landscapes and folding intermediates of the hybrid-1 and -2 types were investigated with parallel tempering metadynamics simulations in the well-tempered ensemble. It was observed that the N-glycosidic conformations of guanines can flip over to accommodate into the cyclic Hoogsteen H-bonding on G-tetrads in which they were not originally involved. Furthermore, a hairpin and a triplex intermediate were identified for the folding of the hybrid-1 type conformation, whereas for the hybrid-2 type, there were no folding intermediates observed from its free energy surface. However, the energy barrier from its native topology to the transition structure is found to be extremely high compared to that of the hybrid-1 type, which is consistent with our stability predictions from the REMD simulations. We hope the insights presented in this work can help to complement current understanding on the stability and dynamics of G-quadruplexes, which is necessary not only to stabilize the structures but also to intervene their formation in genome.

  1. STRUM: structure-based prediction of protein stability changes upon single-point mutation

    PubMed Central

    Quan, Lijun; Lv, Qiang; Zhang, Yang

    2016-01-01

    Motivation: Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. Results: We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability

  2. Folding propensity of intrinsically disordered proteins by osmotic stress

    DOE PAGES

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

    2016-10-11

    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 scatteringmore » (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.« less

  3. Folding propensity of intrinsically disordered proteins by osmotic stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 scatteringmore » (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.« less

  4. Proteome-level interplay between folding and aggregation propensities of proteins.

    PubMed

    Tartaglia, Gian Gaetano; Vendruscolo, Michele

    2010-10-08

    With the advent of proteomics, there is an increasing need of tools for predicting the properties of large numbers of proteins by using the information provided by their amino acid sequences, even in the absence of the knowledge of their structures. One of the most important types of predictions concerns whether proteins will fold or aggregate. Here, we study the competition between these two processes by analyzing the relationship between the folding and aggregation propensity profiles for the human and Escherichia coli proteomes. These profiles are calculated, respectively, using the CamFold method, which we introduce in this work, and the Zyggregator method. Our results indicate that the kinetic behavior of proteins is, to a large extent, determined by the interplay between regions of low folding and high aggregation propensities. Copyright © 2010. Published by Elsevier Ltd.

  5. Developing a molecular dynamics force field for both folded and disordered protein states.

    PubMed

    Robustelli, Paul; Piana, Stefano; Shaw, David E

    2018-05-07

    Molecular dynamics (MD) simulation is a valuable tool for characterizing the structural dynamics of folded proteins and should be similarly applicable to disordered proteins and proteins with both folded and disordered regions. It has been unclear, however, whether any physical model (force field) used in MD simulations accurately describes both folded and disordered proteins. Here, we select a benchmark set of 21 systems, including folded and disordered proteins, simulate these systems with six state-of-the-art force fields, and compare the results to over 9,000 available experimental data points. We find that none of the tested force fields simultaneously provided accurate descriptions of folded proteins, of the dimensions of disordered proteins, and of the secondary structure propensities of disordered proteins. Guided by simulation results on a subset of our benchmark, however, we modified parameters of one force field, achieving excellent agreement with experiment for disordered proteins, while maintaining state-of-the-art accuracy for folded proteins. The resulting force field, a99SB- disp , should thus greatly expand the range of biological systems amenable to MD simulation. A similar approach could be taken to improve other force fields. Copyright © 2018 the Author(s). Published by PNAS.

  6. Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

    PubMed Central

    Aksel, Tural; Barrick, Doug

    2014-01-01

    Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule. PMID:24988356

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

    PubMed

    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.

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

    PubMed

    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.

  9. Multi-stability in folded shells: non-Euclidean origami

    NASA Astrophysics Data System (ADS)

    Evans, Arthur

    2015-03-01

    Both natural and man-made structures benefit from having multiple mechanically stable states, from the quick snapping motion of hummingbird beaks to micro-textured surfaces with tunable roughness. Rather than discuss special fabrication techniques for creating bi-stability through material anisotropy, in this talk I will present several examples of how folding a structure can modify the energy landscape and thus lead to multiple stable states. Using ideas from origami and differential geometry, I will discuss how deforming a non-Euclidean surface can be done either continuously or discontinuously, and explore the effects that global constraints have on the ultimate stability of the surface.

  10. Thermodynamic effects of proline introduction on protein stability.

    PubMed

    Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan

    2007-02-01

    The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.

  11. Mechanical Modeling and Computer Simulation of Protein Folding

    ERIC Educational Resources Information Center

    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…

  12. Predicting folding-unfolding transitions in proteins without a priori knowledge of the folded state

    NASA Astrophysics Data System (ADS)

    Okan, Osman; Turgut, Deniz; Garcia, Angel; Ozisik, Rahmi

    2013-03-01

    The common computational method of studying folding transitions in proteins is to compare simulated conformations against the folded structure, but this method obviously requires the folded structure to be known beforehand. In the current study, we show that the use of bond orientational order parameter (BOOP) Ql [Steinhardt PJ, Nelson DR, Ronchetti M, Phys. Rev. B 1983, 28, 784] is a viable alternative to the commonly adopted root mean squared distance (RMSD) measure in probing conformational transitions. Replica exchange molecular dynamics simulations of the trp-cage protein (with 20 residues) in TIP-3P water were used to compare BOOP against RMSD. The results indicate that the correspondence between BOOP and RMSD time series become stronger with increasing l. We finally show that robust linear models that incorporate different Ql can be parameterized from a given replica run and can be used to study other replica trajectories. This work is partially supported by NSF DUE-1003574.

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

    PubMed

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

    2010-02-23

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

  14. Atomic Interaction Networks in the Core of Protein Domains and Their Native Folds

    PubMed Central

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

    2010-01-01

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

  15. PROTERAN: animated terrain evolution for visual analysis of patterns in protein folding trajectory.

    PubMed

    Zhou, Ruhong; Parida, Laxmi; Kapila, Kush; Mudur, Sudhir

    2007-01-01

    The mechanism of protein folding remains largely a mystery in molecular biology, despite the enormous effort from many groups in the past decades. 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 and so on. In this paper, we present an integrated approach towards understanding the folding process via visual analysis of patterns of these reaction coordinates. The three disparate processes (1) protein folding simulation, (2) pattern elicitation and (3) visualization of patterns, work in tandem. Thus as the protein folds, the changing landscape in the pattern space can be viewed via the visualization tool, PROTERAN, a program we developed for this purpose. We first present an incremental (on-line) trie-based pattern discovery algorithm to elicit the patterns and then describe the terrain metaphor based visualization tool. Using two example small proteins, a beta-hairpin and a designed protein Trp-cage, we next demonstrate that this combined pattern discovery and visualization approach extracts crucial information about protein folding intermediates and mechanism.

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

    PubMed

    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.

  17. On the polymer physics origins of protein folding thermodynamics.

    PubMed

    Taylor, Mark P; Paul, Wolfgang; Binder, Kurt

    2016-11-07

    A remarkable feature of the spontaneous folding of many small proteins is the striking similarity in the thermodynamics of the folding process. This process is characterized by simple two-state thermodynamics with large and compensating changes in entropy and enthalpy and a funnel-like free energy landscape with a free-energy barrier that varies linearly with temperature. One might attribute the commonality of this two-state folding behavior to features particular to these proteins (e.g., chain length, hydrophobic/hydrophilic balance, attributes of the native state) or one might suspect that this similarity in behavior has a more general polymer-physics origin. Here we show that this behavior is also typical for flexible homopolymer chains with sufficiently short range interactions. Two-state behavior arises from the presence of a low entropy ground (folded) state separated from a set of high entropy disordered (unfolded) states by a free energy barrier. This homopolymer model exhibits a funneled free energy landscape that reveals a complex underlying dynamics involving competition between folding and non-folding pathways. Despite the presence of multiple pathways, this simple physics model gives the robust result of two-state thermodynamics for both the cases of folding from a basin of expanded coil states and from a basin of compact globule states.

  18. On the polymer physics origins of protein folding thermodynamics

    NASA Astrophysics Data System (ADS)

    Taylor, Mark P.; Paul, Wolfgang; Binder, Kurt

    2016-11-01

    A remarkable feature of the spontaneous folding of many small proteins is the striking similarity in the thermodynamics of the folding process. This process is characterized by simple two-state thermodynamics with large and compensating changes in entropy and enthalpy and a funnel-like free energy landscape with a free-energy barrier that varies linearly with temperature. One might attribute the commonality of this two-state folding behavior to features particular to these proteins (e.g., chain length, hydrophobic/hydrophilic balance, attributes of the native state) or one might suspect that this similarity in behavior has a more general polymer-physics origin. Here we show that this behavior is also typical for flexible homopolymer chains with sufficiently short range interactions. Two-state behavior arises from the presence of a low entropy ground (folded) state separated from a set of high entropy disordered (unfolded) states by a free energy barrier. This homopolymer model exhibits a funneled free energy landscape that reveals a complex underlying dynamics involving competition between folding and non-folding pathways. Despite the presence of multiple pathways, this simple physics model gives the robust result of two-state thermodynamics for both the cases of folding from a basin of expanded coil states and from a basin of compact globule states.

  19. The role of stabilization centers in protein thermal stability

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Magyar, Csaba; Gromiha, M. Michael; Sávoly, Zoltán

    2016-02-26

    The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilizationmore » of proteins. - Highlights: • Stabilization centers contribute to thermal stabilization of protein structures. • Stabilization center content correlates with melting temperature of proteins. • Exposed stabilization center content correlates with stability even in hyperthermophiles. • Stability changing mutations are frequently found at stabilization centers.« less

  20. The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus, Unlike Its Homologous Neighbours☆

    PubMed Central

    Kwa, Lee Gyan; Wensley, Beth G.; Alexander, Crispin G.; Browning, Stuart J.; Lichman, Benjamin R.; Clarke, Jane

    2014-01-01

    Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape. PMID:24373753

  1. Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions.

    PubMed

    Ramirez-Sarmiento, Cesar A; Komives, Elizabeth A

    2018-04-06

    Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Dynamics of one-state downhill protein folding.

    PubMed

    Li, Peng; Oliva, Fabiana Y; Naganathan, Athi N; Muñoz, Victor

    2009-01-06

    The small helical protein BBL has been shown to fold and unfold in the absence of a free energy barrier according to a battery of quantitative criteria in equilibrium experiments, including probe-dependent equilibrium unfolding, complex coupling between denaturing agents, characteristic DSC thermogram, gradual melting of secondary structure, and heterogeneous atom-by-atom unfolding behaviors spanning the entire unfolding process. Here, we present the results of nanosecond T-jump experiments probing backbone structure by IR and end-to-end distance by FRET. The folding dynamics observed with these two probes are both exponential with common relaxation times but have large differences in amplitude following their probe-dependent equilibrium unfolding. The quantitative analysis of amplitude and relaxation time data for both probes shows that BBL folding dynamics are fully consistent with the one-state folding scenario and incompatible with alternative models involving one or several barrier crossing events. At 333 K, the relaxation time for BBL is 1.3 micros, in agreement with previous folding speed limit estimates. However, late folding events at room temperature are an order of magnitude slower (20 micros), indicating a relatively rough underlying energy landscape. Our results in BBL expose the dynamic features of one-state folding and chart the intrinsic time-scales for conformational motions along the folding process. Interestingly, the simple self-averaging folding dynamics of BBL are the exact dynamic properties required in molecular rheostats, thus supporting a biological role for one-state folding.

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

    PubMed

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

    2014-07-20

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

  4. Analysis of the Free-Energy Surface of Proteins from Reversible Folding Simulations

    PubMed Central

    Allen, Lucy R.; Krivov, Sergei V.; Paci, Emanuele

    2009-01-01

    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 λ-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. PMID:19593364

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

    PubMed

    Allen, Lucy R; Krivov, Sergei V; Paci, Emanuele

    2009-07-01

    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.

  6. Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

    PubMed

    Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul; Özen, Ayşegül; Matthews, C Robert

    2016-03-01

    Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates. © 2015 The Protein Society.

  7. Refolding of urea-denatured α-chymotrypsin by protein-folding liquid chromatography.

    PubMed

    Congyu, Ke; Wujuan, Sun; Qunzheng, Zhang; Xindu, Geng

    2013-04-01

    An approach for re-folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α-chymotrypsin (α-Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α-Chy states - urea-denatured (U state), its folded intermediates (M state) and nature state (N state) - were studied during protein folding. Based on the test by matrix-assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α-Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α-Chy was found to be higher than that of commercial α-Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m; the highest specific bioactivity at urea concentration was 1.0 m, indicating the possibility for re-folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α-Chy. When the urea concentration reached 6.0 m, the unfolded α-Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics.

    PubMed

    Klein, Eili Y; Blumenkrantz, Deena; Serohijos, Adrian; Shakhnovich, Eugene; Choi, Jeong-Mo; Rodrigues, João V; Smith, Brendan D; Lane, Andrew P; Feldman, Andrew; Pekosz, Andrew

    2018-01-01

    Protein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated with in vitro virus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages. IMPORTANCE One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus

  9. Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

    PubMed

    Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver

    2018-06-15

    It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

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

    PubMed

    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.

  11. Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy; Savas, Christopher J.; Kartje, Zachary; Hoops, Geoffrey C.

    2014-01-01

    Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal…

  12. Oxidative Folding and N-terminal Cyclization of Onconase+

    PubMed Central

    Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

    2008-01-01

    Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

  13. Redox-Assisted Protein Folding Systems in Eukaryotic Parasites

    PubMed Central

    Haque, Saikh Jaharul; Majumdar, Tanmay

    2012-01-01

    Abstract Significance: The cysteine (Cys) residues of proteins play two fundamentally important roles. They serve as sites of post-translational redox modifications as well as influence the conformation of the protein through the formation of disulfide bonds. Recent Advances: Redox-related and redox-associated protein folding in protozoan parasites has been found to be a major mode of regulation, affecting myriad aspects of the parasitic life cycle, host-parasite interactions, and the disease pathology. Available genome sequences of various parasites have begun to complement the classical biochemical and enzymological studies of these processes. In this article, we summarize the reversible Cys disulfide (S-S) bond formation in various classes of strategically important parasitic proteins, and its structural consequence and functional relevance. Critical Issues: Molecular mechanisms of folding remain under-studied and often disconnected from functional relevance. Future Directions: The clinical benefit of redox research will require a comprehensive characterization of the various isoforms and paralogs of the redox enzymes and their concerted effect on the structure and function of the specific parasitic client proteins. Antioxid. Redox Signal. 17, 674–683. PMID:22122448

  14. Viroporins, Examples of the Two-Stage Membrane Protein Folding Model.

    PubMed

    Martinez-Gil, Luis; Mingarro, Ismael

    2015-06-26

    Viroporins are small, α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.

  15. Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

    PubMed Central

    Yu, Wookyung; Baxa, Michael C.; Gagnon, Isabelle; Freed, Karl F.; Sosnick, Tobin R.

    2016-01-01

    The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 105 s−1 and a stability of 7.4 kcal·mol−1 at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions. PMID:27078098

  16. Meta-structure correlation in protein space unveils different selection rules for folded and intrinsically disordered proteins.

    PubMed

    Naranjo, Yandi; Pons, Miquel; Konrat, Robert

    2012-01-01

    The number of existing protein sequences spans a very small fraction of sequence space. Natural proteins have overcome a strong negative selective pressure to avoid the formation of insoluble aggregates. Stably folded globular proteins and intrinsically disordered proteins (IDPs) use alternative solutions to the aggregation problem. While in globular proteins folding minimizes the access to aggregation prone regions, IDPs on average display large exposed contact areas. Here, we introduce the concept of average meta-structure correlation maps to analyze sequence space. Using this novel conceptual view we show that representative ensembles of folded and ID proteins show distinct characteristics and respond differently to sequence randomization. By studying the way evolutionary constraints act on IDPs to disable a negative function (aggregation) we might gain insight into the mechanisms by which function-enabling information is encoded in IDPs.

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

    PubMed

    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.

  18. Water promotes the sealing of nanoscale packing defects in folding proteins.

    PubMed

    Fernández, Ariel

    2014-05-21

    A net dipole moment is shown to arise from a non-Debye component of water polarization created by nanoscale packing defects on the protein surface. Accordingly, the protein electrostatic field exerts a torque on the induced dipole, locally impeding the nucleation of ice at the protein-water interface. We evaluate the solvent orientation steering (SOS) as the reversible work needed to align the induced dipoles with the Debye electrostatic field and computed the SOS for the variable interface of a folding protein. The minimization of the SOS is shown to drive protein folding as evidenced by the entrainment of the total free energy by the SOS energy along trajectories that approach a Debye limit state where no torque arises. This result suggests that the minimization of anomalous water polarization at the interface promotes the sealing of packing defects, thereby maintaining structural integrity and committing the protein chain to fold.

  19. Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

    PubMed

    Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim; Turner, Maurice J; Powers, Evan T; Gruebele, Martin; Levy, Yaakov; Kelly, Jeffery W

    2010-11-03

    Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible

  20. Chaperonin-mediated Protein Folding

    PubMed Central

    Horwich, Arthur L.

    2013-01-01

    We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments. PMID:23803606

  1. Homochiral stereochemistry: the missing link of structure to energetics in protein folding.

    PubMed

    Kumar, Anil; Ramakrishnan, Vibin; Ranbhor, Ranjit; Patel, Kirti; Durani, Susheel

    2009-12-24

    The notion is tested that homochiral stereochemistry being ubiquitous to protein structure could be critical to protein folding as well, causing it to become frustrated energetically providing the basis for its solvent- and sequence-mediated control. The proof in support of the notion is found in a consensus of experiment and computation according to which suitable oligopeptides are in their folding-unfolding equilibria, at both macrostate and microstate levels, susceptible to dielectric because of the conflict of peptide-chain electrostatics with interpeptide hydrogen bonds when the structure is poly-L but not when it is alternating-L,D. The argument is thus made that homochiral stereochemistry may in protein folding provide the unifying basis for its solvent- and sequence-mediated control based on screening of peptide-chain electrostatics under conflict with folding of the chain due to homochiral stereochemistry. Dielectric is brought into spotlight as the effect comparatively obscure but presumably critical to the folding in protein structure for its control.

  2. Protein stability: a crystallographer’s perspective

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Deller, Marc C., E-mail: mdeller@stanford.edu; Kong, Leopold; Rupp, Bernhard

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhatmore » practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.« less

  3. STRUM: structure-based prediction of protein stability changes upon single-point mutation.

    PubMed

    Quan, Lijun; Lv, Qiang; Zhang, Yang

    2016-10-01

    Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon

  4. A hybrid MD-kMC algorithm for folding proteins in explicit solvent.

    PubMed

    Peter, Emanuel Karl; Shea, Joan-Emma

    2014-04-14

    We present a novel hybrid MD-kMC algorithm that is capable of efficiently folding proteins in explicit solvent. We apply this algorithm to the folding of a small protein, Trp-Cage. Different kMC move sets that capture different possible rate limiting steps are implemented. The first uses secondary structure formation as a relevant rate event (a combination of dihedral rotations and hydrogen-bonding formation and breakage). The second uses tertiary structure formation events through formation of contacts via translational moves. Both methods fold the protein, but via different mechanisms and with different folding kinetics. The first method leads to folding via a structured helical state, with kinetics fit by a single exponential. The second method leads to folding via a collapsed loop, with kinetics poorly fit by single or double exponentials. In both cases, folding times are faster than experimentally reported values, The secondary and tertiary move sets are integrated in a third MD-kMC implementation, which now leads to folding of the protein via both pathways, with single and double-exponential fits to the rates, and to folding rates in good agreement with experimental values. The competition between secondary and tertiary structure leads to a longer search for the helix-rich intermediate in the case of the first pathway, and to the emergence of a kinetically trapped long-lived molten-globule collapsed state in the case of the second pathway. The algorithm presented not only captures experimentally observed folding intermediates and kinetics, but yields insights into the relative roles of local and global interactions in determining folding mechanisms and rates.

  5. How the folding rates of two- and multistate proteins depend on the amino acid properties.

    PubMed

    Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

    2014-10-01

    Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.

  6. Protein stability: a crystallographer’s perspective

    PubMed Central

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  7. The Energy Landscape, Folding Pathways and the Kinetics of a Knotted Protein

    PubMed Central

    Prentiss, Michael C.; Wales, David J.; Wolynes, Peter G.

    2010-01-01

    The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly. PMID:20617197

  8. Dynamics of partially folded and unfolded proteins investigated with quasielastic neutron spectroscopy

    NASA Astrophysics Data System (ADS)

    Stadler, Andreas M.

    2018-05-01

    Molecular dynamics in proteins animate and play a vital role for biologically relevant processes of these biomacromolecules. Quasielastic incoherent neutron scattering (QENS) is a well-suited experimental method to study protein dynamics from the picosecond to several nanoseconds and in the Ångström length-scale. In QENS experiments of protein solutions hydrogens act as reporters for the motions of methyl groups or amino acids to which they are bound. Neutron Spin-Echo spectroscopy (NSE) offers the highest energy resolution in the field of neutron spectroscopy and allows the study of slow collective motions in proteins up to several hundred nanoseconds and in the nanometer length-scale. In the following manuscript I will review recent studies that stress the relevance of molecular dynamics for protein folding and for conformational transitions of intrinsically disordered proteins (IDPs). During the folding collapse the protein is exploring its accessible conformational space via molecular motions. A large flexibility of partially folded and unfolded proteins, therefore, is mandatory for rapid protein folding. IDPs are a special case as they are largely unstructured under physiological conditions. A large flexibility is a characteristic property of IDPs as it allows, for example, the interaction with various binding partners or the rapid response to different conditions.

  9. An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

    NASA Astrophysics Data System (ADS)

    Alfano, Caterina; Sanfelice, Domenico; Martin, Stephen R.; Pastore, Annalisa; Temussi, Piero Andrea

    2017-05-01

    Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation.

  10. Shaping up the protein folding funnel by local interaction: lesson from a structure prediction study.

    PubMed

    Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

    2006-02-28

    Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of "chimera proteins." In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape.

  11. Faster protein folding using enhanced conformational sampling of molecular dynamics simulation.

    PubMed

    Kamberaj, Hiqmet

    2018-05-01

    In this study, we applied swarm particle-like molecular dynamics (SPMD) approach to enhance conformational sampling of replica exchange simulations. In particular, the approach showed significant improvement in sampling efficiency of conformational phase space when combined with replica exchange method (REM) in computer simulation of peptide/protein folding. First we introduce the augmented dynamical system of equations, and demonstrate the stability of the algorithm. Then, we illustrate the approach by using different fully atomistic and coarse-grained model systems, comparing them with the standard replica exchange method. In addition, we applied SPMD simulation to calculate the time correlation functions of the transitions in a two dimensional surface to demonstrate the enhancement of transition path sampling. Our results showed that folded structure can be obtained in a shorter simulation time using the new method when compared with non-augmented dynamical system. Typically, in less than 0.5 ns using replica exchange runs assuming that native folded structure is known and within simulation time scale of 40 ns in the case of blind structure prediction. Furthermore, the root mean square deviations from the reference structures were less than 2Å. To demonstrate the performance of new method, we also implemented three simulation protocols using CHARMM software. Comparisons are also performed with standard targeted molecular dynamics simulation method. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Application of Time-Resolved Tryptophan Phosphorescence Spectroscopy to Protein Folding Studies.

    NASA Astrophysics Data System (ADS)

    Subramaniam, Vinod

    This thesis presents studies of the protein folding problem, one of the most significant questions in contemporary biophysics. Sensitive biophysical techniques, including room temperature tryptophan phosphorescence, which reports on the local environment of the residue, and the lability of proteins to denaturation, a global parameter, were used to assess the validity of the traditional assumption that the biologically active state of a protein is the 'native' state, and to determine whether the pathways of folding in vitro lead to the folded state achieved in vivo. Phosphorescence techniques have also been extended to study, for the first time, emission from tryptophan residues engineered into specific positions as reporters of protein structure. During in vitro refolding of E. coli alkaline phosphatase and bovine 13-lactoglobulin, significant differences were found between the refolded proteins and the native conformations, which have no apparent effect on the biological functions. Slow conformational transitions, termed 'annealing,' that occur long after the return of enzyme activity of alkaline phosphatase are manifested in the retarded recovery of phosphorescence intensity, lifetime, and protein lability. While 'annealing' is not observed for beta -lactoglobulin, both phosphorescence and lability experiments reveal changes in the structure of the refolded protein, even though its biological activity, retinol binding, is fully recovered. This result suggests that the pathways of folding in vitro need not lead to the structure formed in vivo. We have used phosphorescence techniques to study the refolding of ribonuclease T1, which exhibits slow kinetics characteristic of proline isomerization. Furthermore, the ability to extract structural information from phosphorescent tryptophan probes engineered into selected regions represents an important advance in studying protein structure; we have reported the first such results from a mutant staphylococcal nuclease. The

  13. Molecular basis of the osmolyte effect on protein stability: a lesson from the mechanical unfolding of lysozyme.

    PubMed

    Adamczak, Beata; Wieczór, Miłosz; Kogut, Mateusz; Stangret, Janusz; Czub, Jacek

    2016-10-15

    Osmolytes are a class of small organic molecules that shift the protein folding equilibrium. For this reason, they are accumulated by organisms under environmental stress and find applications in biotechnology where proteins need to be stabilized or dissolved. However, despite years of research, debate continues over the exact mechanisms underpinning the stabilizing and denaturing effect of osmolytes. Here, we simulated the mechanical denaturation of lysozyme in different solvent conditions to study the molecular mechanism by which two biologically relevant osmolytes, denaturing (urea) and stabilizing (betaine), affect the folding equilibrium. We found that urea interacts favorably with all types of residues via both hydrogen bonds and dispersion forces, and therefore accumulates in a diffuse solvation shell around the protein. This not only provides an enthalpic stabilization of the unfolded state, but also weakens the hydrophobic effect, as hydrophobic forces promote the association of urea with nonpolar residues, facilitating the unfolding. In contrast, we observed that betaine is excluded from the protein backbone and nonpolar side chains, but is accumulated near the basic residues, yielding a nonuniform distribution of betaine molecules at the protein surface. Spatially resolved solvent-protein interaction energies further suggested that betaine behaves in a ligand- rather than solvent-like manner and its exclusion from the protein surface arises mostly from the scarcity of favorable binding sites. Finally, we found that, in the presence of betaine, the reduced ability of water molecules to solvate the protein results in an additional enthalpic contribution to the betaine-induced stabilization. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  14. Influence of the native topology on the folding barrier for small proteins

    NASA Astrophysics Data System (ADS)

    Prieto, Lidia; Rey, Antonio

    2007-11-01

    The possibility of downhill instead of two-state folding for proteins has been a very controversial topic which arose from recent experimental studies. From the theoretical side, this question has also been accomplished in different ways. Given the experimental observation that a relationship exists between the native structure topology of a protein and the kinetic and thermodynamic properties of its folding process, Gō-type potentials are an appropriate way to approach this problem. In this work, we employ an interaction potential from this family to get a better insight on the topological characteristics of the native state that may somehow determine the presence of a thermodynamic barrier in the folding pathway. The results presented here show that, indeed, the native topology of a small protein has a great influence on its folding behavior, mostly depending on the proportion of local and long range contacts the protein has in its native structure. Furthermore, when all the interactions present contribute in a balanced way, the transition results to be cooperative. Otherwise, the tendency to a downhill folding behavior increases.

  15. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    DOEpatents

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

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

    NASA Astrophysics Data System (ADS)

    Morrissey, Michael Patrick

    2000-10-01

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

  17. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  18. WeFold: A Coopetition for Protein Structure Prediction

    PubMed Central

    Khoury, George A.; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O.; Faccioli, Rodrigo A.; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A.; Sieradzan, Adam K.; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C. B.; Floudas, Christodoulos A.; Keasar, Chen; Levitt, Michael; Popović, Zoran; Scheraga, Harold A.; Skolnick, Jeffrey; Crivelli, Silvia N.; Players, Foldit

    2014-01-01

    The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by thirteen labs. During the collaboration, the labs were simultaneously competing with each other. Here, we present the first attempt at “coopetition” in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. PMID:24677212

  19. Cleavage Mapping the Topology of Protein Folding Intermediates

    DTIC Science & Technology

    1995-03-13

    1993). 29. Nakano, T., Antonino , L. C, Fox, R. O. & Fink, A. L. Effect of proline mutation on the stability and kinetics of folding of...Biochem. 62, 653-683. Nakano, T., Antonino , L. C., Fox, R. O., & Fink, A. L. (1993) Biochemistry 32, 2534-2541. Nozaki, Y. (1972) Methods Enzymol. 26, 43

  20. Shaping up the protein folding funnel by local interaction: Lesson from a structure prediction study

    PubMed Central

    Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

    2006-01-01

    Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of “chimera proteins.” In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape. PMID:16488978

  1. Novel Protein Folding Pathways for Protein Salvage and Recycling

    DTIC Science & Technology

    2013-08-26

    cooperative protein folding and unfolding reactions. Archaea are primitive microorganisms placed by most taxonomic criteria at the base of the Tree of...Life. The Archaea have many molecular properties that are found universally in modern lineages of both Bacteria and Archaea , and many species are...specialized for survival and growth in extreme conditions, including at or above the normal boiling point of water. These hyperthermophilic archaea

  2. Role of N-terminal residues on folding and stability of C-phycoerythrin: simulation and urea-induced denaturation studies.

    PubMed

    Anwer, Khalid; Sonani, Ravi; Madamwar, Datta; Singh, Parvesh; Khan, Faez; Bisetty, Krishna; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2015-01-01

    The conformational state of biliproteins can be determined by optical properties of the covalently linked chromophores. Recently determined crystal structure of truncated form of α-subunit of cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue provides a new insight into the structure-function relationship of αC-PE. To compare their stabilities, we have measured urea-induced denaturation transitions of the full length αC-PE (FL-αC-PE) and truncated αC-PE (Tr-αC-PE) followed by observing changes in absorbance at 565 nm, fluorescence at 350 and 573 nm, and circular dichroism at 222 nm as a function of [urea], the molar concentration of urea. The transition curve of each protein was analyzed for ΔG(D)(0), the value of Gibbs free energy change on denaturation (ΔG(D)) in the absence of urea; m, the slope (=∂∆G(D)/∂[urea]), and C(m), the midpoint of the denaturation curve, i.e. [urea] at which ΔG(D) = 0. A difference of about 10% in ΔG(D)(0) observed between FL-αC-PE and Tr-αC-PE, suggests that the two proteins are almost equally stable, and the natural deletion of 31 residues from the N-terminal side of the full length protein does not alter its stability. Furthermore, normalization of probes shows that the urea-induced denaturation of both the proteins is a two-state process. Folding of both structural variants (Tr-αC-PE and FL-αC-PE) of P. tenue were also studied using molecular dynamics simulations at 300 K. The results show clearly that the stability of the proteins is evenly distributed over the whole structure indicating no significant role of N-terminal residues in the stability of both proteins.

  3. Processing of Cholinesterase-like α/β-Hydrolase Fold Proteins: Alterations Associated with Congenital Disorders

    PubMed Central

    De Jaco, Antonella; Comoletti, Davide; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2016-01-01

    The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β-hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold. PMID:21933121

  4. Analysis of alpha hemoglobin stabilizing protein overexpression in murine β-thalassemia

    PubMed Central

    Nasimuzzaman, Md; Khandros, Eugene; Wang, Xiaomei; Kong, Yi; Zhao, Huifen; Weiss, David; Rivella, Stefano; Weiss, Mitchell J.; Persons, Derek A.

    2013-01-01

    Excess free α-globin is cytotoxic and contributes to the pathophysiology of β-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free α-globin to promote its folding and inhibit its ability to produce damaging reactive oxygen species. Reduced AHSP levels correlate with increased severity of β-thalassemia in some human cohorts, but causal mechanistic relationships are not established for these associations. We used transgenic and lentiviral gene transfer methods to investigate whether supraphysiologic AHSP levels could mitigate the severity of β-thalassemia intermedia by providing an increased sink for the excess pool of α-globin chains. We tested wild-type AHSP and two mutant versions with amino acid substitutions that confer 3- or 13-fold higher affinity for α-globin. Erythroid overexpression of these AHSP proteins up to 11-fold beyond endogenous levels had no major effects on hematologic parameters in β-thalassemic animals. Our results demonstrate that endogenous AHSP is not limiting for α-globin detoxification in a murine model of β-thalassemia. PMID:20815047

  5. Protein assembly and heat stability in developing thylakoid membranes during greening

    PubMed Central

    Kóta, Zoltán; Horváth, László I.; Droppa, Magdolna; Horváth, Gábor; Farkas, Tibor; Páli, Tibor

    2002-01-01

    The development of the thylakoid membrane was studied during illumination of dark-grown barley seedlings by using biochemical methods, and Fourier transform infrared and spin label electron paramagnetic resonance spectroscopic techniques. Correlated, gross changes in the secondary structure of membrane proteins, conformation, composition, and dynamics of lipid acyl chains, SDS/PAGE pattern, and thermally induced structural alterations show that greening is accompanied with the reorganization of membrane protein assemblies and the protein–lipid interface. Changes in overall membrane fluidity and noncovalent protein–lipid interactions are not monotonic, despite the monotonic accumulation of chlorophyll, LHCII [light-harvesting chlorophyll a/b-binding (polypeptides) associated with photosystem II] apoproteins, and 18:3 fatty acids that follow a similar time course with highest rates between 12–24 h of greening. The 18:3 fatty acid content increases 2.8-fold during greening. This appears to both compensate for lipid immobilization by membrane proteins and facilitate packing of larger protein assemblies. The increase in the amount of protein-solvating immobile lipids, which reaches a maximum at 12 h, is caused by 40% decrease in the membranous mean diameter of protein assemblies at constant protein/lipid mass ratio. Alterations in the SDS/PAGE pattern are most significant between 6–24 h. The size of membrane protein assemblies increases ≈4.5-fold over the 12–48-h period, likely caused by the 2-fold gain in LHCII apoproteins. The thermal stability of thylakoid membrane proteins increases monotonically, as detected by an increasing temperature of partial protein unfolding during greening. Our data suggest that a structural coupling between major protein and lipid components develops during greening. This protein–lipid interaction is required for the development and protection of thylakoid membrane protein assemblies. PMID:12213965

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

    PubMed

    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.

  7. Predictive energy landscapes for folding membrane protein assemblies

    NASA Astrophysics Data System (ADS)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  8. Hierarchical learning architecture with automatic feature selection for multiclass protein fold classification.

    PubMed

    Huang, Chuen-Der; Lin, Chin-Teng; Pal, Nikhil Ranjan

    2003-12-01

    The structure classification of proteins plays a very important role in bioinformatics, since the relationships and characteristics among those known proteins can be exploited to predict the structure of new proteins. The success of a classification system depends heavily on two things: the tools being used and the features considered. For the bioinformatics applications, the role of appropriate features has not been paid adequate importance. In this investigation we use three novel ideas for multiclass protein fold classification. First, we use the gating neural network, where each input node is associated with a gate. This network can select important features in an online manner when the learning goes on. At the beginning of the training, all gates are almost closed, i.e., no feature is allowed to enter the network. Through the training, gates corresponding to good features are completely opened while gates corresponding to bad features are closed more tightly, and some gates may be partially open. The second novel idea is to use a hierarchical learning architecture (HLA). The classifier in the first level of HLA classifies the protein features into four major classes: all alpha, all beta, alpha + beta, and alpha/beta. And in the next level we have another set of classifiers, which further classifies the protein features into 27 folds. The third novel idea is to induce the indirect coding features from the amino-acid composition sequence of proteins based on the N-gram concept. This provides us with more representative and discriminative new local features of protein sequences for multiclass protein fold classification. The proposed HLA with new indirect coding features increases the protein fold classification accuracy by about 12%. Moreover, the gating neural network is found to reduce the number of features drastically. Using only half of the original features selected by the gating neural network can reach comparable test accuracy as that using all the

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

    PubMed

    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.

  10. Natural triple beta-stranded fibrous folds.

    PubMed

    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.

  11. Probing sequence dependence of folding pathway of α-helix bundle proteins through free energy landscape analysis.

    PubMed

    Shao, Qiang

    2014-06-05

    A comparative study on the folding of multiple three-α-helix bundle proteins including α3D, α3W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation-condensation mechanism (e.g., α3D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α3W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.

    PubMed

    Thomas, Sabrina; Bakkes, Patrick J; Smits, Sander H J; Schmitt, Lutz

    2014-09-01

    HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca(2+) binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6-7M urea, relates to Ca(2+) dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Structure of a Trypanosoma Brucei Alpha/Beta--Hydrolase Fold Protein With Unknown Function

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Merritt, E.A.; Holmes, M.; Buckner, F.S.

    2009-05-26

    The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 {angstrom} 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 {alpha}/{beta}-hydrolase fold family. Structural superposition onto representative {alpha}/{beta}-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 similaritymore » at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands {beta}6 and {beta}7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.« less

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

    PubMed

    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

  16. Dynamical Coupling of Intrinsically Disordered Proteins and Their Hydration Water: Comparison with Folded Soluble and Membrane Proteins

    PubMed Central

    Gallat, F.-X.; Laganowsky, A.; Wood, K.; Gabel, F.; van Eijck, L.; Wuttke, J.; Moulin, M.; Härtlein, M.; Eisenberg, D.; Colletier, J.-P.; Zaccai, G.; Weik, M.

    2012-01-01

    Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context. PMID:22828339

  17. Folding pathway of a multidomain protein depends on its topology of domain connectivity

    PubMed Central

    Inanami, Takashi; Terada, Tomoki P.; Sasai, Masaki

    2014-01-01

    How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant. PMID:25267632

  18. Transition Pathway and Its Free-Energy Profile: A Protocol for Protein Folding Simulations

    PubMed Central

    Lee, In-Ho; Kim, Seung-Yeon; Lee, Jooyoung

    2013-01-01

    We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD) method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature. PMID:23917881

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

    PubMed

    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.

  20. Isolation, folding and structural investigations of the amino acid transporter OEP16

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ni, Da Qun; Zook, James; Klewer, Douglas A.

    2011-12-01

    Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on amore » Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of a-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.« less

  1. Isolation, folding and structural investigations of the amino acid transporter OEP16.

    PubMed

    Ni, Da Qun; Zook, James; Klewer, Douglas A; Nieman, Ronald A; Soll, J; Fromme, Petra

    2011-12-01

    Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection. Copyright © 2011. Published by Elsevier Inc.

  2. Effect of osmolytes on the thermal stability of proteins: replica exchange simulations of Trp-cage in urea and betaine solutions.

    PubMed

    Adamczak, Beata; Kogut, Mateusz; Czub, Jacek

    2018-04-25

    Although osmolytes are known to modulate the folding equilibrium, the molecular mechanism of their effect on thermal denaturation of proteins is still poorly understood. Here, we simulated the thermal denaturation of a small model protein (Trp-cage) in the presence of denaturing (urea) and stabilizing (betaine) osmolytes, using the all-atom replica exchange molecular dynamics simulations. We found that urea destabilizes Trp-cage by enthalpically-driven association with the protein, acting synergistically with temperature to induce unfolding. In contrast, betaine is sterically excluded from the protein surface thereby exerting entropic depletion forces that contribute to the stabilization of the native state. In fact, we find that while at low temperatures betaine slightly increases the folding free energy of Trp-cage by promoting another near-native conformation, it protects the protein against temperature-induced denaturation. This, in turn, can be attributed to enhanced exclusion of betaine at higher temperatures that arises from less attractive interactions with the protein surface.

  3. Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels.

    PubMed

    Gloss, L M; Simler, B R; Matthews, C R

    2001-10-05

    The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts. Copyright 2001 Academic Press.

  4. Physical-chemical features of non-detergent sulfobetaines active as protein-folding helpers.

    PubMed

    Expert-Bezançon, Nicole; Rabilloud, Thierry; Vuillard, Laurent; Goldberg, Michel E

    2003-01-01

    Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.

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

    PubMed

    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.

  6. How a Spatial Arrangement of Secondary Structure Elements Is Dispersed in the Universe of Protein Folds

    PubMed Central

    Minami, Shintaro; Sawada, Kengo; Chikenji, George

    2014-01-01

    It has been known that topologically different proteins of the same class sometimes share the same spatial arrangement of secondary structure elements (SSEs). However, the frequency by which topologically different structures share the same spatial arrangement of SSEs is unclear. It is important to estimate this frequency because it provides both a deeper understanding of the geometry of protein folds and a valuable suggestion for predicting protein structures with novel folds. Here we clarified the frequency with which protein folds share the same SSE packing arrangement with other folds, the types of spatial arrangement of SSEs that are frequently observed across different folds, and the diversity of protein folds that share the same spatial arrangement of SSEs with a given fold, using a protein structure alignment program MICAN, which we have been developing. By performing comprehensive structural comparison of SCOP fold representatives, we found that approximately 80% of protein folds share the same spatial arrangement of SSEs with other folds. We also observed that many protein pairs that share the same spatial arrangement of SSEs belong to the different classes, often with an opposing N- to C-terminal direction of the polypeptide chain. The most frequently observed spatial arrangement of SSEs was the 2-layer α/β packing arrangement and it was dispersed among as many as 27% of SCOP fold representatives. These results suggest that the same spatial arrangements of SSEs are adopted by a wide variety of different folds and that the spatial arrangement of SSEs is highly robust against the N- to C-terminal direction of the polypeptide chain. PMID:25243952

  7. Direct folding simulation of helical proteins using an effective polarizable bond force field.

    PubMed

    Duan, Lili; Zhu, Tong; Ji, Changge; Zhang, Qinggang; Zhang, John Z H

    2017-06-14

    We report a direct folding study of seven helical proteins (, Trpcage, , C34, N36, , ) ranging from 17 to 53 amino acids through standard molecular dynamics simulations using a recently developed polarizable force field-Effective Polarizable Bond (EPB) method. The backbone RMSDs, radius of gyrations, native contacts and native helix content are in good agreement with the experimental results. Cluster analysis has also verified that these folded structures with the highest population are in good agreement with their corresponding native structures for these proteins. In addition, the free energy landscape of seven proteins in the two dimensional space comprised of RMSD and radius of gyration proved that these folded structures are indeed of the lowest energy conformations. However, when the corresponding simulations were performed using the standard (nonpolarizable) AMBER force fields, no stable folded structures were observed for these proteins. Comparison of the simulation results based on a polarizable EPB force field and a nonpolarizable AMBER force field clearly demonstrates the importance of polarization in the folding of stable helical structures.

  8. A protein block based fold recognition method for the annotation of twilight zone sequences.

    PubMed

    Suresh, V; Ganesan, K; Parthasarathy, S

    2013-03-01

    The description of protein backbone was recently improved with a group of structural fragments called Structural Alphabets instead of the regular three states (Helix, Sheet and Coil) secondary structure description. Protein Blocks is one of the Structural Alphabets used to describe each and every region of protein backbone including the coil. According to de Brevern (2000) the Protein Blocks has 16 structural fragments and each one has 5 residues in length. Protein Blocks fragments are highly informative among the available Structural Alphabets and it has been used for many applications. Here, we present a protein fold recognition method based on Protein Blocks for the annotation of twilight zone sequences. In our method, we align the predicted Protein Blocks of a query amino acid sequence with a library of assigned Protein Blocks of 953 known folds using the local pair-wise alignment. The alignment results with z-value ≥ 2.5 and P-value ≤ 0.08 are predicted as possible folds. Our method is able to recognize the possible folds for nearly 35.5% of the twilight zone sequences with their predicted Protein Block sequence obtained by pb_prediction, which is available at Protein Block Export server.

  9. Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

    PubMed Central

    Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

    2013-01-01

    The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

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

    PubMed

    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.

  11. Molecular chaperones and protein folding as therapeutic targets in Parkinson's disease and other synucleinopathies.

    PubMed

    Ebrahimi-Fakhari, Darius; Saidi, Laiq-Jan; Wahlster, Lara

    2013-12-05

    Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson's disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson's disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson's disease and related neurodegenerative disorders.

  12. FK506-Binding Protein 22 from a Psychrophilic Bacterium, a Cold Shock-Inducible Peptidyl Prolyl Isomerase with the Ability to Assist in Protein Folding

    PubMed Central

    Budiman, Cahyo; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2011-01-01

    Adaptation of microorganisms to low temperatures remains to be fully elucidated. It has been previously reported that peptidyl prolyl cis-trans isomerases (PPIases) are involved in cold adaptation of various microorganisms whether they are hyperthermophiles, mesophiles or phsycrophiles. The rate of cis-trans isomerization at low temperatures is much slower than that at higher temperatures and may cause problems in protein folding. However, the mechanisms by which PPIases are involved in cold adaptation remain unclear. Here we used FK506-binding protein 22, a cold shock protein from the psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) as a model protein to decipher the involvement of PPIases in cold adaptation. SIB1 FKBP22 is homodimer that assumes a V-shaped structure based on a tertiary model. Each monomer consists of an N-domain responsible for dimerization and a C-catalytic domain. SIB1 FKBP22 is a typical cold-adapted enzyme as indicated by the increase of catalytic efficiency at low temperatures, the downward shift in optimal temperature of activity and the reduction in the conformational stability. SIB1 FKBP22 is considered as foldase and chaperone based on its ability to catalyze refolding of a cis-proline containing protein and bind to a folding intermediate protein, respectively. The foldase and chaperone activites of SIB1 FKBP22 are thought to be important for cold adaptation of Shewanella sp. SIB1. These activities are also employed by other PPIases for being involved in cold adaptation of various microorganisms. Despite other biological roles of PPIases, we proposed that foldase and chaperone activities of PPIases are the main requirement for overcoming the cold-stress problem in microorganisms due to folding of proteins. PMID:21954357

  13. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-27

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

  14. Use of conserved key amino acid positions to morph protein folds.

    PubMed

    Reddy, Boojala V B; Li, Wilfred W; Bourne, Philip E

    2002-07-15

    By using three-dimensional (3D) structure alignments and a previously published method to determine Conserved Key Amino Acid Positions (CKAAPs) we propose a theoretical method to design mutations that can be used to morph the protein folds. The original Paracelsus challenge, met by several groups, called for the engineering of a stable but different structure by modifying less than 50% of the amino acid residues. We have used the sequences from the Protein Data Bank (PDB) identifiers 1ROP, and 2CRO, which were previously used in the Paracelsus challenge by those groups, and suggest mutation to CKAAPs to morph the protein fold. The total number of mutations suggested is less than 40% of the starting sequence theoretically improving the challenge results. From secondary structure prediction experiments of the proposed mutant sequence structures, we observe that each of the suggested mutant protein sequences likely folds to a different, non-native potentially stable target structure. These results are an early indicator that analyses using structure alignments leading to CKAAPs of a given structure are of value in protein engineering experiments. Copyright 2002 Wiley Periodicals, Inc.

  15. Physical and molecular bases of protein thermal stability and cold adaptation.

    PubMed

    Pucci, Fabrizio; Rooman, Marianne

    2017-02-01

    The molecular bases of thermal and cold stability and adaptation, which allow proteins to remain folded and functional in the temperature ranges in which their host organisms live and grow, are still only partially elucidated. Indeed, both experimental and computational studies fail to yield a fully precise and global physical picture, essentially because all effects are context-dependent and thus quite intricate to unravel. We present a snapshot of the current state of knowledge of this highly complex and challenging issue, whose resolution would enable large-scale rational protein design. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Structure and mechanism of maximum stability of isolated alpha-helical protein domains at a critical length scale.

    PubMed

    Qin, Zhao; Fabre, Andrea; Buehler, Markus J

    2013-05-01

    The stability of alpha helices is important in protein folding, bioinspired materials design, and controls many biological properties under physiological and disease conditions. Here we show that a naturally favored alpha helix length of 9 to 17 amino acids exists at which the propensity towards the formation of this secondary structure is maximized. We use a combination of thermodynamical analysis, well-tempered metadynamics molecular simulation and statistical analyses of experimental alpha helix length distributions and find that the favored alpha helix length is caused by a competition between alpha helix folding, unfolding into a random coil and formation of higher-order tertiary structures. The theoretical result is suggested to be used to explain the statistical distribution of the length of alpha helices observed in natural protein structures. Our study provides mechanistic insight into fundamental controlling parameters in alpha helix structure formation and potentially other biopolymers or synthetic materials. The result advances our fundamental understanding of size effects in the stability of protein structures and may enable the design of de novo alpha-helical protein materials.

  17. Statistical mechanics of simple models of protein folding and design.

    PubMed Central

    Pande, V S; Grosberg, A Y; Tanaka, T

    1997-01-01

    It is now believed that the primary equilibrium aspects of simple models of protein folding are understood theoretically. However, current theories often resort to rather heavy mathematics to overcome some technical difficulties inherent in the problem or start from a phenomenological model. To this end, we take a new approach in this pedagogical review of the statistical mechanics of protein folding. The benefit of our approach is a drastic mathematical simplification of the theory, without resort to any new approximations or phenomenological prescriptions. Indeed, the results we obtain agree precisely with previous calculations. Because of this simplification, we are able to present here a thorough and self contained treatment of the problem. Topics discussed include the statistical mechanics of the random energy model (REM), tests of the validity of REM as a model for heteropolymer freezing, freezing transition of random sequences, phase diagram of designed ("minimally frustrated") sequences, and the degree to which errors in the interactions employed in simulations of either folding and design can still lead to correct folding behavior. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:9414231

  18. Extracting features from protein sequences to improve deep extreme learning machine for protein fold recognition.

    PubMed

    Ibrahim, Wisam; Abadeh, Mohammad Saniee

    2017-05-21

    Protein fold recognition is an important problem in bioinformatics to predict three-dimensional structure of a protein. One of the most challenging tasks in protein fold recognition problem is the extraction of efficient features from the amino-acid sequences to obtain better classifiers. In this paper, we have proposed six descriptors to extract features from protein sequences. These descriptors are applied in the first stage of a three-stage framework PCA-DELM-LDA to extract feature vectors from the amino-acid sequences. Principal Component Analysis PCA has been implemented to reduce the number of extracted features. The extracted feature vectors have been used with original features to improve the performance of the Deep Extreme Learning Machine DELM in the second stage. Four new features have been extracted from the second stage and used in the third stage by Linear Discriminant Analysis LDA to classify the instances into 27 folds. The proposed framework is implemented on the independent and combined feature sets in SCOP datasets. The experimental results show that extracted feature vectors in the first stage could improve the performance of DELM in extracting new useful features in second stage. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Characterization of the amino acid contribution to the folding degree of proteins.

    PubMed

    Estrada, Ernesto

    2004-03-01

    The folding degree index (Estrada, Bioinformatics 2002;18:697-704) is extended to account for the contribution of amino acids to folding. First, the mathematical formalism for extending the folding degree index is presented. Then, the amino acid contributions to folding degree of several proteins are used to analyze its relation to secondary structure. The possibilities of using these contributions in helping or checking the assignation of secondary structure to amino acids are also introduced. The influence of external factors to the amino acids contribution to folding degree is studied through the temperature effect on ribonuclease A. Finally, the analysis of 3D protein similarity through the use of amino acid contributions to folding degree is studied by selecting a series of lysozymes. These results are compared to that obtained by sequence alignment (2D similarity) and 3D superposition of the structures, showing the uniqueness of the current approach. Copyright 2004 Wiley-Liss, Inc.

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

    PubMed

    Mallik, Saurav; Kundu, Sudip

    2015-08-04

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

  1. Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry.

    PubMed

    Pan, Hai; Raza, Ashraf S; Smith, David L

    2004-03-05

    Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.

  2. Balancing energy and entropy: A minimalist model for the characterization of protein folding landscapes

    PubMed Central

    Das, Payel; Matysiak, Silvina; Clementi, Cecilia

    2005-01-01

    Coarse-grained models have been extremely valuable in promoting our understanding of protein folding. However, the quantitative accuracy of existing simplified models is strongly hindered either from the complete removal of frustration (as in the widely used Gō-like models) or from the compromise with the minimal frustration principle and/or realistic protein geometry (as in the simple on-lattice models). We present a coarse-grained model that “naturally” incorporates sequence details and energetic frustration into an overall minimally frustrated folding landscape. The model is coupled with an optimization procedure to design the parameters of the protein Hamiltonian to fold into a desired native structure. The application to the study of src-Src homology 3 domain shows that this coarse-grained model contains the main physical-chemical ingredients that are responsible for shaping the folding landscape of this protein. The results illustrate the importance of nonnative interactions and energetic heterogeneity for a quantitative characterization of folding mechanisms. PMID:16006532

  3. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  4. Molecular Simulations of Mutually Exclusive Folding in a Two-Domain Protein Switch

    PubMed Central

    Mills, Brandon M.; Chong, Lillian T.

    2011-01-01

    A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches. PMID:21281591

  5. An alternative view of protein fold space.

    PubMed

    Shindyalov, I N; Bourne, P E

    2000-02-15

    Comparing and subsequently classifying protein structures information has received significant attention concurrent with the increase in the number of experimentally derived 3-dimensional structures. Classification schemes have focused on biological function found within protein domains and on structure classification based on topology. Here an alternative view is presented that groups substructures. Substructures are long (50-150 residue) highly repetitive near-contiguous pieces of polypeptide chain that occur frequently in a set of proteins from the PDB defined as structurally non-redundant over the complete polypeptide chain. The substructure classification is based on a previously reported Combinatorial Extension (CE) algorithm that provides a significantly different set of structure alignments than those previously described, having, for example, only a 40% overlap with FSSP. Qualitatively the algorithm provides longer contiguous aligned segments at the price of a slightly higher root-mean-square deviation (rmsd). Clustering these alignments gives a discreet and highly repetitive set of substructures not detectable by sequence similarity alone. In some cases different substructures represent all or different parts of well known folds indicative of the Russian doll effect--the continuity of protein fold space. In other cases they fall into different structure and functional classifications. It is too early to determine whether these newly classified substructures represent new insights into the evolution of a structural framework important to many proteins. What is apparent from on-going work is that these substructures have the potential to be useful probes in finding remote sequence homology and in structure prediction studies. The characteristics of the complete all-by-all comparison of the polypeptide chains present in the PDB and details of the filtering procedure by pair-wise structure alignment that led to the emergent substructure gallery are discussed

  6. Engineering diverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state.

    PubMed

    Simpson, Emma R; Meldrum, Jill K; Searle, Mark S

    2006-04-04

    Using the N-terminal 17-residue beta-hairpin of ubiquitin as a "host" for mutational studies, we have investigated the influence of the beta-turn sequence on protein stability and folding kinetics by replacing the native G-bulged turn (TLTGK) with more flexible analogues (TG3K and TG5K) and a series of four-residue type I' beta-turn sequences, commonly found in beta-hairpins. Although a statistical analysis of type I' turns demonstrates residue preferences at specific sites, the frequency of occurrence appears to only broadly correlate with experimentally determined protein stabilities. The subsequent engineering of context-dependent non-native tertiary contacts involving turn residues is shown to produce large changes in stability. Relatively few point mutations have been described that probe secondary structure formation in ubiquitin in a manner that is independent of tertiary contacts. To this end, we have used the more rigorous rate-equilibrium free energy relationship (Leffler analysis), rather than the two-point phi value analysis, to show for a family of engineered beta-turn mutants that stability (range of approximately 20 kJ/mol) and folding kinetics (190-fold variation in refolding rate) are linearly correlated (alpha(f) = 0.74 +/- 0.08). The data are consistent with a transition state that is robust with regard to a wide range of statistically favored and disfavored beta-turn mutations and implicate a loosely assembled beta-hairpin as a key template in transition state stabilization with the beta-turn playing a central role.

  7. Towards quantitative classification of folded proteins in terms of elementary functions.

    PubMed

    Hu, Shuangwei; Krokhotin, Andrei; Niemi, Antti J; Peng, Xubiao

    2011-04-01

    A comparative classification scheme provides a good basis for several approaches to understand proteins, including prediction of relations between their structure and biological function. But it remains a challenge to combine a classification scheme that describes a protein starting from its well-organized secondary structures and often involves direct human involvement, with an atomary-level physics-based approach where a protein is fundamentally nothing more than an ensemble of mutually interacting carbon, hydrogen, oxygen, and nitrogen atoms. In order to bridge these two complementary approaches to proteins, conceptually novel tools need to be introduced. Here we explain how an approach toward geometric characterization of entire folded proteins can be based on a single explicit elementary function that is familiar from nonlinear physical systems where it is known as the kink soliton. Our approach enables the conversion of hierarchical structural information into a quantitative form that allows for a folded protein to be characterized in terms of a small number of global parameters that are in principle computable from atomary-level considerations. As an example we describe in detail how the native fold of the myoglobin 1M6C emerges from a combination of kink solitons with a very high atomary-level accuracy. We also verify that our approach describes longer loops and loops connecting α helices with β strands, with the same overall accuracy. ©2011 American Physical Society

  8. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

  9. Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

    PubMed

    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.

  10. Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hsu, P. J.; Lai, S. K., E-mail: sklai@coll.phy.ncu.edu.tw; Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan

    Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1)more » ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such

  11. Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

    NASA Astrophysics Data System (ADS)

    Hsu, P. J.; Cheong, S. A.; Lai, S. K.

    2014-05-01

    Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein

  12. Tackling force-field bias in protein folding simulations: folding of Villin HP35 and Pin WW domains in explicit water.

    PubMed

    Mittal, Jeetain; Best, Robert B

    2010-08-04

    The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly "transferable." Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03( *)) results in an unbiased energy function. We take as examples the 35-residue alpha-helical Villin HP35 and 37 residue beta-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03( *) in explicit solvent fold to within 2.0 A RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

    PubMed

    Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

    2013-02-01

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. The mechanism of folding robustness revealed by the crystal structure of extra-superfolder GFP.

    PubMed

    Choi, Jae Young; Jang, Tae-Ho; Park, Hyun Ho

    2017-01-01

    Stability of green fluorescent protein (GFP) is sometimes important for a proper practical application of this protein. Random mutagenesis and targeted mutagenesis have been used to create better-folded variants of GFP, including recently reported extra-superfolder GFP. Our aim was to determine the crystal structure of extra-superfolder GFP, which is more robustly folded and stable than GFP and superfolder GFP. The structural and structure-based mutagenesis analyses revealed that some of the mutations that created extra-superfolder GFP (F46L, E126K, N149K, and S208L) contribute to folding robustness by stabilizing extra-superfolder GFP with various noncovalent bonds. © 2016 Federation of European Biochemical Societies.

  15. Investigating homology between proteins using energetic profiles.

    PubMed

    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

  16. Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein.

    PubMed Central

    Van Brederode, M E; Hoff, W D; Van Stokkum, I H; Groot, M L; Hellingwerf, K J

    1996-01-01

    Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein. PMID:8804619

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

    PubMed

    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.

  18. Folding of polyglutamine chains

    NASA Astrophysics Data System (ADS)

    Chopra, Manan; Reddy, Allam S.; Abbott, N. L.; de Pablo, J. J.

    2008-10-01

    Long polyglutamine chains have been associated with a number of neurodegenerative diseases. These include Huntington's disease, where expanded polyglutamine (PolyQ) sequences longer than 36 residues are correlated with the onset of symptoms. In this paper we study the folding pathway of a 54-residue PolyQ chain into a β-helical structure. Transition path sampling Monte Carlo simulations are used to generate unbiased reactive pathways between unfolded configurations and the folded β-helical structure of the polyglutamine chain. The folding process is examined in both explicit water and an implicit solvent. Both models reveal that the formation of a few critical contacts is necessary and sufficient for the molecule to fold. Once the primary contacts are formed, the fate of the protein is sealed and it is largely committed to fold. We find that, consistent with emerging hypotheses about PolyQ aggregation, a stable β-helical structure could serve as the nucleus for subsequent polymerization of amyloid fibrils. Our results indicate that PolyQ sequences shorter than 36 residues cannot form that nucleus, and it is also shown that specific mutations inferred from an analysis of the simulated folding pathway exacerbate its stability.

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

    PubMed

    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.

  20. Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

    PubMed Central

    Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

    2014-01-01

    Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

  1. Invariant patterns in crystal lattices: Implications for protein folding algorithms

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

    Crystal lattices are infinite periodic graphs that occur naturally in a variety of geometries and which are of fundamental importance in polymer science. Discrete models of protein folding use crystal lattices to define the space of protein conformations. Because various crystal lattices provide discretizations of the same physical phenomenon, it is reasonable to expect that there will exist invariants across lattices related to fundamental properties of the protein folding process. This paper considers whether performance-guaranteed approximability is such an invariant for HP lattice models. The authors define a master approximation algorithm that has provable performance guarantees provided that a specificmore » sublattice exists within a given lattice. They describe a broad class of crystal lattices that are approximable, which further suggests that approximability is a general property of HP lattice models.« less

  2. Protein Folding and the Challenges of Maintaining Endoplasmic Reticulum Proteostasis in Idiopathic Pulmonary Fibrosis.

    PubMed

    Romero, Freddy; Summer, Ross

    2017-11-01

    Alveolar epithelial type II (AEII) cells are "professional" secretory cells that synthesize and secrete massive quantities of proteins to produce pulmonary surfactant and maintain airway immune defenses. To facilitate this high level of protein synthesis, AEII cells are equipped with an elaborate endoplasmic reticulum (ER) structure and possess an abundance of the machinery needed to fold, assemble, and secrete proteins. However, conditions that suddenly increase the quantity of new proteins entering the ER or that impede the capacity of the ER to fold proteins can cause misfolded or unfolded proteins to accumulate in the ER lumen, also called ER stress. To minimize this stress, AEII cells adapt by (1) reducing the quantity of proteins entering the ER, (2) increasing the amount of protein-folding machinery, and (3) removing misfolded proteins when they accumulate. Although these adaptive responses, aptly named the unfolded protein response, are usually effective in reducing ER stress, chronic aggregation of misfolded proteins is recognized as a hallmark feature of AEII cells in patients with idiopathic pulmonary fibrosis (IPF). Although mutations in surfactant proteins are linked to the development of ER stress in some rare IPF cases, the mechanisms causing protein misfolding in most cases are unknown. In this article, we review the mechanisms regulating ER proteostasis and highlight specific aspects of protein folding and the unfolded protein response that are most vulnerable to failure. Then, we postulate mechanisms other than genetic mutations that might contribute to protein aggregation in the alveolar epithelium of IPF lung.

  3. An ensemble approach to protein fold classification by integration of template-based assignment and support vector machine classifier.

    PubMed

    Xia, Jiaqi; Peng, Zhenling; Qi, Dawei; Mu, Hongbo; Yang, Jianyi

    2017-03-15

    Protein fold classification is a critical step in protein structure prediction. There are two possible ways to classify protein folds. One is through template-based fold assignment and the other is ab-initio prediction using machine learning algorithms. Combination of both solutions to improve the prediction accuracy was never explored before. We developed two algorithms, HH-fold and SVM-fold for protein fold classification. HH-fold is a template-based fold assignment algorithm using the HHsearch program. SVM-fold is a support vector machine-based ab-initio classification algorithm, in which a comprehensive set of features are extracted from three complementary sequence profiles. These two algorithms are then combined, resulting to the ensemble approach TA-fold. We performed a comprehensive assessment for the proposed methods by comparing with ab-initio methods and template-based threading methods on six benchmark datasets. An accuracy of 0.799 was achieved by TA-fold on the DD dataset that consists of proteins from 27 folds. This represents improvement of 5.4-11.7% over ab-initio methods. After updating this dataset to include more proteins in the same folds, the accuracy increased to 0.971. In addition, TA-fold achieved >0.9 accuracy on a large dataset consisting of 6451 proteins from 184 folds. Experiments on the LE dataset show that TA-fold consistently outperforms other threading methods at the family, superfamily and fold levels. The success of TA-fold is attributed to the combination of template-based fold assignment and ab-initio classification using features from complementary sequence profiles that contain rich evolution information. http://yanglab.nankai.edu.cn/TA-fold/. yangjy@nankai.edu.cn or mhb-506@163.com. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  4. Can natural proteins designed with 'inverted' peptide sequences adopt native-like protein folds?

    PubMed

    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.

  5. Electrostatically Accelerated Coupled Binding and Folding of Intrinsically Disordered Proteins

    PubMed Central

    Ganguly, Debabani; Otieno, Steve; Waddell, Brett; Iconaru, Luigi; Kriwacki, Richard W.; Chen, Jianhan

    2012-01-01

    Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27Kip1 (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering), but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition. PMID:22721951

  6. Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

    PubMed

    Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

    2013-10-29

    During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

  7. Structural basis for the enhanced stability of protein model compounds and peptide backbone unit in ammonium ionic liquids.

    PubMed

    Vasantha, T; Attri, Pankaj; Venkatesu, Pannuru; Devi, R S Rama

    2012-10-04

    Protein folding/unfolding is a fascinating study in the presence of cosolvents, which protect/disrupt the native structure of protein, respectively. The structure and stability of proteins and their functional groups may be modulated by the addition of cosolvents. Ionic liquids (ILs) are finding a vast array of applications as novel cosolvents for a wide variety of biochemical processes that include protein folding. Here, the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of protein model compounds from water to ILs through solubility measurements as a function of IL concentration at 25 °C have been exploited to quantify and interpret biomolecular interactions between model compounds of glycine peptides (GPs) with ammonium based ILs. The investigated aqueous systems consist of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), tetraglycine (Gly(4)), and cyclic glycylglycine (c(GG)) in the presence of six ILs such as diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). We have observed positive values of ΔG'(tr) for GPs from water to ILs, indicating that interactions between ILs and GPs are unfavorable, which leads to stabilization of the structure of model protein compounds. Moreover, our experimental data ΔG'(tr) is used to obtain transfer free energies (Δg'(tr)) of the peptide backbone unit (or glycyl unit) (-CH(2)C═ONH-), which is the most numerous group in globular proteins, from water to IL solutions. To obtain the mechanism events of the ILs' role in enhancing the stability of the model compounds, we have further obtained m-values for GPs from solubility limits. These results explicitly elucidate that all alkyl ammonium ILs act as stabilizers for model compounds through the exclusion of ILs from model compounds of

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

    PubMed

    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.

  9. Using Chou's general PseAAC to analyze the evolutionary relationship of receptor associated proteins (RAP) with various folding patterns of protein domains.

    PubMed

    Muthu Krishnan, S

    2018-05-14

    The receptor-associated protein (RAP) is an inhibitor of endocytic receptors that belong to the lipoprotein receptor gene family. In this study, a computational approach was tried to find the evolutionarily related fold of the RAP proteins. Through the structural and sequence-based analysis, found various protein folds that are very close to the RAP folds. Remote homolog datasets were used potentially to develop a different support vector machine (SVM) methods to recognize the homologous RAP fold. This study helps in understanding the relationship of RAP homologs folds based on the structure, function and evolutionary history. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

  11. Information and redundancy in the burial folding code of globular proteins within a wide range of shapes and sizes.

    PubMed

    Ferreira, Diogo C; van der Linden, Marx G; de Oliveira, Leandro C; Onuchic, José N; de Araújo, Antônio F Pereira

    2016-04-01

    Recent ab initio folding simulations for a limited number of small proteins have corroborated a previous suggestion that atomic burial information obtainable from sequence could be sufficient for tertiary structure determination when combined to sequence-independent geometrical constraints. Here, we use simulations parameterized by native burials to investigate the required amount of information in a diverse set of globular proteins comprising different structural classes and a wide size range. Burial information is provided by a potential term pushing each atom towards one among a small number L of equiprobable concentric layers. An upper bound for the required information is provided by the minimal number of layers L(min) still compatible with correct folding behavior. We obtain L(min) between 3 and 5 for seven small to medium proteins with 50 ≤ Nr ≤ 110 residues while for a larger protein with Nr = 141 we find that L ≥ 6 is required to maintain native stability. We additionally estimate the usable redundancy for a given L ≥ L(min) from the burial entropy associated to the largest folding-compatible fraction of "superfluous" atoms, for which the burial term can be turned off or target layers can be chosen randomly. The estimated redundancy for small proteins with L = 4 is close to 0.8. Our results are consistent with the above-average quality of burial predictions used in previous simulations and indicate that the fraction of approachable proteins could increase significantly with even a mild, plausible, improvement on sequence-dependent burial prediction or on sequence-independent constraints that augment the detectable redundancy during simulations. © 2016 Wiley Periodicals, Inc.

  12. Role of Urea-Aromatic Stacking Interactions in Stabilizing the Aromatic Residues of the Protein in Urea-Induced Denatured State.

    PubMed

    Goyal, Siddharth; Chattopadhyay, Aditya; Kasavajhala, Koushik; Priyakumar, U Deva

    2017-10-25

    A delicate balance of different types of intramolecular interactions makes the folded states of proteins marginally more stable than the unfolded states. Experiments use thermal, chemical, or mechanical stress to perturb the folding equilibrium for examining protein stability and the protein folding process. Elucidation of the mechanism by which chemical denaturants unfold proteins is crucial; this study explores the nature of urea-aromatic interactions relevant in urea-assisted protein denaturation. Free energy profiles corresponding to the unfolding of Trp-cage miniprotein in the presence and absence of urea at three different temperatures demonstrate the distortion of the hydrophobic core to be a crucial step. Exposure of the Trp6 residue to the solvent is found to be favored in the presence of urea. Previous experiments showed that urea has a high affinity for aromatic groups of proteins. We show here that this is due to the remarkable ability of urea to form stacking and NH-π interactions with aromatic groups of proteins. Urea-nucleobase stacking interactions have been shown to be crucial in urea-assisted RNA unfolding. Examination of these interactions using microsecond-long unrestrained simulations shows that urea-aromatic stacking interactions are stabilizing and long lasting. Further MD simulations, thermodynamic integration, and quantum mechanical calculations on aromatic model systems reveal that such interactions are possible for all the aromatic amino acid side-chains. Finally, we validate the ubiquitous nature of urea-aromatic stacking interactions by analyzing experimental structures of urea transporters and proteins crystallized in the presence of urea or urea derivatives.

  13. Topology-based modeling of intrinsically disordered proteins: balancing intrinsic folding and intermolecular interactions.

    PubMed

    Ganguly, Debabani; Chen, Jianhan

    2011-04-01

    Coupled binding and folding is frequently involved in specific recognition of so-called intrinsically disordered proteins (IDPs), a newly recognized class of proteins that rely on a lack of stable tertiary fold for function. Here, we exploit topology-based Gō-like modeling as an effective tool for the mechanism of IDP recognition within the theoretical framework of minimally frustrated energy landscape. Importantly, substantial differences exist between IDPs and globular proteins in both amino acid sequence and binding interface characteristics. We demonstrate that established Gō-like models designed for folded proteins tend to over-estimate the level of residual structures in unbound IDPs, whereas under-estimating the strength of intermolecular interactions. Such systematic biases have important consequences in the predicted mechanism of interaction. A strategy is proposed to recalibrate topology-derived models to balance intrinsic folding propensities and intermolecular interactions, based on experimental knowledge of the overall residual structure level and binding affinity. Applied to pKID/KIX, the calibrated Gō-like model predicts a dominant multistep sequential pathway for binding-induced folding of pKID that is initiated by KIX binding via the C-terminus in disordered conformations, followed by binding and folding of the rest of C-terminal helix and finally the N-terminal helix. This novel mechanism is consistent with key observations derived from a recent NMR titration and relaxation dispersion study and provides a molecular-level interpretation of kinetic rates derived from dispersion curve analysis. These case studies provide important insight into the applicability and potential pitfalls of topology-based modeling for studying IDP folding and interaction in general. Copyright © 2011 Wiley-Liss, Inc.

  14. Fold independent structural comparisons of protein-ligand binding sites for exploring functional relationships.

    PubMed

    Gold, Nicola D; Jackson, Richard M

    2006-02-03

    The rapid growth in protein structural data and the emergence of structural genomics projects have increased the need for automatic structure analysis and tools for function prediction. Small molecule recognition is critical to the function of many proteins; therefore, determination of ligand binding site similarity is important for understanding ligand interactions and may allow their functional classification. Here, we present a binding sites database (SitesBase) that given a known protein-ligand binding site allows rapid retrieval of other binding sites with similar structure independent of overall sequence or fold similarity. However, each match is also annotated with sequence similarity and fold information to aid interpretation of structure and functional similarity. Similarity in ligand binding sites can indicate common binding modes and recognition of similar molecules, allowing potential inference of function for an uncharacterised protein or providing additional evidence of common function where sequence or fold similarity is already known. Alternatively, the resource can provide valuable information for detailed studies of molecular recognition including structure-based ligand design and in understanding ligand cross-reactivity. Here, we show examples of atomic similarity between superfamily or more distant fold relatives as well as between seemingly unrelated proteins. Assignment of unclassified proteins to structural superfamiles is also undertaken and in most cases substantiates assignments made using sequence similarity. Correct assignment is also possible where sequence similarity fails to find significant matches, illustrating the potential use of binding site comparisons for newly determined proteins.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region.

    PubMed

    Hagihara, Yoshihisa; Mine, Shouhei; Uegaki, Koichi

    2007-12-14

    We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.

  17. Internal friction controls the speed of protein folding from a compact configuration.

    PubMed

    Pabit, Suzette A; Roder, Heinrich; Hagen, Stephen J

    2004-10-05

    Several studies have found millisecond protein folding reactions to be controlled by the viscosity of the solvent: Reducing the viscosity allows folding to accelerate. In the limit of very low solvent viscosity, however, one expects a different behavior. Internal interactions, occurring within the solvent-excluded interior of a compact molecule, should impose a solvent-independent upper limit to folding speed once the bulk diffusional motions become sufficiently rapid. Why has this not been observed? We have studied the effect of solvent viscosity on the folding of cytochrome c from a highly compact, late-stage intermediate configuration. Although the folding rate accelerates as the viscosity declines, it tends toward a finite limiting value approximately 10(5) s(-1) as the viscosity tends toward zero. This limiting rate is independent of the cosolutes used to adjust solvent friction. Therefore, interactions within the interior of a compact denatured polypeptide can limit the folding rate, but the limiting time scale is very fast. It is only observable when the solvent-controlled stages of folding are exceedingly rapid or else absent. Interestingly, we find a very strong temperature dependence in these "internal friction"-controlled dynamics, indicating a large energy scale for the interactions that govern reconfiguration within compact, near-native states of a protein.

  18. An improved method to detect correct protein folds using partial clustering.

    PubMed

    Zhou, Jianjun; Wishart, David S

    2013-01-16

    Structure-based clustering is commonly used to identify correct protein folds among candidate folds (also called decoys) generated by protein structure prediction programs. However, traditional clustering methods exhibit a poor runtime performance on large decoy sets. We hypothesized that a more efficient "partial" clustering approach in combination with an improved scoring scheme could significantly improve both the speed and performance of existing candidate selection methods. We propose a new scheme that performs rapid but incomplete clustering on protein decoys. Our method detects structurally similar decoys (measured using either C(α) RMSD or GDT-TS score) and extracts representatives from them without assigning every decoy to a cluster. We integrated our new clustering strategy with several different scoring functions to assess both the performance and speed in identifying correct or near-correct folds. Experimental results on 35 Rosetta decoy sets and 40 I-TASSER decoy sets show that our method can improve the correct fold detection rate as assessed by two different quality criteria. This improvement is significantly better than two recently published clustering methods, Durandal and Calibur-lite. Speed and efficiency testing shows that our method can handle much larger decoy sets and is up to 22 times faster than Durandal and Calibur-lite. The new method, named HS-Forest, avoids the computationally expensive task of clustering every decoy, yet still allows superior correct-fold selection. Its improved speed, efficiency and decoy-selection performance should enable structure prediction researchers to work with larger decoy sets and significantly improve their ab initio structure prediction performance.

  19. An improved method to detect correct protein folds using partial clustering

    PubMed Central

    2013-01-01

    Background Structure-based clustering is commonly used to identify correct protein folds among candidate folds (also called decoys) generated by protein structure prediction programs. However, traditional clustering methods exhibit a poor runtime performance on large decoy sets. We hypothesized that a more efficient “partial“ clustering approach in combination with an improved scoring scheme could significantly improve both the speed and performance of existing candidate selection methods. Results We propose a new scheme that performs rapid but incomplete clustering on protein decoys. Our method detects structurally similar decoys (measured using either Cα RMSD or GDT-TS score) and extracts representatives from them without assigning every decoy to a cluster. We integrated our new clustering strategy with several different scoring functions to assess both the performance and speed in identifying correct or near-correct folds. Experimental results on 35 Rosetta decoy sets and 40 I-TASSER decoy sets show that our method can improve the correct fold detection rate as assessed by two different quality criteria. This improvement is significantly better than two recently published clustering methods, Durandal and Calibur-lite. Speed and efficiency testing shows that our method can handle much larger decoy sets and is up to 22 times faster than Durandal and Calibur-lite. Conclusions The new method, named HS-Forest, avoids the computationally expensive task of clustering every decoy, yet still allows superior correct-fold selection. Its improved speed, efficiency and decoy-selection performance should enable structure prediction researchers to work with larger decoy sets and significantly improve their ab initio structure prediction performance. PMID:23323835

  20. Some physical approaches to protein folding

    NASA Astrophysics Data System (ADS)

    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

  1. On the effect of hydrostatic pressure on the conformational stability of globular proteins.

    PubMed

    Graziano, Giuseppe

    2015-12-01

    The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245-14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure- volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure-volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc.

  2. Mapping the Geometric Evolution of Protein Folding Motor.

    PubMed

    Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

    2016-01-01

    Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

  3. The complex folding pathways of protein A suggest a multiple-funnelled energy landscape

    NASA Astrophysics Data System (ADS)

    St-Pierre, Jean-Francois; Mousseau, Normand; Derreumaux, Philippe

    2008-01-01

    Folding proteins into their native states requires the formation of both secondary and tertiary structures. Many questions remain, however, as to whether these form into a precise order, and various pictures have been proposed that place the emphasis on the first or the second level of structure in describing folding. One of the favorite test models for studying this question is the B domain of protein A, which has been characterized by numerous experiments and simulations. Using the activation-relaxation technique coupled with a generic energy model (optimized potential for efficient peptide structure prediction), we generate more than 50 folding trajectories for this 60-residue protein. While the folding pathways to the native state are fully consistent with the funnel-like description of the free energy landscape, we find a wide range of mechanisms in which secondary and tertiary structures form in various orders. Our nonbiased simulations also reveal the presence of a significant number of non-native β and α conformations both on and off pathway, including the visit, for a non-negligible fraction of trajectories, of fully ordered structures resembling the native state of nonhomologous proteins.

  4. The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

    PubMed Central

    Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

    2010-01-01

    Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

  5. Investigation of protein folding by coarse-grained molecular dynamics with the UNRES force field.

    PubMed

    Maisuradze, Gia G; Senet, Patrick; Czaplewski, Cezary; Liwo, Adam; Scheraga, Harold A

    2010-04-08

    Coarse-grained molecular dynamics simulations offer a dramatic extension of the time-scale of simulations compared to all-atom approaches. In this article, we describe the use of the physics-based united-residue (UNRES) force field, developed in our laboratory, in protein-structure simulations. We demonstrate that this force field offers about a 4000-times extension of the simulation time scale; this feature arises both from averaging out the fast-moving degrees of freedom and reduction of the cost of energy and force calculations compared to all-atom approaches with explicit solvent. With massively parallel computers, microsecond folding simulation times of proteins containing about 1000 residues can be obtained in days. A straightforward application of canonical UNRES/MD simulations, demonstrated with the example of the N-terminal part of the B-domain of staphylococcal protein A (PDB code: 1BDD, a three-alpha-helix bundle), discerns the folding mechanism and determines kinetic parameters by parallel simulations of several hundred or more trajectories. Use of generalized-ensemble techniques, of which the multiplexed replica exchange method proved to be the most effective, enables us to compute thermodynamics of folding and carry out fully physics-based prediction of protein structure, in which the predicted structure is determined as a mean over the most populated ensemble below the folding-transition temperature. By using principal component analysis of the UNRES folding trajectories of the formin-binding protein WW domain (PDB code: 1E0L; a three-stranded antiparallel beta-sheet) and 1BDD, we identified representative structures along the folding pathways and demonstrated that only a few (low-indexed) principal components can capture the main structural features of a protein-folding trajectory; the potentials of mean force calculated along these essential modes exhibit multiple minima, as opposed to those along the remaining modes that are unimodal. In addition

  6. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    PubMed Central

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

  7. Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

    PubMed Central

    Belgrano, Fabricio S.; de Abreu da Silva, Isabel C.; Bastos de Oliveira, Francisco M.; Fantappié, Marcelo R.; Mohana-Borges, Ronaldo

    2013-01-01

    High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling. PMID:24255708

  8. Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family*

    PubMed Central

    De Jaco, Antonella; Lin, Michael Z.; Dubi, Noga; Comoletti, Davide; Miller, Meghan T.; Camp, Shelley; Ellisman, Mark; Butko, Margaret T.; Tsien, Roger Y.; Taylor, Palmer

    2010-01-01

    Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems. PMID:20615874

  9. Neuroligin trafficking deficiencies arising from mutations in the alpha/beta-hydrolase fold protein family.

    PubMed

    De Jaco, Antonella; Lin, Michael Z; Dubi, Noga; Comoletti, Davide; Miller, Meghan T; Camp, Shelley; Ellisman, Mark; Butko, Margaret T; Tsien, Roger Y; Taylor, Palmer

    2010-09-10

    Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

  10. Controlling protein molecular dynamics: How to accelerate folding while preserving the native state

    NASA Astrophysics Data System (ADS)

    Jensen, Christian H.; Nerukh, Dmitry; Glen, Robert C.

    2008-12-01

    The dynamics of peptides and proteins generated by classical molecular dynamics (MD) is described by using a Markov model. The model is built by clustering the trajectory into conformational states and estimating transition probabilities between the states. Assuming that it is possible to influence the dynamics of the system by varying simulation parameters, we show how to use the Markov model to determine the parameter values that preserve the folded state of the protein and at the same time, reduce the folding time in the simulation. We investigate this by applying the method to two systems. The first system is an imaginary peptide described by given transition probabilities with a total folding time of 1μs. We find that only small changes in the transition probabilities are needed to accelerate (or decelerate) the folding. This implies that folding times for slowly folding peptides and proteins calculated using MD cannot be meaningfully compared to experimental results. The second system is a four residue peptide valine-proline-alanine-leucine in water. We control the dynamics of the transitions by varying the temperature and the atom masses. The simulation results show that it is possible to find the combinations of parameter values that accelerate the dynamics and at the same time preserve the native state of the peptide. A method for accelerating larger systems without performing simulations for the whole folding process is outlined.

  11. Stability of halophilic proteins: from dipeptide attributes to discrimination classifier.

    PubMed

    Zhang, Guangya; Huihua, Ge; Yi, Lin

    2013-02-01

    To investigate the molecular features responsible for protein halophilicity is of great significance for understanding the structure basis of protein halo-stability and would help to develop a practical strategy for designing halophilic proteins. In this work, we have systematically analyzed the dipeptide composition of the halophilic and non-halophilic protein sequences. We observed the halophilic proteins contained more DA, RA, AD, RR, AP, DD, PD, EA, VG and DV at the expense of LK, IL, II, IA, KK, IS, KA, GK, RK and AI. We identified some macromolecular signatures of halo-adaptation, and thought the dipeptide composition might contain more information than amino acid composition. Based on the dipeptide composition, we have developed a machine learning method for classifying halophilic and non-halophilic proteins for the first time. The accuracy of our method for the training dataset was 100.0%, and for the 10-fold cross-validation was 93.1%. We also discussed the influence of some specific dipeptides on prediction accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Modeling chain folding in protein-constrained circular DNA.

    PubMed Central

    Martino, J A; Olson, W K

    1998-01-01

    An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems. PMID:9591675

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

    PubMed

    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.

  14. Protein Folding Simulations Combining Self-Guided Langevin Dynamics and Temperature-Based Replica Exchange

    DTIC Science & Technology

    2010-01-01

    formulations of molecular dynamics (MD) and Langevin dynamics (LD) simulations for the prediction of thermodynamic folding observables of the Trp-cage...ad hoc force term in the SGLD model. Introduction Molecular dynamics (MD) simulations of small proteins provide insight into the mechanisms and... molecular dynamics (MD) and Langevin dynamics (LD) simulations for the prediction of thermodynamic folding observables of the Trp-cage mini-protein. All

  15. Folding thermodynamics of model four-strand antiparallel beta-sheet proteins.

    PubMed Central

    Jang, Hyunbum; Hall, Carol K; Zhou, Yaoqi

    2002-01-01

    The thermodynamic properties for three different types of off-lattice four-strand antiparallel beta-strand protein models interacting via a hybrid Go-type potential have been investigated. Discontinuous molecular dynamic simulations have been performed for different sizes of the bias gap g, an artificial measure of a model protein's preference for its native state. The thermodynamic transition temperatures are obtained by calculating the squared radius of gyration R(g)(2), the root-mean-squared pair separation fluctuation Delta(B), the specific heat C(v), the internal energy of the system E, and the Lindemann disorder parameter Delta(L). Despite these models' simplicity, they exhibit a complex set of protein transitions, consistent with those observed in experimental studies on real proteins. Starting from high temperature, these transitions include a collapse transition, a disordered-to-ordered globule transition, a folding transition, and a liquid-to-solid transition. The high temperature transitions, i.e., the collapse transition and the disordered-to-ordered globule transition, exist for all three beta-strand proteins, although the native-state geometry of the three model proteins is different. However the low temperature transitions, i.e., the folding transition and the liquid-to-solid transition, strongly depend on the native-state geometry of the model proteins and the size of the bias gap. PMID:11806908

  16. Effects of monohydric alcohols and polyols on the thermal stability of a protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murakami, Shota; Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp

    2016-03-28

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either puremore » water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance

  17. Effects of monohydric alcohols and polyols on the thermal stability of a protein

    NASA Astrophysics Data System (ADS)

    Murakami, Shota; Kinoshita, Masahiro

    2016-03-01

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

  18. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

    PubMed Central

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

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

  19. Folding behavior of ribosomal protein S6 studied by modified Go¯ -like model

    NASA Astrophysics Data System (ADS)

    Wu, L.; Zhang, J.; Wang, J.; Li, W. F.; Wang, W.

    2007-03-01

    Recent experimental and theoretical studies suggest that, although topology is the determinant factor in protein folding, especially for small single-domain proteins, energetic factors also play an important role in the folding process. The ribosomal protein S6 has been subjected to intensive studies. A radical change of the transition state in its circular permutants has been observed, which is believed to be caused by a biased distribution of contact energies. Since the simplistic topology-only Gō -like model is not able to reproduce such an observation, we modify the model by introducing variable contact energies between residues based on their physicochemical properties. The modified Gō -like model can successfully reproduce the Φ -value distributions, folding nucleus, and folding pathways of both the wild-type and circular permutants of S6. Furthermore, by comparing the results of the modified and the simplistic models, we find that the hydrophobic effect constructs the major force that balances the loop entropies. This may indicate that nature maintains the folding cooperativity of this protein by carefully arranging the location of hydrophobic residues in the sequence. Our study reveals a strategy or mechanism used by nature to get out of the dilemma when the native structure, possibly required by biological function, conflicts with folding cooperativity. Finally, the possible relationship between such a design of nature and amyloidosis is also discussed.

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

    PubMed

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

    2017-10-20

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

  1. Self-Complementarity within Proteins: Bridging the Gap between Binding and Folding

    PubMed Central

    Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

    2012-01-01

    Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. PMID:22713576

  2. Self-complementarity within proteins: bridging the gap between binding and folding.

    PubMed

    Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

    2012-06-06

    Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. The double life of the ribosome: When its protein folding activity supports prion propagation.

    PubMed

    Voisset, Cécile; Blondel, Marc; Jones, Gary W; Friocourt, Gaëlle; Stahl, Guillaume; Chédin, Stéphane; Béringue, Vincent; Gillet, Reynald

    2017-03-04

    It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

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

    NASA Technical Reports Server (NTRS)

    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

  5. Chaperones and protein folding in the archaea.

    PubMed

    Large, Andrew T; Goldberg, Martin D; Lund, Peter A

    2009-02-01

    A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.

  6. Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease.

    PubMed

    Goswami, Arvind Vittal; Samaddar, Madhuja; Sinha, Devanjan; Purushotham, Jaya; D'Silva, Patrick

    2012-08-01

    Parkinson's disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with 'mitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

  7. M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding

    PubMed Central

    Son, Ahyun; Choi, Seong Il; Han, Gyoonhee; Seong, Baik L

    2015-01-01

    It is one of the fundamental questions in biology how proteins efficiently fold into their native conformations despite off-pathway events such as misfolding and aggregation in living cells. Although molecular chaperones have been known to assist the de novo folding of certain types of proteins, the role of a binding partner (or a ligand) in the folding and in-cell solubility of its interacting protein still remains poorly defined. RNase P is responsible for the maturation of tRNAs as adaptor molecules of amino acids in ribosomal protein synthesis. The RNase P from Escherichia coli, composed of M1 RNA and C5 protein, is a prototypical ribozyme in which the RNA subunit contains the catalytic activity. Using E. coli RNase P, we demonstrate that M1 RNA plays a pivotal role in the in-cell solubility of C5 protein both in vitro and in vivo. Mutations in either the C5 protein or M1 RNA that affect their interactions significantly abolished the folding of C5 protein. Moreover, we find that M1 RNA provides quality insurance of interacting C5 protein, either by promoting the degradation of C5 mutants in the presence of functional proteolytic machinery, or by abolishing their solubility if the machinery is non-functional. Our results describe a crucial role of M1 RNA in the folding, in-cell solubility, and, consequently, the proteostasis of the client C5 protein, giving new insight into the biological role of RNAs as chaperones and mediators that ensure the quality of interacting proteins. PMID:26517763

  8. Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

    PubMed

    Pagano, Bruno; Jama, Abdullah; Martinez, Pierre; Akanho, Ester; Bui, Tam T T; Drake, Alex F; Fraternali, Franca; Nikolova, Penka V

    2013-01-01

    The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

  9. Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean1[OPEN

    PubMed Central

    Okuda, Aya; Masuda, Taro; Koishihara, Katsunori; Mita, Ryuta; Iwasaki, Kensuke; Hara, Kumiko; Naruo, Yurika; Hirose, Akiho; Tsuchi, Yuichiro

    2016-01-01

    Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants. PMID:26645455

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

    PubMed

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

    2017-12-15

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

  11. Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin Nanodispersion and Its Transport via a Caco-2 Monolayer.

    PubMed

    Shen, Xue; Zhao, Changhui; Lu, Jing; Guo, Mingruo

    2018-02-14

    Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and polymerized whey protein (PWP) through an emulsification-evaporation technique. The physicochemical properties of the astaxanthin nanodispersion were evaluated, and the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small particle size (121 ± 4.9 and 80.4 ± 5.9 nm, respectively), negative ζ potential (-19.3 ± 1.5 and -35.0 ± 2.2 mV, respectively), and high encapsulation efficiency (92.1 ± 2.9 and 93.5 ± 2.4%, respectively). Differential scanning calorimetry curves indicated that amorphous astaxanthin existed in both astaxanthin nanodispersions. Whey-protein-stabilized astaxanthin nanodispersion showed resistance to pepsin digestion but readily released astaxanthin after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at a protein concentration below 10 mg/mL. WPI- and PWP-stabilized nanodispersions improved the apparent permeability coefficient (P app ) of Caco-2 cells to astaxanthin by 10.3- and 16.1-fold, respectively. The results indicated that whey-protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive compounds, such as astaxanthin, and to improve their bioavailability.

  12. How Adequate are One- and Two-Dimensional Free Energy Landscapes for Protein Folding Dynamics?

    NASA Astrophysics Data System (ADS)

    Maisuradze, Gia G.; Liwo, Adam; Scheraga, Harold A.

    2009-06-01

    The molecular dynamics trajectories of protein folding or unfolding, generated with the coarse-grained united-residue force field for the B domain of staphylococcal protein A, were analyzed by principal component analysis (PCA). The folding or unfolding process was examined by using free-energy landscapes (FELs) in PC space. By introducing a novel multidimensional FEL, it was shown that the low-dimensional FELs are not always sufficient for the description of folding or unfolding processes. Similarities between the topographies of FELs along low- and high-indexed principal components were observed.

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

    PubMed

    Oshima, Hiraku; Kinoshita, Masahiro

    2015-04-14

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oshima, Hiraku; Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp

    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 modelsmore » 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

  15. Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

    PubMed Central

    Charlier, Cyril; Alderson, T. Reid; Courtney, Joseph M.; Ying, Jinfa; Anfinrud, Philip

    2018-01-01

    In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state. PMID:29666248

  16. Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

    PubMed Central

    Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Cenacchi, Lucia; Ciofi-Baffoni, Simone; Felli, Isabella Caterina; Gallo, Angelo; Gonnelli, Leonardo; Luchinat, Enrico; Sideris, Dionisia; Tokatlidis, Kostas

    2010-01-01

    Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation. PMID:21059946

  17. Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations

    PubMed Central

    Kannan, Srinivasaraghavan; Zacharias, Martin

    2014-01-01

    The 20 residue Trp-cage mini-protein is one of smallest proteins that adopt a stable folded structure containing also well-defined secondary structure elements. The hydrophobic core is arranged around a single central Trp residue. Despite several experimental and simulation studies the detailed folding mechanism of the Trp-cage protein is still not completely understood. Starting from fully extended as well as from partially folded Trp-cage structures a series of molecular dynamics simulations in explicit solvent and using four different force fields was performed. All simulations resulted in rapid collapse of the protein to on average relatively compact states. The simulations indicate a significant dependence of the speed of folding to near-native states on the side chain rotamer state of the central Trp residue. Whereas the majority of intermediate start structures with the central Trp side chain in a near-native rotameric state folded successfully within less than 100 ns only a fraction of start structures reached near-native folded states with an initially non-native Trp side chain rotamer state. Weak restraining of the Trp side chain dihedral angles to the state in the folded protein resulted in significant acceleration of the folding both starting from fully extended or intermediate conformations. The results indicate that the side chain conformation of the central Trp residue can create a significant barrier for controlling transitions to a near native folded structure. Similar mechanisms might be of importance for the folding of other protein structures. PMID:24563686

  18. In silico study of amyloid -protein folding and oligomerization

    NASA Astrophysics Data System (ADS)

    Urbanc, B.; Cruz, L.; Yun, S.; Buldyrev, S. V.; Bitan, G.; Teplow, D. B.; Stanley, H. E.

    2004-12-01

    Experimental findings suggest that oligomeric forms of the amyloid protein (A) play a critical role in Alzheimer's disease. Thus, elucidating their structure and the mechanisms of their formation is critical for developing therapeutic agents. We use discrete molecular dynamics simulations and a four-bead protein model to study oligomerization of two predominant alloforms, A40 and A42, at the atomic level. The four-bead model incorporates backbone hydrogen-bond interactions and amino acid-specific interactions mediated through hydrophobic and hydrophilic elements of the side chains. During the simulations we observe monomer folding and aggregation of monomers into oligomers of variable sizes. A40 forms significantly more dimers than A42, whereas pentamers are significantly more abundant in A42 relative to A40. Structure analysis reveals a turn centered at Gly-37-Gly-38 that is present in a folded A42 monomer but not in a folded A40 monomer and is associated with the first contacts that form during monomer folding. Our results suggest that this turn plays an important role in A42 pentamer formation. A pentamers have a globular structure comprising hydrophobic residues within the pentamer's core and hydrophilic N-terminal residues at the surface of the pentamer. The N termini of A40 pentamers are more spatially restricted than A42 pentamers. A40 pentamers form a -strand structure involving Ala-2-Phe-4, which is absent in A42 pentamers. These structural differences imply a different degree of hydrophobic core exposure between pentamers of the two alloforms, with the hydrophobic core of the Aβ42 pentamer being more exposed and thus more prone to form larger oligomers.

  19. Optimization of rotamers prior to template minimization improves stability predictions made by computational protein design.

    PubMed

    Davey, James A; Chica, Roberto A

    2015-04-01

    Computational protein design (CPD) predictions are highly dependent on the structure of the input template used. However, it is unclear how small differences in template geometry translate to large differences in stability prediction accuracy. Herein, we explored how structural changes to the input template affect the outcome of stability predictions by CPD. To do this, we prepared alternate templates by Rotamer Optimization followed by energy Minimization (ROM) and used them to recapitulate the stability of 84 protein G domain β1 mutant sequences. In the ROM process, side-chain rotamers for wild-type (WT) or mutant sequences are optimized on crystal or nuclear magnetic resonance (NMR) structures prior to template minimization, resulting in alternate structures termed ROM templates. We show that use of ROM templates prepared from sequences known to be stable results predominantly in improved prediction accuracy compared to using the minimized crystal or NMR structures. Conversely, ROM templates prepared from sequences that are less stable than the WT reduce prediction accuracy by increasing the number of false positives. These observed changes in prediction outcomes are attributed to differences in side-chain contacts made by rotamers in ROM templates. Finally, we show that ROM templates prepared from sequences that are unfolded or that adopt a nonnative fold result in the selective enrichment of sequences that are also unfolded or that adopt a nonnative fold, respectively. Our results demonstrate the existence of a rotamer bias caused by the input template that can be harnessed to skew predictions toward sequences displaying desired characteristics. © 2014 The Protein Society.

  20. Conformational plasticity of DM43, a metalloproteinase inhibitor from Didelphis marsupialis: chemical and pressure-induced equilibrium (un)folding studies.

    PubMed

    Chapeaurouge, Alex; Martins, Samantha M; Holub, Oliver; Rocha, Surza L G; Valente, Richard H; Neves-Ferreira, Ana G C; Ferreira, Sérgio T; Domont, Gilberto B; Perales, Jonas

    2009-10-01

    We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.

  1. Folding molecular dynamics simulations accurately predict the effect of mutations on the stability and structure of a vammin-derived peptide.

    PubMed

    Koukos, Panagiotis I; Glykos, Nicholas M

    2014-08-28

    Folding molecular dynamics simulations amounting to a grand total of 4 μs of simulation time were performed on two peptides (with native and mutated sequences) derived from loop 3 of the vammin protein and the results compared with the experimentally known peptide stabilities and structures. The simulations faithfully and accurately reproduce the major experimental findings and show that (a) the native peptide is mostly disordered in solution, (b) the mutant peptide has a well-defined and stable structure, and (c) the structure of the mutant is an irregular β-hairpin with a non-glycine β-bulge, in excellent agreement with the peptide's known NMR structure. Additionally, the simulations also predict the presence of a very small β-hairpin-like population for the native peptide but surprisingly indicate that this population is structurally more similar to the structure of the native peptide as observed in the vammin protein than to the NMR structure of the isolated mutant peptide. We conclude that, at least for the given system, force field, and simulation protocol, folding molecular dynamics simulations appear to be successful in reproducing the experimentally accessible physical reality to a satisfactory level of detail and accuracy.

  2. Entropic (de)stabilization of surface-bound peptides conjugated with polymers

    NASA Astrophysics Data System (ADS)

    Carmichael, Scott P.; Shell, M. Scott

    2015-12-01

    In many emerging biotechnologies, functional proteins must maintain their native structures on or near interfaces (e.g., tethered peptide arrays, protein coated nanoparticles, and amphiphilic peptide micelles). Because the presence of a surface is known to dramatically alter the thermostability of tethered proteins, strategies to stabilize surface-bound proteins are highly sought. Here, we show that polymer conjugation allows for significant control over the secondary structure and thermostability of a model surface-tethered peptide. We use molecular dynamics simulations to examine the folding behavior of a coarse-grained helical peptide that is conjugated to polymers of various lengths and at various conjugation sites. These polymer variations reveal surprisingly diverse behavior, with some stabilizing and some destabilizing the native helical fold. We show that ideal-chain polymer entropies explain these varied effects and can quantitatively predict shifts in folding temperature. We then develop a generic theoretical model, based on ideal-chain entropies, that predicts critical lengths for conjugated polymers to effect changes in the folding of a surface-bound protein. These results may inform new design strategies for the stabilization of surface-associated proteins important for a range technological applications.

  3. Entropic (de)stabilization of surface-bound peptides conjugated with polymers.

    PubMed

    Carmichael, Scott P; Shell, M Scott

    2015-12-28

    In many emerging biotechnologies, functional proteins must maintain their native structures on or near interfaces (e.g., tethered peptide arrays, protein coated nanoparticles, and amphiphilic peptide micelles). Because the presence of a surface is known to dramatically alter the thermostability of tethered proteins, strategies to stabilize surface-bound proteins are highly sought. Here, we show that polymer conjugation allows for significant control over the secondary structure and thermostability of a model surface-tethered peptide. We use molecular dynamics simulations to examine the folding behavior of a coarse-grained helical peptide that is conjugated to polymers of various lengths and at various conjugation sites. These polymer variations reveal surprisingly diverse behavior, with some stabilizing and some destabilizing the native helical fold. We show that ideal-chain polymer entropies explain these varied effects and can quantitatively predict shifts in folding temperature. We then develop a generic theoretical model, based on ideal-chain entropies, that predicts critical lengths for conjugated polymers to effect changes in the folding of a surface-bound protein. These results may inform new design strategies for the stabilization of surface-associated proteins important for a range technological applications.

  4. Increasing protein stability by improving beta-turns.

    PubMed

    Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

    2009-11-15

    Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

  5. INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

    PubMed Central

    Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

  6. A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3

    PubMed Central

    Dietmann, Sabine; Park, Jong; Notredame, Cedric; Heger, Andreas; Lappe, Michael; Holm, Liisa

    2001-01-01

    The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families. PMID:11125048

  7. Hierarchy of folding and unfolding events of protein G, CI2, and ACBP from explicit-solvent simulations

    NASA Astrophysics Data System (ADS)

    Camilloni, Carlo; Broglia, Ricardo A.; Tiana, Guido

    2011-01-01

    The study of the mechanism which is at the basis of the phenomenon of protein folding requires the knowledge of multiple folding trajectories under biological conditions. Using a biasing molecular-dynamics algorithm based on the physics of the ratchet-and-pawl system, we carry out all-atom, explicit solvent simulations of the sequence of folding events which proteins G, CI2, and ACBP undergo in evolving from the denatured to the folded state. Starting from highly disordered conformations, the algorithm allows the proteins to reach, at the price of a modest computational effort, nativelike conformations, within a root mean square deviation (RMSD) of approximately 1 Å. A scheme is developed to extract, from the myriad of events, information concerning the sequence of native contact formation and of their eventual correlation. Such an analysis indicates that all the studied proteins fold hierarchically, through pathways which, although not deterministic, are well-defined with respect to the order of contact formation. The algorithm also allows one to study unfolding, a process which looks, to a large extent, like the reverse of the major folding pathway. This is also true in situations in which many pathways contribute to the folding process, like in the case of protein G.

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

    PubMed

    Sen, Siddhartha; Voorheis, H Paul

    2014-12-21

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-04-19

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

  11. Structural stability of proteins in aqueous and nonpolar environments

    NASA Astrophysics Data System (ADS)

    Yasuda, Satoshi; Oshima, Hiraku; Kinoshita, Masahiro

    2012-10-01

    A protein folds into its native structure with the α-helix and/or β-sheet in aqueous solution under the physiological condition. The relative content of these secondary structures largely varies from protein to protein. However, such structural variability is not exhibited in nonaqueous environment. For example, there is a strong trend that alcohol induces a protein to form α-helices, and many of the membrane proteins within the lipid bilayer consists of α-helices. Here we investigate the structural stability of proteins in aqueous and nonpolar environments using our recently developed free-energy function F = (Λ - TS)/(kBT0) = Λ/(kBT0) - S/kB (T0 = 298 K and the absolute temperature T is set at T0) which is based on statistical thermodynamics. Λ/(kBT0) and S/kB are the energetic and entropic components, respectively, and kB is Boltzmann's constant. A smaller value of the positive quantity, -S, represents higher efficiency of the backbone and side-chain packing promoted by the entropic effect arising from the translational displacement of solvent molecules or the CH2, CH3, and CH groups which constitute nonpolar chains of lipid molecules. As for Λ, in aqueous solution, a transition to a more compact structure of a protein accompanies the break of protein-solvent hydrogen bonds: As the number of donors and acceptors buried without protein intramolecular hydrogen bonding increases, Λ becomes higher. In nonpolar solvent, lower Λ simply implies more intramolecular hydrogen bonds formed. We find the following. The α-helix and β-sheet are advantageous with respect to -S as well as Λ and to be formed as much as possible. In aqueous solution, the solvent-entropy effect on the structural stability is so strong that the close packing of side chains is dominantly important, and the α-helix and β-sheet contents are judiciously adjusted to accomplish it. In nonpolar solvent, the solvent-entropy effect is substantially weaker than in aqueous solution. Λ is

  12. Folding processes of the B domain of protein A to the native state observed in all-atom ab initio folding simulations

    NASA Astrophysics Data System (ADS)

    Lei, Hongxing; Wu, Chun; Wang, Zhi-Xiang; Zhou, Yaoqi; Duan, Yong

    2008-06-01

    Reaching the native states of small proteins, a necessary step towards a comprehensive understanding of the folding mechanisms, has remained a tremendous challenge to ab initio protein folding simulations despite the extensive effort. In this work, the folding process of the B domain of protein A (BdpA) has been simulated by both conventional and replica exchange molecular dynamics using AMBER FF03 all-atom force field. Started from an extended chain, a total of 40 conventional (each to 1.0 μs) and two sets of replica exchange (each to 200.0 ns per replica) molecular dynamics simulations were performed with different generalized-Born solvation models and temperature control schemes. The improvements in both the force field and solvent model allowed successful simulations of the folding process to the native state as demonstrated by the 0.80 A˚ Cα root mean square deviation (RMSD) of the best folded structure. The most populated conformation was the native folded structure with a high population. This was a significant improvement over the 2.8 A˚ Cα RMSD of the best nativelike structures from previous ab initio folding studies on BdpA. To the best of our knowledge, our results demonstrate, for the first time, that ab initio simulations can reach the native state of BdpA. Consistent with experimental observations, including Φ-value analyses, formation of helix II/III hairpin was a crucial step that provides a template upon which helix I could form and the folding process could complete. Early formation of helix III was observed which is consistent with the experimental results of higher residual helical content of isolated helix III among the three helices. The calculated temperature-dependent profile and the melting temperature were in close agreement with the experimental results. The simulations further revealed that phenylalanine 31 may play critical to achieve the correct packing of the three helices which is consistent with the experimental observation

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

    PubMed Central

    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

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

    PubMed

    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.

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

    ERIC Educational Resources Information Center

    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…

  16. Coupled Protein Diffusion and Folding in the Cell

    PubMed Central

    Guo, Minghao; Gelman, Hannah; Gruebele, Martin

    2014-01-01

    When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control's diffusion coefficient, PGK's diffusion coefficient is a non-monotonic function of temperature, signaling ‘sticking’ of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates. PMID:25436502

  17. Coupled protein diffusion and folding in the cell.

    PubMed

    Guo, Minghao; Gelman, Hannah; Gruebele, Martin

    2014-01-01

    When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control's diffusion coefficient, PGK's diffusion coefficient is a non-monotonic function of temperature, signaling 'sticking' of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates.

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

    PubMed

    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.

  19. Competition between protein folding and aggregation: A three-dimensional lattice-model simulation

    NASA Astrophysics Data System (ADS)

    Bratko, D.; Blanch, H. W.

    2001-01-01

    Aggregation of protein molecules resulting in the loss of biological activity and the formation of insoluble deposits represents a serious problem for the biotechnology and pharmaceutical industries and in medicine. Considerable experimental and theoretical efforts are being made in order to improve our understanding of, and ability to control, the process. In the present work, we describe a Monte Carlo study of a multichain system of coarse-grained model proteins akin to lattice models developed for simulations of protein folding. The model is designed to examine the competition between intramolecular interactions leading to the native protein structure, and intermolecular association, resulting in the formation of aggregates of misfolded chains. Interactions between the segments are described by a variation of the Go potential [N. Go and H. Abe, Biopolymers 20, 1013 (1981)] that extends the recognition between attracting types of segments to pairs on distinct chains. For the particular model we adopt, the global free energy minimum of a pair of protein molecules corresponds to a dimer of native proteins. When three or more molecules interact, clusters of misfolded chains can be more stable than aggregates of native folds. A considerable fraction of native structure, however, is preserved in these cases. Rates of conformational changes rapidly decrease with the size of the protein cluster. Within the timescale accessible to computer simulations, the folding-aggregation balance is strongly affected by kinetic considerations. Both the native form and aggregates can persist in metastable states, even if conditions such as temperature or concentration favor a transition to an alternative form. Refolding yield can be affected by the presence of an additional polymer species mimicking the function of a molecular chaperone.

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

    PubMed Central

    Sarkar, Aurijit; Kellogg, Glen E.

    2009-01-01

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

  1. Sequentially distant but structurally similar proteins exhibit fold specific patterns based on their biophysical properties.

    PubMed

    Rajendran, Senthilnathan; Jothi, Arunachalam

    2018-05-16

    The Three-dimensional structure of a protein depends on the interaction between their amino acid residues. These interactions are in turn influenced by various biophysical properties of the amino acids. There are several examples of proteins that share the same fold but are very dissimilar at the sequence level. For proteins to share a common fold some crucial interactions should be maintained despite insignificant sequence similarity. Since the interactions are because of the biophysical properties of the amino acids, we should be able to detect descriptive patterns for folds at such a property level. In this line, the main focus of our research is to analyze such proteins and to characterize them in terms of their biophysical properties. Protein structures with sequence similarity lesser than 40% were selected for ten different subfolds from three different mainfolds (according to CATH classification) and were used for this analysis. We used the normalized values of the 49 physio-chemical, energetic and conformational properties of amino acids. We characterize the folds based on the average biophysical property values. We also observed a fold specific correlational behavior of biophysical properties despite a very low sequence similarity in our data. We further trained three different binary classification models (Naive Bayes-NB, Support Vector Machines-SVM and Bayesian Generalized Linear Model-BGLM) which could discriminate mainfold based on the biophysical properties. We also show that among the three generated models, the BGLM classifier model was able to discriminate protein sequences coming under all beta category with 81.43% accuracy and all alpha, alpha-beta proteins with 83.37% accuracy. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. The Est3 protein associates with yeast telomerase through an OB-fold domain

    PubMed Central

    Lee, Jaesung S.; Mandell, Edward K.; Tucey, Timothy M.; Morris, Danna K.; Victoria, Lundblad

    2009-01-01

    The Est3 protein is a small regulatory subunit of yeast telomerase which is dispensable for enzyme catalysis but essential for telomere replication in vivo. Using structure prediction combined with in vivo characterization, we show here that Est3 consists of a predicted OB (oligo-saccharide/oligo-nucleotide binding) fold. Mutagenesis of predicted surface residues was used to generate a functional map of one surface of Est3, which identified a site that mediates association with the telomerase complex. Surprisingly, the predicted OB-fold of Est3 is structurally similar to the OB-fold of the mammalian TPP1 protein, despite the fact that Est3 and TPP1, as components of telomerase and a telomere capping complex, respectively, perform functionally distinct tasks at chromosome ends. The analysis performed on Est3 may be instructive in generating comparable missense mutations on the surface of the OB-fold domain of TPP1. PMID:19172754

  3. Measuring internal friction of an ultrafast-folding protein.

    PubMed

    Cellmer, Troy; Henry, Eric R; Hofrichter, James; Eaton, William A

    2008-11-25

    Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein.

  4. Search for Functional Flexible Regions in the G-protein Family: New Reading of the FoldUnfold Program.

    PubMed

    Galzitskaya, Oxana; Deryusheva, Eugenia; Machulin, Andrey; Nemashkalova, Ekaterina; Glyakina, Anna

    2018-06-21

    High prediction accuracy of flexible loops in different protein families is a challenge because of the crucial functions associated with these regions. Results of the currently available programs for prediction of loops vary from protein to protein. For prediction of flexible regions in the G-domain for 23 representatives of G-proteins with the known 3D structure we have used eight programs. The results of predictions demonstrate that the FoldUnfold program predicts better loop positions than the PONDR, RОNN, DisEMBL, IUPred, GlobPlot 2, FoldIndex, and MobiDB programs. When classifying the predicted loops (rigid/flexible) according to the Debye-Waller fluctuation factors, our data reveal the existing weak correlation between the B-factors and the average number of closed residues according to the FoldUnfold program; the percentage of overlapping characteristics (residue fold/unfold status) of the protein residues from the two methods is about 60-70%. According to the FoldUnfold program, for G-proteins with the posttranslational modifications, the surrounding binding site residues by disordered-promoting glycine and alanine residues conduces to a more flexible position of the binding sites for fatty acid, while methionine, cysteine and isoleucine residues provide more rigid binding sites. Thus, our research demonstrates additional possibilities of the FoldUnfold program for prediction of flexible regions and characteristics of individual residues in a different protein family. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins.

    PubMed

    Heuck, Alexander; Schleiffer, Alexander; Clausen, Tim

    2011-03-11

    β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Statistical thermodynamics of protein folding: Comparison of a mean-field theory with Monte Carlo simulations

    NASA Astrophysics Data System (ADS)

    Hao, Ming-Hong; Scheraga, Harold A.

    1995-01-01

    A comparative study of protein folding with an analytical theory and computer simulations, respectively, is reported. The theory is based on an improved mean-field formalism which, in addition to the usual mean-field approximations, takes into account the distributions of energies in the subsets of conformational states. Sequence-specific properties of proteins are parametrized in the theory by two sets of variables, one for the energetics of mean-field interactions and one for the distribution of energies. Simulations are carried out on model polypeptides with different sequences, with different chain lengths, and with different interaction potentials, ranging from strong biases towards certain local chain states (bond angles and torsional angles) to complete absence of local conformational preferences. Theoretical analysis of the simulation results for the model polypeptides reveals three different types of behavior in the folding transition from the statistical coiled state to the compact globular state; these include a cooperative two-state transition, a continuous folding, and a glasslike transition. It is found that, with the fitted theoretical parameters which are specific for each polypeptide under a different potential, the mean-field theory can describe the thermodynamic properties and folding behavior of the different polypeptides accurately. By comparing the theoretical descriptions with simulation results, we verify the basic assumptions of the theory and, thereby, obtain new insights about the folding transitions of proteins. It is found that the cooperativity of the first-order folding transition of the model polypeptides is determined mainly by long-range interactions, in particular the dipolar orientation; the local interactions (e.g., bond-angle and torsion-angle potentials) have only marginal effect on the cooperative characteristic of the folding, but have a large impact on the difference in energy between the folded lowest-energy structure and

  7. Identification of kinetically hot residues in proteins.

    PubMed Central

    Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

    1998-01-01

    A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

  8. Atomic-level description of ubiquitin folding

    PubMed Central

    Piana, Stefano; Lindorff-Larsen, Kresten; Shaw, David E.

    2013-01-01

    Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. PMID:23503848

  9. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    NASA Astrophysics Data System (ADS)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  10. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  11. Folding free energy surfaces of three small proteins under crowding: validation of the postprocessing method by direct simulation

    NASA Astrophysics Data System (ADS)

    Qin, Sanbo; Mittal, Jeetain; Zhou, Huan-Xiang

    2013-08-01

    We have developed a ‘postprocessing’ method for modeling biochemical processes such as protein folding under crowded conditions (Qin and Zhou 2009 Biophys. J. 97 12-19). In contrast to the direct simulation approach, in which the protein undergoing folding is simulated along with crowders, the postprocessing method requires only the folding simulation without crowders. The influence of the crowders is then obtained by taking conformations from the crowder-free simulation and calculating the free energies of transferring to the crowders. This postprocessing yields the folding free energy surface of the protein under crowding. Here the postprocessing results for the folding of three small proteins under ‘repulsive’ crowding are validated by those obtained previously by the direct simulation approach (Mittal and Best 2010 Biophys. J. 98 315-20). This validation confirms the accuracy of the postprocessing approach and highlights its distinct advantages in modeling biochemical processes under cell-like crowded conditions, such as enabling an atomistic representation of the test proteins.

  12. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

    NASA Astrophysics Data System (ADS)

    Faure, Guilhem; Koonin, Eugene V.

    2015-05-01

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

  13. Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

    PubMed

    Korkosh, V S; Zhorov, B S; Tikhonov, D B

    2016-05-01

    The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

  14. Universal partitioning of the hierarchical fold network of 50-residue segments in proteins

    PubMed Central

    Ito, Jun-ichi; Sonobe, Yuki; Ikeda, Kazuyoshi; Tomii, Kentaro; Higo, Junichi

    2009-01-01

    Background Several studies have demonstrated that protein fold space is structured hierarchically and that power-law statistics are satisfied in relation between the numbers of protein families and protein folds (or superfamilies). We examined the internal structure and statistics in the fold space of 50 amino-acid residue segments taken from various protein folds. We used inter-residue contact patterns to measure the tertiary structural similarity among segments. Using this similarity measure, the segments were classified into a number (Kc) of clusters. We examined various Kc values for the clustering. The special resolution to differentiate the segment tertiary structures increases with increasing Kc. Furthermore, we constructed networks by linking structurally similar clusters. Results The network was partitioned persistently into four regions for Kc ≥ 1000. This main partitioning is consistent with results of earlier studies, where similar partitioning was reported in classifying protein domain structures. Furthermore, the network was partitioned naturally into several dozens of sub-networks (i.e., communities). Therefore, intra-sub-network clusters were mutually connected with numerous links, although inter-sub-network ones were rarely done with few links. For Kc ≥ 1000, the major sub-networks were about 40; the contents of the major sub-networks were conserved. This sub-partitioning is a novel finding, suggesting that the network is structured hierarchically: Segments construct a cluster, clusters form a sub-network, and sub-networks constitute a region. Additionally, the network was characterized by non-power-law statistics, which is also a novel finding. Conclusion Main findings are: (1) The universe of 50 residue segments found here was characterized by non-power-law statistics. Therefore, the universe differs from those ever reported for the protein domains. (2) The 50-residue segments were partitioned persistently and universally into some dozens (ca

  15. A limited universe of membrane protein families and folds

    PubMed Central

    Oberai, Amit; Ihm, Yungok; Kim, Sanguk; Bowie, James U.

    2006-01-01

    One of the goals of structural genomics is to obtain a structural representative of almost every fold in nature. A recent estimate suggests that 70%–80% of soluble protein domains identified in the first 1000 genome sequences should be covered by about 25,000 structures—a reasonably achievable goal. As no current estimates exist for the number of membrane protein families, however, it is not possible to know whether family coverage is a realistic goal for membrane proteins. Here we find that virtually all polytopic helical membrane protein families are present in the already known sequences so we can make an estimate of the total number of families. We find that only ∼700 polytopic membrane protein families account for 80% of structured residues and ∼1700 cover 90% of structured residues. While apparently a finite and reachable goal, we estimate that it will likely take more than three decades to obtain the structures needed for 90% residue coverage, if current trends continue. PMID:16815920

  16. SPLINTS: small-molecule protein ligand interface stabilizers.

    PubMed

    Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

    2016-04-01

    Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. What amyloidoses may tell us about normal protein folding: The Alzheimer's disease story

    NASA Astrophysics Data System (ADS)

    Teplow, David B.

    2002-03-01

    Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by severe neuronal injury and death. A prominent histopathologic feature of AD is disseminated parenchymal and vascular amyloid deposition. The fibrils in these deposits are composed of the amyloid β-protein (Aβ), a peptide of 4 kDa mass. In vitro and in vivo studies of Aβ fibril formation have shown that both oligomeric and polymeric Aβ assemblies have neurotoxic activity. Understanding how these assemblies form thus could be of direct therapeutic relevance. However, the aggregation and fibril-forming propensities of Aβ have complicated structure determination. Nevertheless, careful morphologic, spectroscopic, protein chemical, and physiologic analyses of the time-dependent changes in Aβ conformation, assembly state, and biological activity which occur during fibrillogenesis have significantly advanced our understanding of this clinically important process. Here, I will discuss recent findings about the pathway(s) of Aβ folding and assembly and about key structural features of Aβ which control the associated kinetics. Interestingly, the amyloidogenic folding pathway of Aβ is in some respects the mirror image of that through which natively folded amyloidogenic proteins proceed.

  18. Protein folding: complex potential for the driving force in a two-dimensional space of collective variables.

    PubMed

    Chekmarev, Sergei F

    2013-10-14

    Using the Helmholtz decomposition of the vector field of folding fluxes in a two-dimensional space of collective variables, a potential of the driving force for protein folding is introduced. The potential has two components. One component is responsible for the source and sink of the folding flows, which represent respectively, the unfolded states and the native state of the protein, and the other, which accounts for the flow vorticity inherently generated at the periphery of the flow field, is responsible for the canalization of the flow between the source and sink. The theoretical consideration is illustrated by calculations for a model β-hairpin protein.

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

    PubMed

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

    2013-03-01

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

  20. Exploring the protein folding free energy landscape: coupling replica exchange method with P3ME/RESPA algorithm.

    PubMed

    Zhou, Ruhong

    2004-05-01

    A highly parallel replica exchange method (REM) that couples with a newly developed molecular dynamics algorithm particle-particle particle-mesh Ewald (P3ME)/RESPA has been proposed for efficient sampling of protein folding free energy landscape. The algorithm is then applied to two separate protein systems, beta-hairpin and a designed protein Trp-cage. The all-atom OPLSAA force field with an explicit solvent model is used for both protein folding simulations. Up to 64 replicas of solvated protein systems are simulated in parallel over a wide range of temperatures. The combined trajectories in temperature and configurational space allow a replica to overcome free energy barriers present at low temperatures. These large scale simulations reveal detailed results on folding mechanisms, intermediate state structures, thermodynamic properties and the temperature dependences for both protein systems.