蛋白质折叠机理的理论研究

简要综述了近年来蛋白质折叠机理的理论研究。首先回顾了蛋白质折叠理论的发展历程,然后对折叠中间体的研究现状作了较详细的介绍。同时,对折叠机理理论研究中的几种理论模型和模拟算法作了细致评述,分析了其现状和存在的问题。最后,总结和讨论了折叠机理理论研究的现存问题及研究热点,并展望了该领域研究的发展趋势。     

Rapid folding of the prion protein captured by pressure-jump

The conversion of the cellular form of the prion protein (PrP^C) to an altered disease state, generally denoted as scrapie isoform (PrP^Sc), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before. D. C. Jenkins and D. S. Pearson contributed equally.     

N-terminal extension changes the folding mechanism of the FK506-binding protein

Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.     

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.     

Stability and folding studies of the N-domain of troponin C. Evidence for the formation of an intermediate

We report here on the stability and folding of the 91 residue alpha-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC(1-91)F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC(1-91)F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed.     

Predicting protein folding cores by empirical potential functions

Theoretical and in vitro experiments suggest that protein folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14 (2006) 1401), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.     

Entropic criteria for protein folding derived from recurrences: six residues patch as the basic protein word

Some research has suggested that patches of six constitute an important amino acid window length in proteins for conveying information. We present database evidence that supports this conjecture, as well as additional recurrence-based data that characterization and quantification of these words affect the folding/aggregation features of proteins. Other indirect evidence is presented and discussed.     

Bromodomains shake the hegemony of pan‐acetyl antibodies

Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan‐acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch‐to‐batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470–1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes.     

Sulfate stabilizes the folding intermediate more than the native structure of endostatin

Endostatin, a potent angiogenesis inhibitor, is an acid resistant protein with compact tertiary structure. Nuclear magnetic resonance, circular dichroism, and tryptophan emission fluorescence were used to monitor the structural changes of endostatin during acid-, heat-, and urea-induced unfolding processes. Results show that sulfate anions sensitize endostatin to acid, but specifically stabilize it against heat or urea. Moreover, the disappearance of the tertiary structure and the formation of the folding intermediate of endostatin at pH 3.0 are sulfate concentration dependent. These phenomena indicate that sulfate anions stabilize the folding intermediate more than the native structure of endostatin. In addition, heparin shows stronger effect than sodium sulfate on sensitizing endostatin against acid, and very limited stabilizing effect against urea. The loose structure of endostatin upon heparin binding may imply that the physiologically favorable structure for endostatin exerting its biological functions is not as compact as what was reported.     

Structural features of protein folding nuclei

A crucial event of protein folding is the formation of a folding nucleus. We demonstrate the presence of a considerable coincidence between the location of folding nuclei and the location of so-called "root structural motifs", which have unique overall folds and handedness. In the case of proteins with a single root structural motif, the involvement in the formation of a folding nucleus is in average significantly higher for amino acids residues that are in root structural motifs, compared to residues in other parts of the protein. The tests carried out revealed that the observed difference is statistically reliable. Thus, a structural feature that corresponds to the protein folding nucleus is now found.     

Protein folding: then and now

Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field. Specifically, we discuss the progress in our understanding of protein folding thermodynamics and kinetics, the properties of evasive intermediates, and unfolded states. We also discuss how some abnormalities in protein folding lead to protein aggregation and human diseases.     

Mechanisms of protein folding

Understanding the mechanism by which a polypeptide chain folds into its native structure is a central problem of modern biophysics. The collaborative efforts of experimental and theoretical studies recently raised the tantalizing possibility to define a unifying mechanism for protein folding. In this review we summarize some of these intriguing advances and analyze them together with a discussion on the new findings concerning the so-called downhill folding.     

Correlation of folding kinetics with the number and isomerization states of prolines in three homologous proteins of the RNase family

Several studies attribute the slower phases in protein folding to prolyl isomerizations, and several others do not. A correlation exists between the number of prolines in a protein and the complexity of the mechanism with which it folds. In this study, we have demonstrated a direct correlation between the number of cis-prolyl bonds in a native protein and the complexity with which it folds via slower phases by studying the folding of three structurally homologous proteins of the ribonuclease family, namely RNase A, onconase and angiogenin, which differ in the number and isomerization states of their proline residues.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

The nature of protein folding pathways: The classical versus the new view

Summary Pulsed hydrogen exchange and other studies of the kinetic refolding pathways of several small proteins have established that folding intermediates with native-like secondary structures are well populated, but these studies have also shown that the folding kinetics are not well synchronized. Older studies of the kinetics of formation of the native protein, monitored by optical probes, indicate that the folding kinetics should be synchronized. The model commonly used in these studies is the simple sequential model, which postulates a unique folding pathway with defined and sequential intermediates. Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates accumulate because of kinetic traps caused by partial misfolding. Recent experiments with cytochrome c lend support to this new view of folding pathways. These different views of the folding process are discussed. Misfolding and consequent slowing down of the folding process as a result of cis-trans isomerization about prolyl peptide bonds in the unfolded protein are well known; isomerization occurs before refolding is initiated. The occurrence of equilibrium intermediates on the kinetic folding pathways of some proteins, such as -lactalbumin and apomyoglobin, argues that these intermediates are not caused by kinetic traps but rather are stable intermediates under certain conditions, and this conclusion is consistent with a sequential model of folding. Folding reactions with successive kinetic intermediates, in which late intermediates are more highly folded than early intermediates, indicate that folding is hierarchical. New experiments that test the predictions of the classical and the new views are needed.     

Inclusion body formation by interleukin-1β depends on the thermal sensitivity of a folding intermediate

We show that sequence and growth temperature effects on IB formation in the small, monomeric β-barrel protein interleukin-1β (IL-1β) can be quantitatively reproduced in an in vitro system in which IL-1β is refolded from denaturant at different temperatures. The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors. We also report striking correlations of IB formation with mutation-dependent changes in residue hydrophobicity. The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure.     

Signatures of co-translational folding

Global and co-translational protein folding may both occur in vivo, and understanding the relationship between these folding mechanisms is pivotal to our understanding of protein-structure formation. Within this study, over 1.5 million hydrophobic-polar sequences were classified based on their ability to attain a unique, but not necessarily minimal energy conformation through co-translational folding. The sequence and structure properties of the sets were then compared to elucidate signatures of co-translational folding. The strongest signature of co-translational folding is a reduced number of possible favorable contacts in the amino terminus. There is no evidence of fewer contacts, more local contacts, or less-compact structures. Co-translational folding produces a more compact amino- than carboxy-terminal region and an amino-terminal-biased set of core residues. In real proteins these signatures are also observed and found most strongly in proteins of the alpha/beta structural class of proteins (SCOP) where 71 % have an amino-terminal set of core residues. The prominence of co-translational features in experimentally determined protein structures suggests that the importance of co-translational folding is currently underestimated.     

Relationships between the folding rate constant and the topological parameters of small two-state proteins based on general random walk model

In this paper, we propose an analytically tractable model of protein folding based on one-dimensional general random walk. A second-order differential equation for the mean folding time of a single protein is constructed which can be used to derive the observed relationship between the folding rate constant and the number of native contacts. The parameters appearing in the model can be determined by fitting the theoretical prediction to the experimental result. In addition, taking into account the fact that the number of native contacts is almost proportional to the relative contact order, we can also explain the observed relationship between the folding rate constant and the relative contact order.     

Reducing the computational complexity of protein folding via fragment folding and assembly

Understanding, and ultimately predicting, how a 1-D protein chain reaches its native 3-D fold has been one of the most challenging problems during the last few decades. Data increasingly indicate that protein folding is a hierarchical process. Hence, the question arises as to whether we can use the hierarchical concept to reduce the practically intractable computational times. For such a scheme to work, the first step is to cut the protein sequence into fragments that form local minima on the polypeptide chain. The conformations of such fragments in solution are likely to be similar to those when the fragments are embedded in the native fold, although alternate conformations may be favored during the mutual stabilization in the combinatorial assembly process. Two elements are needed for such cutting: (1) a library of (clustered) fragments derived from known protein structures and (2) an assignment algorithm that selects optimal combinations to "cover" the protein sequence. The next two steps in hierarchical folding schemes, not addressed here, are the combinatorial assembly of the fragments and finally, optimization of the obtained conformations. Here, we address the first step in a hierarchical protein-folding scheme. The input is a target protein sequence and a library of fragments created by clustering building blocks that were generated by cutting all protein structures. The output is a set of cutout fragments. We briefly outline a graph theoretic algorithm that automatically assigns building blocks to the target sequence, and we describe a sample of the results we have obtained.