Cooperative folding mechanism of a beta-hairpin peptide studied by a multicanonical replica-exchange molecular dynamics simulation

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.     

Role of side-chains in the cooperative beta-hairpin folding of the short C-terminal fragment derived from streptococcal protein G

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.     

Bergerac-SH3: "frustation" induced by stabilizing the folding nucleus

The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha-spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour.     

Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stable beta-hairpin: atomic details of trimer dissociation and local beta-hairpin stability from residual dipolar couplings

The C-terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. Its native structure consists of a trimeric beta-hairpin propeller. At low pH, the foldon trimer disintegrates into a monomeric (A-state) form that has similar properties as that of an early intermediate of the trimer folding pathway. The formation of this A-state monomer from the trimer, its structure, thermodynamic stability, equilibrium association and folding dynamics have been characterized to atomic detail by modern high-resolution NMR techniques. The foldon A-state monomer forms a beta-hairpin with intact and stable H-bonds that is similar to the monomer in the foldon trimer, but lacks a defined structure in its N and C-terminal parts. Its thermodynamic stability in pure water is comparable to designed hairpins stabilized in alcohol/water mixtures. Details of the thermal unfolding of the foldon A-state have been characterized by chemical shifts and residual dipolar couplings (RDCs) detected in inert, mechanically stretched polyacrylamide gels. At the onset of the thermal transition, uniform relative changes in RDC values indicate a uniform decrease of local N-HN and Calpha-Halpha order parameters for the hairpin strand residues. In contrast, near-turn residues show particular thermal stability in RDC values and hence in local order parameters. This coincides with increased transition temperatures of the beta-turn residues observed by chemical shifts. At high temperatures, the RDCs converge to non-zero average values consistent with predictions from random chain polymer models. Residue-specific deviations above the unfolding transition reveal the persistence of residual order around proline residues, large hydrophobic residues and at the beta-turn.     

The folding landscape of an alpha-lytic protease variant reveals the role of a conserved beta-hairpin in the development of kinetic stability

Most secreted bacterial proteases, including alpha-lytic protease (alphaLP), are synthesized with covalently attached pro regions necessary for their folding. The alphaLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the alphaLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although alphaLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved beta-hairpin in the C-terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an alphaLP variant with a more favorable beta-hairpin turn conformation (alphaLP(beta-turn)). Indeed, alphaLP(beta-turn) pro region-catalyzed folding is faster than that for alphaLP. However, instead of accelerating spontaneous folding, alphaLP(beta-turn) actually unfolds more slowly than alphaLP. Our data support a model where the beta-hairpin is formed early, but its packing with a loop in the N-terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of alphaLP(beta-turn), to nearly the same degree as the change between alphaLP and a faster folding homolog. However, alphaLP(beta-turn) has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties.     

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.     

Conformational studies of the C-terminal 16-amino-acid-residue fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

The structure and stability of the 16-amino-acid-residue fragment [IG(46-61)] corresponding to the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus was investigated by means of CD and NMR spectroscopy and by differential scanning calorimetry. The CD and 2D NMR experiments were carried out (i) in water at different temperatures and (ii) at one temperature (305 K), with only CD, at different TFE concentrations. Our results show that the IG(46-61) peptide possesses organized three-dimensional structure at all investigated temperatures. The three-dimensional structure of the IG(46-61) peptide resembles the general shape of a beta-hairpin that is also observed for this peptide in the experimental structure of the B3 domain in the whole G protein; the structure is stabilized by hydrophobic interactions between nonpolar side chains. Our study shows that the melting temperature of the IG(46-61) peptide is about 320 K which supports the hypothesis that the investigated peptide can serve as a folding initiation site of the B3 domain of the immunoglobulin binding protein G.     

Structural and dynamics studies of the D54A mutant of human T cell leukemia virus-1 capsid protein

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.     

Folding time predictions from all-atom replica exchange simulations

We present an approach to predicting the folding time distribution from all-atom replica exchange simulations. This is accomplished by approximating the multidimensional folding process as stochastic reaction-coordinate dynamics for which effective drift velocities and diffusion coefficients are determined from the short-time replica exchange simulations. Our approach is applied to the folding of the second beta-hairpin of the B domain of protein G. The folding time prediction agrees quite well with experimental measurements. Therefore, we have in hand a fast numerical tool for calculating the folding kinetic properties from all-atom "first-principles" models.     

Folding and aggregation kinetics of a beta-hairpin

We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.     

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.     

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.     

A breakdown of symmetry in the folding transition state of protein L

The 62 residue IgG binding domain of protein L consists of a central alpha-helix packed on a four-stranded beta-sheet formed by N and C-terminal beta-hairpins. The overall topology of the protein is quite symmetric: the beta-hairpins have similar lengths and make very similar interactions with the central helix. Characterization of the effects of 70 point mutations distributed throughout the protein on the kinetics of folding and unfolding reveals that this symmetry is completely broken during folding; the first beta-hairpin is largely structured while the second beta-hairpin and helix are largely disrupted in the folding transition state ensemble. The results are not consistent with a "hydrophobic core first" picture of protein folding; the first beta-hairpin appears to be at least as ordered at the rate limiting step in folding as the hydrophobic core.     

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.     

Molecular dynamics study of the proposed beta-hairpin form of the switch domain from HIV1 gp120 alone and complexed with an inhibitor of CD4 binding

The strong tendency of beta-hairpin peptides to aggregate can prevent their structural resolution. The polar form of the switch peptide (LAV 15mer) at the CD4-binding domain of HIV1 gp120 is such a peptide, and NMR investigations of its interaction with a class of CD4-binding inhibitors developed in this laboratory have been hindered. Detailed knowledge of the interaction is required for the development of more potent switch inhibitors, that act by disrupting the cooperative folding transition necessary for binding to the CD4 receptor. In carrying out molecular dynamics simulation of the free peptide under polar conditions, we found that the properties of the resulting structure agree closely with those observed by circular dichroism. The same conditions, used to model the peptide/ inhibitor complex, produced a stable bimolecular structure with specific interactions between the inhibitor and side chains on the peptide, (e.g., Trp12 and the LPCR tetrad), known to control the folding transition. These help explain existing data on the relative potency of inhibitor derivatives and provide a basis for improved inhibitor design.     

The solution structure of a chimeric LEKTI domain reveals a chameleon sequence

The conversion of an alpha-helical to a beta-strand conformation and the presence of chameleon sequences are fascinating from the perspective that such structural features are implicated in the induction of amyloid-related fatal diseases. In this study, we have determined the solution structure of a chimeric domain (Dom1PI) from the multidomain Kazal-type serine proteinase inhibitor LEKTI using multidimensional NMR spectroscopy. This chimeric protein was constructed to investigate the reasons for differences in the folds of the homologous LEKTI domains 1 and 6 [Lauber, T., et al. (2003) J. Mol. Biol. 328, 205-219]. In Dom1PI, two adjacent phenylalanine residues (F28 and F29) of domain 1 were substituted with proline and isoleucine, respectively, as found in the corresponding P4' and P5' positions of domain 6. The three-dimensional structure of Dom1PI is significantly different from the structure of domain 1 and closely resembles the structure of domain 6, despite the sequence being identical to that of domain 1 except for the two substituted phenylalanine residues and being only 31% identical to the sequence of domain 6. The mutation converted a short 3(10)-helix into an extended loop conformation and parts of the long COOH-terminal alpha-helix of domain 1 into a beta-hairpin structure. The latter conformational change occurs in a sequence stretch distinct from the region containing the substituted residues. Therefore, this switch from an alpha-helical structure to a beta-hairpin structure indicates a chameleon sequence of seven residues. We conclude that the secondary structure of Dom1PI is determined not only by the local protein sequence but also by nonlocal interactions.     

Kinetic pathways of beta-hairpin (un)folding in explicit solvent

We examine the dynamical (un)folding pathways of the C-terminal beta-hairpin of protein G-B1 at room temperature in explicit solvent, by employing transition path sampling algorithms. The path ensembles contain information on the folding kinetics, including solvent motion. We determine the transition state ensembles for the two main transitions: 1), the hydrophobic collapse; and 2), the backbone hydrogen bond formation. In both cases the transition state ensembles are characterized by a layer (1) or a strip (2) of water molecules in between the two hairpin strands, supporting the hypothesis of the solvent as lubricant in the folding process. The transition state ensembles do not correspond with saddle points in the equilibrium free-energy landscapes. The kinetic pathways are thus not completely determined by the free-energy landscape. This phenomenon can occur if the order parameters obey different timescales. Using the transition interface sampling technique, we calculate the rate constants for (un)folding and find them in reasonable agreement with experiments, thus supporting the validation of using all-atom force fields to study protein folding.     

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.     

Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations

Full sequence design protein FSD-1 is a designed protein based on the motif of zinc finger protein. In this work, its folding mechanism and thermal stability are investigated using the replica exchange molecular dynamics model with the water molecules being treated explicitly. The results show that the folding of the FSD-1 is initiated by the hydrophobic collapse, which is accompanied with the formation of the C-terminal alpha-helix. Then the folding proceeds with the formation of the beta-hairpin and the further package of the hydrophobic core. Compared with the beta-hairpin, the alpha-helix has much higher stability. It is also found that the N-capping motif adopted by the FSD-1 contributes to the stability of the alpha-helix dramatically. The hydrophobic contacts made by the side chain of Tyr3 in the native state are essential for the stabilization of the beta-hairpin. It is also found that the folding of the N-terminal beta-hairpin and the C-terminal alpha-helix exhibits weak cooperativity, which is consistent with the experimental data. Meanwhile, the folding pathway is compared between the FSD-1 and the target zinc finger peptide, and the possible role of the zinc ion on the folding pathway of zinc finger is proposed. Proteins 2007. (c) 2007 Wiley-Liss, Inc.