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

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.     

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.     

A conserved folding mechanism for PDZ domains

An important question in protein folding is whether the folding mechanism is sequence dependent and conserved for homologous proteins. In this work we compared the kinetic folding mechanism of five postsynaptic density protein-95, disc-large tumor suppressor protein, zonula occludens-1 (PDZ) domains, sharing similar topology but having different primary structures. Investigation of the different proteins under various experimental conditions revealed that the folding kinetics of each member of the PDZ family can be described by a model with two transition states separated by an intermediate. Moreover, the positions of the two transition states along the reaction coordinate (as given by their beta(T)-values) are fairly constant for the five PDZ domains.     

The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding.

The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter phi. There are two extreme values of phi that are often found in practice and may be interpreted in a simple manner. A value of phi = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, phi = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of phi are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of phi found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of phi = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the double-mutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.     

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.     

Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus

The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (phi) values, and polarized transition states, which instead display distinct substructures with very high phi-values. Apo-and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine phi-value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular phi-values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo-and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.     

Interpretation of protein folding psi values

The structural characterization of transition states is essential for understanding the mechanism of protein folding. Analyzing the effect of mutations on protein stability and folding kinetics in phi-value analysis is commonly used to gain information about the presence of side-chain interactions in transition states. Recently, specific binding of ligands to engineered binding sites was applied to monitor the formation of local structures in transition states (psi analysis). A surprising result from psi analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between phi and psi values measured in the same protein. Here, we show that psi values cannot be analyzed in the same way as other rate-equilibrium free energy relationships due to the involvement of bimolecular reactions that may have different dissociation constants for the native, unfolded and transition state. As a consequence, psi values reflect the relative binding energy (kappa) of the transition state only for the extreme values of kappa=0 or kappa=1. In all other cases, non-linear rate-equilibrium free-energy relationships (Leffler plots) are observed. This apparently indicates the presence of parallel folding pathways even if folding occurs over a single homogeneous transition state. Consequently, the results from Leffler plots do not yield information about the structural properties of the transition state. This explains the lack of agreement between results from psi analysis and other methods used to characterize protein folding transition states. We further show that the same considerations apply for the analysis of the effect of pH on protein folding.     

Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions

The N-terminal beta-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 is also significantly populated in the transition state (phi(F)-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Ubeta4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.     

An error analysis for two-state protein-folding kinetic parameters and phi-values: progress toward precision by exploring pH dependencies on Leffler plots

The interpretation of φ-values has led to an understanding of the folding transition state ensemble of a variety of proteins. Although the main guidelines and equations for calculating φ are well established, there remains some controversy about the quality of the numerical values obtained. By analyzing a complete set of results from kinetic experiments with the SH3 domain of α-spectrin (Spc-SH3) and applying classical error methods and error-propagation formulas, we evaluated the uncertainties involved in two-state-folding kinetic experimental parameters and the corresponding calculated φ-values. We show that kinetic constants in water and m values can be properly estimated from a judicious weighting of fitting errors and describe some procedures to calculate the errors in Gibbs energies and φ-values from a traditional two-point Leffler analysis. Furthermore, on the basis of general assumptions made with the protein engineering method, we show how to generate multipoint Leffler plots via the analysis of pH dependencies of kinetic parameters. We calculated the definitive φ-values for a collection of single mutations previously designed to characterize the folding transition state of the α-spectrin SH3 domain. The effectiveness of the pH-scanning procedure is also discussed in the context of error analysis. Judging from the magnitudes of the error bars obtained from two-point and multipoint Leffler plots, we conclude that the precision obtained for φ-values should be ∼25%, a reasonable limit that takes into account the propagation of experimental errors.     

On prediction of folding nuclei in globular proteins

The approach described in this paper on the prediction of folding nuclei in globular proteins with known three dimensional structures is based on a search of the lowest saddle points through the barrier separating the unfolded state from the native structure on the free-energy landscape of protein chain. This search is performed by a dynamic programming method. Comparison of theoretical results with experimental data on the folding nuclei of two dozen of proteins shows that our model provides good phi value predictions for proteins whose structures have been determined by X-ray analysis, with a less limited success for proteins whose structures have been determined by NMR techniques only. Consideration of a full ensemble of transition states results in more successful prediction than consideration of only the transition states with the minimal free energy. In conclusion we have predicted the localization of folding nuclei for three dimensional protein structures for which kinetics of folding is studied now but the localization of folding nuclei is still unknown.     

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

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.     

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

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.     

The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

The protein engineering analysis of the alpha-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop beta-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Br?nsted plot analysis confirms the straight phi(doubledagger-U) results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the alpha-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.     

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.     

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.     

The analysis of protein folding kinetic data produced in protein engineering experiments

Over the past decade, the "protein engineering method" has been used to investigate the folding pathways of more than 20 different proteins. This method involves measuring the folding and unfolding rates of mutant proteins with single amino acid substitutions spread across the sequence. Comparison of folding rates of the mutant proteins to that of the wild-type protein allows the calculation of the phi value, which can be used to evaluate the stabilizing contribution of an amino acid side chain to the structure of the folding transition state. Here, we review the methodology for analysing data collected in protein engineering folding kinetics studies. We discuss the calculation of folding rates and kinetic m values, the estimation of errors in folding kinetics experiments, phi value calculation including potential pitfalls of the analysis, Br?nsted plots, detecting Hammond behaviour, and the analysis of curved chevron plots.     

Comparison of the transition state ensembles for folding of Im7 and Im9 determined using all-atom molecular dynamics simulations with phi value restraints

Delineation of the structural properties of transition states is key to deriving models for protein folding. Here we describe the structures of the transition states of the bacterial immunity proteins Im7 and Im9 obtained by all-atom molecular dynamics simulations with phi value restraints derived from protein engineering experiments. This pair of proteins is of special interest because, at pH 7 and 10 degrees C, Im7 folds via an intermediate while Im9 folds with a two-state transition. The structures of the transition states for Im7 and Im9, together with their radii of gyration and distances from the native state, are similar. The typical distance between any two members of the transition state ensemble of both proteins is large, with that of Im9 nearly twice that of Im7. Thus, a broad range of structures make up the transition state ensembles of these proteins. The ensembles satisfy the set of rather low phi values and yet are consistent with high beta(T) values (> 0.85 for both proteins). For both Im7 and Im9 the inter-helical angles are highly variable in the transition state ensembles, although the native contacts between helices I and IV are well conserved. By measuring the distribution of the accessible surface area for each residue we show that the hydrophobic residues that are buried in the native state remain buried in the transition state, corresponding to a hydrophobic collapse to a relatively ordered globule. The data provide new insights into the structural properties of the transition states of these proteins at an atomic level of detail and show that molecular dynamics simulations with phi value restraints can significantly enhance the knowledge of the transition state ensembles (TSE) provided by the experimental phi values alone.     

Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101.     

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.     

Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.