Understanding the mechanism of beta-sheet folding from a chemical and biological perspective

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.     

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.     

Toward quantification of protein backbone-backbone hydrogen bonding energies: An energetic analysis of an amide-to-ester mutation in an alpha-helix within a protein

Amide-to-ester backbone mutagenesis enables a specific backbone-backbone hydrogen bond (H-bond) in a protein to be eliminated in order to quantify its energetic contribution to protein folding. To extract a H-bonding free energy from an amide-to-ester perturbation free energy (DeltaG (folding,wt) - DeltaG (folding,mut)), it is necessary to correct for the putative introduction of a lone pair-lone pair electrostatic repulsion, as well as for the transfer free energy differences that may arise between the all amide sequence and the predominantly amide sequence harboring an ester bond. Mutation of the 9-10 amide bond within the V9F variant of the predominantly helical villin headpiece subdomain (HP35) to an ester or an E-olefin backbone bond results in a less stable but defined wild-type fold, an attribute required for this study. Comparing the folding free energies of the ester and E-olefin mutants, with correction for the desolvation free energy differences (ester and E-olefin) and the loss of an n-to-pi* interaction (E-olefin), yields an experimentally based estimate of +0.4 kcal/mol for the O-O repulsion energy in an alpha-helical context, analogous to our previous experimentally based estimate of the O-O repulsion free energy in the context of a beta-sheet. The small O-O repulsion energy indicates that amide-to-ester perturbation free energies can largely be attributed to the deletion of the backbone H-bonds after correction for desolvation differences. Quantitative evaluation of H-bonding in an alpha-helix should now be possible, an important step toward deciphering the balance of forces that enable spontaneous protein folding.     

Comparative analysis of two different amide-to-ester bond mutations in the beta-sheet of 4-oxalocrotonate tautomerase

Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.     

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.     

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.     

Critical role of beta-hairpin formation in protein G folding

Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the approximately 60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed beta-hairpins. We demonstrate that, as in the case of protein L, one of the two beta-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second beta-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain-main chain hydrogen bond in the second beta-turn slows the folding rate approximately 20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact beta-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated beta-hairpins.     

Beta-sheet folding mechanisms from perturbation energetics

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.     

Phi-analysis of the folding of the B domain of protein A using multiple optical probes

We examined the co-operativity of ultra-fast folding of a protein and whether the Phi-value analysis of its transition state depended on the location of the optical probe. We incorporated in turn a tryptophan residue into each of the three helices of the B domain of Protein A. Each Trp mutant of the three-helix bundle protein was used as a pseudo-wild-type parent for Phi-analysis in which the intrinsic Trp fluorescence probed the formation of each helix during the transition state. Apart from local effects in the immediate vicinity of the probe, the three separate sets of Phi-values were in excellent agreement, demonstrating the overall co-operativity of folding and the robustness of the Phi-analysis. The transition state of folding of Protein A contains the second helix being well formed with many stabilizing tertiary hydrophobic interactions. In contrast, the first and the third helices are more poorly structured in the transition state. The mechanism of folding thus involves the concurrent formation of secondary and tertiary interactions, and is towards the nucleation-condensation extreme in the nucleation-condensation-framework continuum of mechanism, with helix 2 being the nucleus. We provide an error analysis of Phi-values derived purely from the kinetics of two-state chevron plots.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.     

Insights into stabilizing weak interactions in designed peptide beta-hairpins

beta-Hairpin peptides (two anti-parallel strands linked by a reverse beta-turn) have emerged as the simplest systems for probing weak interactions in beta-sheet folding. We describe a model 16-residue hairpin system (peptide beta1: KKYTVSINGKKITVSI) designed around the anti-parallel beta-sheet DNA binding motif of the Met repressor dimer in which two beta-strand sequences are linked through an Asn-Gly type I' beta-turn. The peptide is significantly folded in aqueous solution and has a well-defined conformation as evident from an abundance of NOE data. We review a number of analogues of beta1 designed to estimate the energetic contribution of electrostatic (ion pairing) interactions to hairpin stability, to examine effects of cooperativity and preorganization in determining the energetics of weak interactions, and examine the effects on stability and conformation of incorporation of a three-histidine motif on one face of the hairpin capable of zinc complexation.     

Evidence for partial secondary structure formation in the transition state for arc repressor refolding and dimerization

Structure formation and dimerization are concerted processes in the refolding of Arc repressor. The integrity of secondary structure in the transition state of Arc refolding has been investigated here by determining the changes in equilibrium stability and refolding/unfolding kinetics for a set of Ala --> Gly mutations at residues that are solvent-exposed in the native Arc dimer. At some sites, reduced stability was caused primarily by faster unfolding, indicating that secondary structure at these positions is largely absent in the transition state. However, most of the Ala --> Gly substitutions in the alpha-helices of Arc and a triple mutant in the beta-sheet also resulted in decreased refolding rates, in some cases, accounting for the major fraction of thermodynamic destabilization. Overall, these results suggest that some regions of native secondary structure are present but incompletely formed in the transition state of Arc refolding and dimerization. Consolidation of this secondary structure, like close packing of the hydrophobic core, seems to occur later in the folding process. On average, Phi(F) values for the Ala --> Gly mutations were significantly larger than Phi(F) values previously determined for alanine-substitution mutants, suggesting that backbone interactions in the transition state may be stronger than side chain interactions. Mutations causing significant reductions in the Arc refolding rate were found to cluster in the central turn of alpha-helix A and in the first two turns of alpha-helix B. In the Arc dimer, these elements pack together in a compact structure, which might serve as nucleus for further folding.     

Beta-hairpin folding simulations in atomistic detail using an implicit solvent model

We have used distributed computing techniques and a supercluster of thousands of computer processors to study folding of the C-terminal beta-hairpin from protein G in atomistic detail using the GB/SA implicit solvent model at 300 K. We have simulated a total of nearly 38 micros of folding time and obtained eight complete and independent folding trajectories. Starting from an extended state, we observe relaxation to an unfolded state characterized by non-specific, temporary hydrogen bonding. This is followed by the appearance of interactions between hydrophobic residues that stabilize a bent intermediate. Final formation of the complete hydrophobic core occurs cooperatively at the same time that the final hydrogen bonding pattern appears. The folded hairpin structures we observe all contain a closely packed hydrophobic core and proper beta-sheet backbone dihedral angles, but they differ in backbone hydrogen bonding pattern. We show that this is consistent with the existing experimental data on the hairpin alone in solution. Our analysis also reveals short-lived semi-helical intermediates which define a thermodynamic trap. Our results are consistent with a three-state mechanism with a single rate-limiting step in which a varying final hydrogen bond pattern is apparent, and semi-helical off-pathway intermediates may appear early in the folding process. We include details of the ensemble dynamics methodology and a discussion of our achievements using this new computational device for studying dynamics at the atomic level.     

Contribution of a buried hydrogen bond to lambda repressor folding kinetics.

A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of lambda6-85, wild type and the faster folding G46A/G48A (WT), show similar effects. Diffusion-collision correctly predicts the behavior of WT but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14-77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion-collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.     

Time-resolved backbone desolvation and mutational hot spots in folding proteins

A method is presented to identify hot mutational spots and predict the extent of surface burial at the transition state relative to the native fold in two-state folding proteins. The method is based on ab initio simulations of folding histories in which transitions between coarsely defined conformations and pairwise interactions are dependent on the solvent environments created by the chain. The highly conserved mammalian ubiquitin is adopted as a study case to make predictions. The evolution in time of the chain topology suggests a nucleation process with a critical point signaled by a sudden quenching of structural fluctuations. The occurrence of this nucleus is shown to be concurrent with a sudden escalation in the number of three-body correlations whereby hydrophobic units approach residue pairs engaged in amide-carbonyl hydrogen bonding. These correlations determine a pattern designed to structure the surrounding solvent, protecting intramolecular hydrogen bonds from water attack. Such correlations are shown to be required to stabilize the nucleus, with kinetic consequences for the folding process. Those nuclear residues that adopt the dual role of protecting and being protected while engaged in hydrogen bonds are predicted to be the hottest mutational spots. Some such residues are shown not to retain the same protecting role in the native fold. This kinetic treatment of folding nucleation is independently validated vis-a-vis a Phi-value analysis on chymotrypsin inhibitor 2, a protein for which extensive mutational data exists.     

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.     

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy,protein engineering and molecular dynamics simulations

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.     

Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.     

The protein folding transition state: what are Phi-values really telling us?

Protein engineering-based studies of the folding transition state have accelerated significantly in the last decade, and more than a half dozen proteins have been subjected to extensive Phi-value analysis. A general picture is emerging from these studies of a transition state in which the large majority of experimentally characterized side chains participate in relatively homogeneous and energetically weak interactions playing only a relatively small role in defining relative folding rates.     

Acceleration of the refolding of Arc repressor by nucleic acids and other polyanions.

The refolding rate of the Arc repressor dimer can be accelerated 30-fold or more by negatively charged polymers including single-stranded and double-stranded DNA, RNA, and polyvinylsulfate but not by neutral or positively charged polymers. The salt-dependence of the polyanion-mediated process and mutant studies indicate that electrostatic interactions are important in the rate acceleration. Urea-dependence studies suggest that Arc is relatively unstructured in the transition state for polyanion-stimulated refolding. At low ionic strength, the observed kinetics of refolding are consistent with a model in which denatured Arc monomers bind rapidly and nonspecifically to the polyanion and complete folding in the bound state.