Common Structural Transitions in Explicit-Solvent Simulations of Villin Headpiece Folding

Molecular dynamics simulations of protein folding can provide very high-resolution data on the folding process; however, due to computational challenges most studies of protein folding have been limited to small peptides, or made use of approximations such as Gō potentials or implicit solvent models. We have performed a set of molecular dynamics simulations totaling >50 μs on the villin headpiece subdomain, one of the most stable and fastest-folding naturally occurring proteins, in explicit solvent. We find that the wild-type villin headpiece reliably folds to a native conformation on timescales similar to experimentally observed folding, but that a fast folding double-norleucine mutant shows significantly more heterogeneous behavior. Along with other recent simulation studies, we note the occurrence of nonnative structures intermediates, which may yield a nativelike signal in the fluorescence measurements typically used to study villin folding. Based on the wild-type simulations, we propose alternative approaches to measure the formation of the native state.     

Simulation of folding of a small alpha-helical protein in atomistic detail using worldwide-distributed computing

By employing thousands of PCs and new worldwide-distributed computing techniques, we have simulated in atomistic detail the folding of a fast-folding 36-residue alpha-helical protein from the villin headpiece. The total simulated time exceeds 300 micros, orders of magnitude more than previous simulations of a molecule of this size. Starting from an extended state, we obtained an ensemble of folded structures, which is on average 1.7A and 1.9A away from the native state in C(alpha) distance-based root-mean-square deviation (dRMS) and C(beta) dRMS sense, respectively. The folding mechanism of villin is most consistent with the hydrophobic collapse view of folding: the molecule collapses non-specifically very quickly ( approximately 20ns), which greatly reduces the size of the conformational space that needs to be explored in search of the native state. The conformational search in the collapsed state appears to be rate-limited by the formation of the aromatic core: in a significant fraction of our simulations, the C-terminal phenylalanine residue packs improperly with the rest of the hydrophobic core. We suggest that the breaking of this interaction may be the rate-determining step in the course of folding. On the basis of our simulations we estimate the folding rate of villin to be approximately 5micros. By analyzing the average features of the folded ensemble obtained by simulation, we see that the mean folded structure is more similar to the native fold than any individual folded structure. This finding highlights the need for simulating ensembles of molecules and averaging the results in an experiment-like fashion if meaningful comparison between simulation and experiment is to be attempted. Moreover, our results demonstrate that (1) the computational methodology exists to simulate the multi-microsecond regime using distributed computing and (2) that potential sets used to describe interatomic interactions may be sufficiently accurate to reach the folded state, at least for small proteins. We conclude with a comparison between our results and current protein-folding theory.     

Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain

The villin headpiece subdomain is a cooperatively folded 36-residue, three-alpha-helix protein. The domain is one of the smallest naturally occurring sequences which has been shown to fold. Recent experimental studies have shown that it folds on the 10-micros time scale. Its small size, simple topology, and very rapid folding have made it an attractive target for computational studies of protein folding. We present temperature-dependent NMR studies that provide evidence for significant structure in the denatured state of the headpiece subdomain. A set of peptide fragments derived from the headpiece were also characterized in order to determine if there is a significant tendency to form a locally stabilized structure in the denatured state. Peptides corresponding to each of the three isolated helices and to the connection between the first and second helices were largely unstructured. A longer peptide fragment which contains the first and second helices shows considerable structure, as judged by NMR and CD. Concentration-dependent CD measurements and analytical ultracentrifugation experiments indicate that the structure is not due to self-association. NMR studies indicate that the structure is stabilized by tertiary interactions involving phenylalanines and Val 50. A peptide in which two of the three phenylalanines are changed to leucine is considerably less structured, confirming the importance of the phenylalanines. This work indicates that there is significant structure in the denatured state of this rapidly folding protein.     

High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity

Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 A resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 A resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.     

Experimental tests of villin subdomain folding simulations

We have used laser temperature-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(+/-0.6) micros, making it the fastest folding, naturally occurring protein, with a rate close to the theoretical speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) micros by Zagrovic et al. from atomistic molecular dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equilibrium constant. Implications of this result for the validity of the simulated folding mechanism are discussed.     

Multistate folding of the villin headpiece domain

The villin headpiece (HP67) is a 67 residue, monomeric protein derived from the C-terminal domain of villin. Wild-type HP67 (WT HP67) is the smallest fragment of villin that retains strong in vitro actin-binding activity. WT HP67 is made up of two subdomains, which form a tightly packed interface. The C-terminal subdomain of WT HP67, denoted HP35, is rich in helical structure, folds in isolation, and has been widely used as a model system for folding studies. In contrast, very little is known about the folding of the intact villin headpiece domain. Here, NMR, CD and H/2H amide exchange measurements are used to follow the pH, thermal and urea-induced unfolding of WT HP67 and a mutant (HP67 H41Y) in which a buried conserved histidine in the N-terminal subdomain, His41, has been mutated to Tyr. Although most small proteins display two-state equilibrium unfolding, the results presented here demonstrate that unfolding of the villin headpiece is a multistate process. The presence of a folded N-terminal subdomain is shown to stabilize the C-terminal subdomain, increasing the midpoints of the thermal and urea-induced unfolding transitions and increasing protection factors for H/2H exchange. Histidine 41 has been shown to act as a pH-dependent switch in wild-type HP67: the N-terminal subdomain is unfolded when His41 is protonated, while the C-terminal subdomain remains folded irrespective of the protonation state of His41. Mutation of His41 to Tyr eliminates the segmental pH-dependent unfolding of the headpiece. The mutation stabilizes both domains, but folding is still multistate, indicating that His41 is not solely responsible for the unusual equilibrium unfolding behavior of villin headpiece domain.     

NMR characterization of a peptide model provides evidence for significant structure in the unfolded state of the villin headpiece helical subdomain

The villin headpiece subdomain (HP36) is the smallest naturally occurring protein that folds cooperatively. The protein folds on a microsecond time scale. Its small size and very rapid folding have made it a popular target for biophysical studies of protein folding. Temperature-dependent one-dimensional (1D) NMR studies of the full-length protein together with CD and 1D NMR studies of the 21-residue peptide fragment (HP21) derived from HP36 have shown that there is significant structure in the unfolded state of HP36 and have demonstrated that HP21 is a good model of these interactions. Here, we characterized the model peptide HP21 in detail by two-dimensional NMR. Strongly upfield shifted C(alpha) protons, the magnitude of the 3J(NH,alpha) coupling constants, and the pattern of backbone-backbone and backbone-side chain NOEs indicate that the ensemble of structures populated by HP21 contains alpha-helical structure and native as well as non-native hydrophobic contacts. The hydrogen-bonded secondary structure inferred from the NOEs is, however, not sufficient to confer significant protection against amide H-D exchange. These studies indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36. The implications for the folding of HP36 are discussed.     

The isolated sixth gelsolin repeat and headpiece domain of villin bundle F-actin in the presence of calcium and are linked by a 40-residue unstructured sequence

Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal "headpiece" domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actin and, at higher calcium concentrations, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of the headpiece to the core has not been determined. To address these issues, we employ a 208-residue modular fragment of villin, D6-HP, which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstrate that this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca2+-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonance assignments and 15N relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence. The headpiece domain in D6-HP retains its structure and interacts with D6 only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for D6 in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent of the core domain.     

Determination of ultrafast protein folding rates from loop formation dynamics

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.     

Equilibrium Kinetic Network of the Villin Headpiece in Implicit Solvent

We applied the single-replica multiple-state transition-interface sampling method to elucidate the equilibrium kinetic network of the 35-residue-fragment (HP-35) villin headpiece in implicit water at room temperature. Starting from the native Protein Data Bank structure, nine (meta)stable states of the system were identified, from which the kinetic network was built by sampling pathways between these states. Application of transition path theory allowed analysis of the (un)folding mechanism. The resulting (un)folding rates agree well with experiments. This work demonstrates that high (un)folding barriers can now be studied.     

All-atom fast protein folding simulations: the villin headpiece

We provide a fast folding simulation using an all-atom solute, implicit solvent method that eliminates the need for treating solvent degrees of freedom. The folding simulations for the 36-residue villin headpiece exhibit close correspondence with the landmark all-atom explicit solvent molecular dynamics simulations by Duan and Kollman (Duan & Kollman, Science 1998;282:740-744; Duan, Wang, & Kollman, Proc Natl Acad Sci USA 1998;95:9897-9902). Our implicit solvent approach uses only an entry-level single CPU PC with comparable throughput ( approximately 4 nsec/day) to the DK supercomputer simulation. The native state is shown to be stable. Our 200-nsec folding trajectory agrees with the DK simulation in displaying a burst phase, a rapid initial shrinkage, a highly native-like binding site structure, and more.     

The unusual internal motion of the villin headpiece subdomain

The thermostable 36‐residue subdomain of the villin headpiece (HP36) is the smallest known cooperatively folding protein. Although the folding and internal dynamics of HP36 and close variants have been extensively studied, there has not been a comprehensive investigation of side‐chain motion in this protein. Here, the fast motion of methyl‐bearing amino acid side chains is explored over a range of temperatures using site‐resolved solution nuclear magnetic resonance deuterium relaxation. The squared generalized order parameters of methyl groups extensively spatially segregate according to motional classes. This has not been observed before in any protein studied using this methodology. The class segregation is preserved from 275 to 305 K. Motions detected in Helix 3 suggest a fast timescale of conformational heterogeneity that has not been previously observed but is consistent with a range of folding and dynamics studies. Finally, a comparison between the order parameters in solution with previous results based on solid‐state nuclear magnetic resonance deuterium line shape analysis of HP36 in partially hydrated powders shows a clear disagreement for half of the sites. This result has significant implications for the interpretation of data derived from a variety of approaches that rely on partially hydrated protein samples.     

Differential stabilization of two hydrophobic cores in the transition state of the villin 14T folding reaction

We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.     

Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity, and trapping in the ribosome

Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.     

Sub-microsecond protein folding

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.     

Population of on-pathway intermediates in the folding of ubiquitin

The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.     

Heterogeneity even at the speed limit of folding: large-scale molecular dynamics study of a fast-folding variant of the villin headpiece

We have performed molecular dynamics simulations on a set of nine unfolded conformations of the fastest-folding protein yet discovered, a variant of the villin headpiece subdomain (HP-35 NleNle). The simulations were generated using a new distributed computing method, yielding hundreds of trajectories each on a time scale comparable to the experimental folding time, despite the large (10,000 atom) size of the simulation system. This strategy eliminates the need to assume a two-state kinetic model or to build a Markov state model. The relaxation to the folded state at 300 K from the unfolded configurations (generated by simulation at 373 K) was monitored by a method intended to reflect the experimental observable (quenching of tryptophan by histidine). We also monitored the relaxation to the native state by directly comparing structural snapshots with the native state. The rate of relaxation to the native state and the number of resolvable kinetic time scales both depend upon starting structure. Moreover, starting structures with folding rates most similar to experiment show some native-like structure in the N-terminal helix (helix 1) and the phenylalanine residues constituting the hydrophobic core, suggesting that these elements may exist in the experimentally relevant unfolded state. Our large-scale simulation data reveal kinetic complexity not resolved in the experimental data. Based on these findings, we propose additional experiments to further probe the kinetics of villin folding.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.