A residue in helical conformation in the native state adopts a β-strand conformation in the folding transition state despite its high and canonical Φ-value

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state.     

Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.     

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.     

Hydrophobic core packing in the SH3 domain folding transition state.

How tightly packed is the hydrophobic core of a folding transition state structure? We have addressed this question by characterizing the effects on folding kinetics of > 40 substitutions of both large and small amino acids in the hydrophobic core of the Fyn SH3 domain. Our results show that residues at three positions, which we designate as the 'core folding nucleus', are tightly packed in the transition state, and substitutions at these positions cause the largest changes in the folding rate. The other six positions examined appear to be loosely packed; thus, substitutions at these positions with larger hydrophobic residues generally accelerate folding, presumably by increasing the rate of nonspecific hydrophobic collapse. Surprisingly, the folding rate can be greatly accelerated by residues that also significantly destabilize the native state structure. Furthermore, mutants with identical thermodynamic stability can differ by up to 55-fold in their folding rates. These results highlight the importance of hydrophobic core composition, as opposed to only topology, in determining the folding rate of a protein. They also provide a new explanation for the 'abnormal' phi-values observed in many protein folding kinetics studies.     

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.     

Thermodynamics and folding kinetics analysis of the SH3 domain form discrete molecular dynamics

We perform a detailed analysis of the thermodynamics and folding kinetics of the SH3 domain fold with discrete molecular dynamic simulations. We propose a protein model that reproduces some of the experimentally observed thermodynamic and folding kinetic properties of proteins. Specifically, we use our model to study the transition state ensemble of the SH3 fold family of proteins, a set of unstable conformations that fold to the protein native state with probability 1/2. We analyze the participation of each secondary structure element formed at the transition state ensemble. We also identify the folding nucleus of the SH3 fold and test extensively its importance for folding kinetics. We predict that a set of amino acid contacts between the RT-loop and the distal hairpin are the critical folding nucleus of the SH3 fold and propose a hypothesis that explains this result.     

Exploring local and non-local interactions for protein stability by structural motif engineering

In order to probe the relative contribution of local and non-local interactions to the thermodynamic stability of proteins, we have devised an experimental approach based on a combination of motif engineering and sequence shuffling. Candidate chain segments in an immunoglobulin V(L) domain were identified whose conformation is proposed to be dominated by non-local interactions. Locally interacting structural motifs of a different conformation were then constructed as replacements, by introducing motif consensus sequences. We find that all nine replacements we constructed systematically reduce the folding cooperativity. By comparing this destabilising effect with the folding transitions of shuffled sequences for three of these motifs, we estimate the contribution of local, native interactions to the free energy of folding. Our results suggest that local and non-local interactions contribute to stability by an approximately equal amount, but that local interactions stabilise by increasing the resistance to denaturation while non-local interactions increase folding cooperativity. The systematic loss of stability by sequence shuffling in these host-guest experiments suggests that the designed interactions indeed are present in the native state, thus consensus sequence engineering may be a useful tool in structure design, but non-local interactions must be taken into account for global stability engineering. Statistical approaches are powerful tools for engineering protein structure and stability, but an analysis based on local sequence propensities alone does not adequately represent the balance of sequence and context in protein structures.     

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure.     

Experiment and theory highlight role of native state topology in SH3 folding.

We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded beta-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.     

Cooperativity in two-state protein folding kinetics

We present a solvable model that predicts the folding kinetics of two-state proteins from their native structures. The model is based on conditional chain entropies. It assumes that folding processes are dominated by small-loop closure events that can be inferred from native structures. For CI2, the src SH3 domain, TNfn3, and protein L, the model reproduces two-state kinetics, and it predicts well the average Phi-values for secondary structures. The barrier to folding is the formation of predominantly local structures such as helices and hairpins, which are needed to bring nonlocal pairs of amino acids into contact.     

NMR elucidation of early folding hierarchy in HIV-1 protease

Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, HN-Halpha coupling constants, 1H-15N nuclear Overhauser effects, and 15N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 m guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.     

Detecting hidden sequence propensity for amyloid fibril formation

The preponderance of evidence implicates protein misfolding in many unrelated human diseases. In all cases, normal correctly folded proteins transform from their proper native structure into an abnormal beta-rich structure known as amyloid fibril. Here we introduce a computational algorithm to detect nonnative (hidden) sequence propensity for amyloid fibril formation. Analyzing sequence-structure relationships in terms of tertiary contact (TC), we find that the hidden beta-strand propensity of a query local sequence can be quantitatively estimated from the secondary structure preferences of template sequences of known secondary structure found in regions of high TC. The present method correctly pinpoints the minimal peptide fragment shown experimentally as the likely local mediator of amyloid fibril formation in beta-amyloid peptide, islet amyloid polypeptide (hIAPP), alpha-synuclein, and human acetylcholinesterase (AChE). It also found previously unrecognized beta-strand propensities in the prototypical helical protein myoglobin that has been reported as amyloidogenic. Analysis of 2358 nonhomologous protein domains provides compelling evidence that most proteins contain sequences with significant hidden beta-strand propensity. The present method may find utility in many medically relevant applications, such as the engineering of protein sequences and the discovery of therapeutic agents that specifically target these sequences for the prevention and treatment of amyloid diseases.     

Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540-548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540-544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.     

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.     

Multiple folding pathways of the SH3 domain

Experimental observations suggest that proteins follow different folding pathways under different environmental conditions. We perform molecular dynamics simulations of a model of the c-Crk SH3 domain over a broad range of temperatures, and identify distinct pathways in the folding transition. We determine the kinetic partition temperature-the temperature for which the c-Crk SH3 domain undergoes a rapid folding transition with minimal kinetic barriers-and observe that below this temperature the model protein may undergo a folding transition by multiple folding pathways via only one or two intermediates. Our findings suggest the hypothesis that the SH3 domain, a protein fold for which only two-state folding kinetics was observed in previous experiments, may exhibit intermediate states under conditions that strongly stabilize the native state.     

Transition states for protein folding have native topologies despite high structural variability

We present a structural analysis of the folding transition states of three SH3 domains. Our results reveal that the secondary structure is not yet fully formed at this stage of folding and that the solvent is only partially excluded from the interior of the protein. Comparison of the members of the transition state ensemble with a database of native folds shows that, despite substantial local variability, the transition state structures can all be classified as having the topology characteristic of an SH3 domain. Our results suggest a mechanism for folding in which the formation of a network of interactions among a subset of hydrophobic residues ensures that the native topology is generated. Such a mechanism enables high fidelity in folding while minimizing the need to establish a large number of specific interactions in the conformational search.     

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.     

Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains

Protein engineering experiments and Phi(F)-value analysis of SH3 domains reveal that their transition state ensemble (TSE) is conformationally restricted, i.e. the fluctuations in the transition state (TS) structures are small. In the TS of src SH3 and alpha-spectrin SH3 the distal loop and the associated hairpin are fully structured, while the rest of the protein is relatively disordered. If native structure predominantly determines the folding mechanism, the findings for SH3 folds raise the question: What are the features of the native topology that determine the nature of the TSE? We propose that the presence of stiff loops in the native state that connect local structural elements (such as the distal hairpin in SH3 domains) conformationally restricts TSE. We validate this hypothesis using the simulations of a "control" system (16 residue beta-hairpin forming C-terminal fragment of the GBl protein) and its variants. In these fragments the role of bending rigidity in determining the nature of the TSE can be directly examined without complications arising from interactions with the rest of the protein. The TSE structures in the beta-hairpins are determined computationally using cluster analysis and limited Phi(F)-value analysis. Both techniques prove that the conformational heterogeneity decreases as the bending rigidity of the loop increases. To extend this finding to SH3 domains a measure of bending rigidity based on loop curvature, which utilizes native structures in the Protein Data Bank (PDB), is introduced. Using this measure we show that, with few exceptions, the ordering of stiffness of the distal, n-src, and RT loops in the 29 PDB structures of SH3 domains is conserved. Combining the simulation results for beta-hairpins and the analysis of PDB structures for SH3 domains, we propose that the stiff distal loop restricts the conformational fluctuations in the TSE. We also predict that constraining the distal loop to be preformed in the denatured ensemble should not alter the nature of TSE. On the other hand, if the amino and carboxy terminals are cross-linked to form a circular polypeptide chain, the pathways and TSs are altered. These contrasting scenarios are illustrated using simulations of cross-linked WT beta-hairpin fragments. Computations of bending rigidities for immunoglobulin-like domain proteins reveal no clear separation in the stiffness of their loops. In the beta-sandwich proteins, which have large fractions of non-local native contacts, the nature of the TSE cannot be apparently determined using purely local structural characteristics. Nevertheless, the measure of loop stiffness still provides qualitative predictions of the ordered regions in the TSE of Ig27 and TenFn3.     

The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue-residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.