Residues participating in the protein folding nucleus do not exhibit preferential evolutionary conservation

To what extent does natural selection act to optimize the details of protein folding kinetics? In an effort to address this question, the relationship between an amino acid's evolutionary conservation and its role in protein folding kinetics has been investigated intensively. Despite this effort, no consensus has been reached regarding the degree to which residues involved in native-like transition state structure (the folding nucleus) are conserved. Here we report the results of an exhaustive, systematic study of sequence conservation among residues known to participate in the experimentally (Phi-value) defined folding nuclei of all of the appropriately characterized proteins reported to date. We observe no significant evidence that these residues exhibit any anomalous sequence conservation. We do observe, however, a significant bias in the existing kinetic data: the mean sequence conservation of the residues that have been the subject of kinetic characterization is greater than the mean sequence conservation of all residues in 13 of 14 proteins studied. This systematic experimental bias gives rise to the previous observation that the median conservation of residues reported to participate in the folding nucleus is greater than the median conservation of all of the residues in a protein. When this bias is corrected (by comparing, for example, the conservation of residues known to participate in the folding nucleus with that of other, kinetically characterized residues) the previously reported preferential conservation is effectively eliminated. In contrast to well-established theoretical expectations, both poorly and highly conserved residues are apparently equally likely to participate in the protein-folding nucleus.     

Sequence optimization for native state stability determines the evolution and folding kinetics of a small protein

Investigating the relative importance of protein stability, function, and folding kinetics in driving protein evolution has long been hindered by the fact that we can only compare modern natural proteins, the products of the very process we seek to understand, to each other, with no external references or baselines. Through a large-scale all-atom simulation of protein evolution, we have created a large diverse alignment of SH3 domain sequences which have been selected only for native state stability, with no other influencing factors. Although the average pairwise identity between computationally evolved and natural sequences is only 17%, the residue frequency distributions of the computationally evolved sequences are similar to natural SH3 sequences at 86% of the positions in the domain, suggesting that optimization for the native state structure has dominated the evolution of natural SH3 domains. Additionally, the positions which play a consistent role in the transition state of three well-characterized SH3 domains (by phi-value analysis) are structurally optimized for the native state, and vice versa. Indeed, we see a specific and significant correlation between sequence optimization for native state stability and conservation of transition state structure.     

The fast folding pathway in human lysozyme and its blockage by appropriate mutagenesis: a sequential stopped-flow fluorescence study

In this work we were able to show that human lysozyme refolds along two parallel pathways: a fast path followed by 13% of the molecules that leads directly from a collapsed state to the native protein and a slow one for the remaining molecules that involves a partially unfolded intermediate state. However, in the refolding process of LYLA1, a chimera of human lysozyme which possesses the Ca2+-binding loop and helix C of bovine alpha-lactalbumin, the direct pathway is no longer accessible. This indicates that these structural elements, which are located in the interface region between the alpha- and beta-domain of the protein, and their interaction with the environment play an important role in the fast folding of the molecules.These results also shed some light on the conservation of folding patterns amongst structurally homologous proteins. In recent years it was often stated that structurally homologous proteins with high sequence identity follow the same folding pattern. Human lysozyme and LYLA1 have a sequence identity of 87%. However, we have shown that their folding patterns are different. Therefore, a high degree of sequence identity for two proteins belonging to the same family is not a guarantee for an identical folding pattern.     

The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family

Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Φ-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Φ-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Φ values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Φ values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains.     

The family feud: do proteins with similar structures fold via the same pathway?

Theoretical and experimental studies of protein folding have suggested that the topology of the native state may be the most important factor determining the folding pathway of a protein, independent of its specific amino acid sequence. To test this concept, many experimental studies have been carried out with the aim of comparing the folding pathways of proteins that possess similar tertiary structures, but divergent sequences. Many of these studies focus on quantitative comparisons of folding transition state structures, as determined by Phi(f) value analysis of folding kinetic data. In some of these studies, folding transition state structures are found to be highly conserved, whereas in others they are not. We conclude that folds displaying more conserved transition state structures may have the most restricted number of possible folding pathways and that folds displaying low transition state structural conservation possess many potential pathways for reaching the native state.     

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.     

Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: similarities and differences in the folding of homologous proteins

The bacterial immunity proteins Im7 and Im9 fold with mechanisms of different kinetic complexity. Whilst Im9 folds in a two-state transition at pH 7.0 and 10 degrees C, Im7 populates an on-pathway intermediate under these conditions. In order to assess the role of sequence versus topology in the folding of these proteins, and to analyse the effect of populating an intermediate on the landscape for folding, we have determined the conformational properties of the rate-limiting transition state for Im9 folding/unfolding using Phi(F)-value analysis and have compared the results with similar data obtained previously for Im7. The data show that the rate-limiting transition states for Im9 and Im7 folding/unfolding are similar: both are compact (beta(T)=0.94 and 0.89, respectively) and contain three of the four native helices docked around a specific hydrophobic core. Significant differences are observed, however, in the magnitude of the Phi(F)-values obtained for the two proteins. Of the 20 residues studied in both proteins, ten have Phi(F)-values in Im7 that exceed those in Im9 by more than 0.2, and of these five differ by more than 0.4. The data suggest that the population of an intermediate in Im7 results in folding via a transition state ensemble that is conformationally restricted relative to that of Im9. The data are consistent with the view that topology is an important determinant of folding. Importantly, however, they also demonstrate that while the folding transition state may be conserved in homologous proteins that fold with two and three-state kinetics, the population of an intermediate can have a significant effect on the breadth of the transition state ensemble.     

Sensitivity of the Folding Pathway to the Details of Amino Acid Sequence

Experimental data on the structure of the transition state demonstrate that proteins with the same topology as a rule have similar folding nuclei (the structured formed part of the transition state). In this review discussed are the experimental works showing that the position of folding nuclei is different among proteins with the same topology. These facts emphasize that the folding pathway is sensitive to the details of amino acid sequence.     

Sensitivity of the folding pathway to the details of amino acid sequence

Experimental data on the structure of transition state demonstrate that the proteins with the same topology as a rule have similar folding nuclei (the structured formed part of the transition state). In this review we discuss the experimental works which show that the position of folding nuclei is different among proteins with the same topology. These facts underline that the folding pathway is sensitive to the details of amino-acid sequence.     

The SH3-fold family: experimental evidence and prediction of variations in the folding pathways

To investigate the relationships between protein topology, amino acid sequence and folding mechanisms, the folding transition state of the Sso7d protein has been characterised both experimentally and theoretically. Although Sso7d protein has a similar topology to that of the SH3 domains, the structure of its transition state is different from that of alpha-spectrin and src SH3 domains previously studied. The folding algorithm, Fold-X, including an energy function with specific sequence features, accounts for these differences and reproduces with a good agreement the set of experimental phi(double dagger-U) values obtained for the three proteins. Our analysis shows that taking into account sequence features underlying protein topology is critical for an accurate prediction of the folding process.     

Sequence and structure conservation in a protein core

In order to study structural aspects of sequence conservation in families of homologous proteins, we have analyzed structurally aligned sequences of 585 proteins grouped into 128 homologous families. The conservation of a residue in a family is defined as the average residue similarity in a given position of aligned sequences. The residue similarities were expressed in the form of log-odd substitution tables that take into account the environments of amino acids in three-dimensional structures. The protein core is defined as those residues that have less then 7% solvent accessibility. The density of a protein core is described in terms of atom packing, which is investigated as a criterion for residue substitution and conservation. Although there is no significant correlation between sequence conservation and average atom packing around nonpolar residues such as leucine, valine and isoleucine, a significant correlation is observed for polar residues in the protein core. This may be explained by the hydrogen bonds in which polar residues are involved; the better their protection from water access the more stable should be the structure in that position. Proteins 33:358–366, 1998. © 1998 Wiley-Liss, Inc.     

Analycys: a database for conservation and conformation of disulphide bonds in homologous protein domains

Disulphide bonds in proteins are known to play diverse roles ranging from folding to structure to function. Thorough knowledge of the conservation status and structural state of the disulphide bonds will help in understanding of the differences in homologous proteins. Here we present a database for the analysis of conservation and conformation of disulphide bonds in SCOP structural families. This database has a wide range of applications including mapping of disulphide bond mutation patterns, identification of disulphide bonds important for folding and stabilization, modeling of protein tertiary structures and in protein engineering. The database can be accessed at: http://bioinformatics.univ-reunion.fr/analycys/.     

Prediction of folding transition-state position (betaT) of small, two-state proteins from local secondary structure content

Folding kinetics of proteins is governed by the free energy and position of transition states. But attempts to predict the position of folding transition state on reaction pathway from protein structure have been met with only limited success, unlike the folding-rate prediction. Here, we find that the folding transition-state position is related to the secondary structure content of native two-state proteins. We present a simple method for predicting the transition-state position from their alpha-helix, turn and polyproline secondary structures. The method achieves 81% correlation with experiment over 24 small, two-state proteins, suggesting that the local secondary structure content, especially for content of alpha-helix, is a determinant of the solvent accessibility of the transition state ensemble and size of folding nucleus.     

Accurate prediction of protein folding rates from sequence and sequence‐derived residue flexibility and solvent accessibility

Protein folding rates vary by several orders of magnitude and they depend on the topology of the fold and the size and composition of the sequence. Although recent works show that the rates can be predicted from the sequence, allowing for high‐throughput annotations, they consider only the sequence and its predicted secondary structure. We propose a novel sequence‐based predictor, PFR‐AF, which utilizes solvent accessibility and residue flexibility predicted from the sequence, to improve predictions and provide insights into the folding process. The predictor includes three linear regressions for proteins with two‐state, multistate, and unknown (mixed‐state) folding kinetics. PFR‐AF on average outperforms current methods when tested on three datasets. The proposed approach provides high‐quality predictions in the absence of similarity between the predicted and the training sequences. The PFR‐AF's predictions are characterized by high (between 0.71 and 0.95, depending on the dataset) correlation and the lowest (between 0.75 and 0.9) mean absolute errors with respect to the experimental rates, as measured using out‐of‐sample tests. Our models reveal that for the two‐state chains inclusion of solvent‐exposed Ala may accelerate the folding, while increased content of Ile may reduce the folding speed. We also demonstrate that increased flexibility of coils facilitates faster folding and that proteins with larger content of solvent‐exposed strands may fold at a slower pace. The increased flexibility of the solvent‐exposed residues is shown to elongate folding, which also holds, with a lower correlation, for buried residues. Two case studies are included to support our findings. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Investigation of an Anomalously Accelerating Substitution in the Folding of a Prototypical Two-State Protein

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.     

Calculation of mutational free energy changes in transition states for protein folding

Recent advances in experimental and computational methods have made it possible to determine with considerable accuracy the structures whose formation is rate limiting for the folding of some small proteins-the transition state ensemble, or TSE. We present a method to analyze and validate all-atom models of such structures. The method is based on the comparison of experimental data with the computation of the change in free energy of the TSE resulting from specific mutations. Each mutation is modeled individually in all members of an ensemble of transition state structures using a method originally developed to predict mutational changes in the stability of native proteins. We first apply this method to six proteins for which we have determined the TSEs with a technique that uses experimental mutational data (Phi-values) as restraints in the structure determination and find a highly significant correlation between the calculated free energy changes and those derived from experimental kinetic data. We then use the procedure to analyze transition state structures determined by molecular dynamics simulations of unfolding, again finding a high correlation. Finally, we use the method to estimate changes in folding rates of several hydrophobic core mutants of Fyn SH3. Taken together, these results show that the procedure developed here is a tool of general validity for analyzing, assessing, and improving the quality of the structures of transition states for protein folding.     

Studying the folding process of the acylphosphatase from Sulfolobus solfataricus. A comparative analysis with other proteins from the same superfamily

The folding process of the acylphosphatase from Sulfolobus solfataricus (Sso AcP) has been followed, starting from the fully unfolded state, using a variety of spectroscopic probes, including intrinsic fluorescence, circular dichroism, and ANS binding. The results indicate that an ensemble of partially folded or misfolded species form rapidly on the submillisecond time scale after initiation of folding. This conformational ensemble produces a pronounced downward curvature in the Chevron plot, appears to possess a content of secondary structure similar to that of the native state, as revealed by far-UV circular dichroism, and appears to have surface-exposed hydrophobic clusters, as indicated by the ability of this ensemble to bind to 8-anilino-1-naphthalenesulfonic acid (ANS). Sso AcP folds from this conformational state with a rate constant of ca. 5 s(-1) at pH 5.5 and 37 degrees C. A minor slow exponential phase detected during folding (rate constant of 0.2 s(-1) under these conditions) is accelerated by cyclophilin A and is absent in a mutant of Sso AcP in which alanine replaces the proline residue at position 50. This indicates that for a lower fraction of Sso AcP molecules the folding process is rate-limited by the cis-trans isomerism of the peptide bond preceding Pro50. A comparative analysis with four other homologous proteins from the acylphosphatase superfamily shows that sequence hydrophobicity is an important determinant of the conformational stability of partially folded states that may accumulate during folding of a protein. A low net charge and a high propensity to form alpha-helical structure also emerge as possibly important determinants of the stability of partially folded states. A significant correlation is also observed between folding rate and hydrophobic content of the sequence within this superfamily, lending support to the idea that sequence hydrophobicity, in addition to relative contact order and conformational stability of the native state, is a key determinant of folding rate.     

Effects of Topology and Sequence in Protein Folding Linked via Conformational Fluctuations

Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-β, whereas the folding of more local, all-α proteins typically exhibits a stronger sequence dependence. Here, we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3α, 4β + α, and β-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free-energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2000 sequence variants representing all possible hydrophobic or polar single- and double-point mutations. From an analysis of the subset of stability-neutral mutations, we find that folding is perturbed in a topology-dependent manner, with the β-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the β-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.     

Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway.

BACKGROUND: Are folding pathways conserved in protein families? To test this explicitly and ask to what extent structure specifies folding pathways requires comparison of proteins with a common fold. Our strategy is to choose members of a highly diverse protein family with no conservation of function and little or no sequence identity, but with structures that are essentially the same. The immunoglobulin-like fold is one of the most common structural families, and is subdivided into superfamilies with no detectable evolutionary or functional relationship. RESULTS: We compared the folding of a number of immunoglobulin-like proteins that have a common structural core and found a strong correlation between folding rate and stability. The results suggest that the folding pathways of these immunoglobulin-like proteins share common features. CONCLUSIONS: This study is the first to compare the folding of structurally related proteins that are members of different superfamilies. The most likely explanation for the results is that interactions that are important in defining the structure of immunoglobulin-like proteins are also used to guide folding.     

Mapping the folding pathway of an immunoglobulin domain: structural detail from Phi value analysis and movement of the transition state

BACKGROUND: Do proteins that have the same structure fold by the same pathway even when they are unrelated in sequence? To address this question, we are comparing the folding of a number of different immunoglobulin-like proteins. Here, we present a detailed protein engineering phi value analysis of the folding pathway of TI I27, an immunoglobulin domain from human cardiac titin. RESULTS: TI I27 folds rapidly via a kinetic intermediate that is destabilized by most mutations. The transition state for folding is remarkably native-like in terms of solvent accessibility. We use phi value analysis to map this transition state and show that it is highly structured; only a few residues close to the N-terminal region of the protein remain completely unfolded. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of obtaining additional structural information about the transition state. CONCLUSIONS: The residues that are involved in nucleating the folding of TI I27 are structurally equivalent to the residues that form the folding nucleus in an evolutionary unrelated fibronectin type III protein. These residues form part of the common structural core of Ig-like domains. The data support the hypothesis that interactions essential for defining the structure of these beta sandwich proteins are also important in nucleation of folding.