Calorimetric evidence for a two-state unfolding of the beta-hairpin peptide trpzip4

beta-Sheets are a common secondary structural element found in proteins. The difficulty in studying beta-sheet folding and stability is that their formation is often dependent on the tertiary structural environment within the protein. However, the discovery of water-soluble beta-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of beta-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model beta-hairpin peptide to study beta-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV-CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.     

Direct observation of the folding and unfolding of a beta-hairpin in explicit water through computer simulation

The cooperative folding and unfolding of a beta-hairpin structure are observed in explicit water at native folding conditions through self-guided molecular dynamics simulation. The folded structure agrees excellently with the NMR NOE data. After going through a fully hydrated state, the peptide folds into a beta-hairpin structure in a highly cooperative process. During the folding process it is observed that side chain interaction occurs first, while intrapeptide hydrogen bonds only form at the final stage. On the contrary, the unfolding process starts with the breaking of interstrand hydrogen bonds. Energetic analysis indicates that the driving force of the folding is the intrapeptide interaction, while the solvent interaction opposes the folding.     

Characterization of the stereochemical selectivity of beta-hairpin formation by molecular dynamics simulations

The stability of secondary structure motifs found in proteins is influenced by the choice of the configuration of the chiral centers present in the amino acid residues (i.e., D vs L). Experimental studies showed that the structural properties of the tetrapeptide (L)V(L)P(L)A(L)L (all-L) are drastically altered upon mutating the L-proline and the L-alanine by their d-enantiomers [J. Am. Chem. Soc. 1996, 118, 6975]. The all-L diastereomer is unstructured, experiencing little or no beta-hairpin formation, while the (L)V(D)P(D)A(L)L peptide exhibits a substantial population of beta-hairpin conformation. In this study, we perform molecular dynamics simulations to investigate the folding propensity of these two model peptides. The results confirm the experimental findings, namely, that the presence of d-amino acids in the loop region strongly induces beta-hairpin formation (a population increase from about 1.5% to 50% is observed). The major factor determining the different behavior is found to be the large difference in energy between the two diastereomers, approximately 22 kJ/mol, when they adopt a beta-hairpin structure. The higher energy observed for the all-L peptide is a consequence of none-ideal hydrogen bond formation and of steric repulsions. The results suggest that selective incorporation of D-amino acids in proteins can be used to enhance certain secondary structure elements. The kinetic behavior of the folding process observed in the simulations is also investigated. We find that the decay rate of the folded structure fits to a biexponential function, suggesting that the folding/unfolding process of a beta-hairpin is governed by two different mechanisms.     

Thermally reversible hydrogels via intramolecular folding and consequent self-assembly of a de novo designed peptide

A small de novo designed peptide (MAX3) is described that exhibits complete thermoreversible self-assembly into a hydrogel network. Importantly, a prerequisite to hydrogelation is that the peptide must first fold into a conformation conducive to self-assembly. At ambient temperature, MAX3 is unfolded, resulting in a low viscosity aqueous solution. On increasing the temperature, the peptide undergoes a unimolecular folding event, affording an amphiphilic beta-hairpin that consequently self-assembles into a hydrogel network. Increasing the temperature serves to dehydrate the nonpolar residues of the unfolded peptide and trigger folding via hydrophobic collapse. Cooling the resultant hydrogel results in beta-hairpin unfolding and consequent complete dissolution of the hydrogel. The temperature at which folding and consequent self-assembly into a rigid hydrogel occur can be tuned by altering the hydrophobicity of the peptides.     

Beta-hairpin folding by a model amyloid peptide in solution and at an interface

The development of specific agents against amyloidoses requires an understanding of the conformational distribution of fibrillogenic peptides at a microscopic level. Here, I present molecular dynamics simulations of the model amyloid peptide LSFD with sequence LSFDNSGAITIG-NH2 in explicit water and at a water/vapor interface for a total time scale of approximately 1.8 micros. An extended structure was used as initial peptide configuration. At approximately 290 K, solvated LSFD was kinetically trapped in diverse misfolded beta-sheet/coil conformations. At 350 K, in contrast, the same type II' beta-hairpin in equilibrium with less ordered but also U-shaped conformations was observed for the core residues DNSGAITI in solution and at the interface in multiple independent simulations. The most stable structural unit of the beta-hairpin was the two residue turn (GA). The core residues exhibited a well-defined folded state in which the beta-hairpin was stabilized by a hydrogen bond between the side chain of Asn-385 and the main chain carbonyl group of Gly-387. My results suggest that beta-sheet conformations indicated from previous Fourier-transform infrared spectroscopy measurements immediately after preparation of the peptide solution may not arise from protofilaments as speculated by others but are a property of LSFD monomers. In addition, combined with previous results from X-ray scattering, my findings suggest that interfacial aggregation of LSFD implies a transition from U-shaped to extended peptide conformations. This work including the first simulations of reversible beta-hairpin folding at an interface is an essential step toward a microscopic understanding of interfacial peptide folding and self-assembly. Knowledge of the main conformation of the peptide core may facilitate the design of possible inhibitors of LSFD aggregation as a test ground for future computational therapeutic strategies against amyloid diseases.     

Theoretical characterization of alpha-helix and beta-hairpin folding kinetics

By means of the conformational free energy surface and corresponding diffusion coefficients, as obtained by long time scale atomistic molecular dynamics simulations (mus time scale), we model the folding kinetics of alpha-helix and beta-hairpin peptides as a diffusive process over the free energy surface. The two model systems studied in this paper (the alpha-helical temporin L and the beta-hairpin prion protein H1 peptide) exhibit a funnel-like almost barrierless free energy profile, leading to nonexponential folding kinetics matching rather well the available experimental data. Moreover, using the free energy profile provided by Mu?oz et al. [Mu?oz et al. Nature 1997, 390: 196-199], this model was also applied to reproduce the two-state folding kinetics of the C-terminal beta-hairpin of protein GB1, yielding an exponential folding kinetics with a time constant (approximately 5 micros) in excellent agreement with the experimentally observed one (approximately 6 micros). Finally, the folding kinetics obtained by solving the diffusion equation, considering either a one-dimensional or a two-dimensional free energy surface, are also compared in order to understand the relevance of the possible kinetic coupling between conformational degrees of freedom in the folding process.     

Infrared study of the stability and folding kinetics of a 15-residue beta-hairpin

The thermal stability and folding kinetics of a 15-residue beta-hairpin (SESYINPDGTWTVTE) have been studied by using infrared (IR) spectroscopy coupled with laser-induced temperature-jump (T-jump) technique for rapid folding-unfolding initiation. An alternative method based on analyzing IR difference spectra was also introduced to obtain thermodynamic properties of beta-sheets, which complements the commonly used circular dichroism (CD) and fluorescence techniques. Equilibrium IR measurements indicate that the thermal unfolding of this beta-hairpin is fairly broad. However, it can be described by a two-state transition with a thermal melting temperature of approximately 29 degrees C. Time-resolved IR measurements following a T-jump, probed at 1634 cm(-1), indicate that the folding of this beta-hairpin follows first-order kinetics and is amazingly fast. At 300 K, the folding time is approximately 0.8 micros, which is only 2-3 times slower than that of alpha-helix formation. Additionally, the energetic barrier for folding is small (approximately 2 kcal mol(-1)). These results, in conjunction with results from other studies, support a view that the details of native contacts play a dominant role in the kinetics of beta-hairpin folding.     

Thermodynamic analysis of beta-hairpin-forming peptides from the thermal dependence of (1)H NMR chemical shifts

The temperature dependence of the (1)H chemical shifts of six designed peptides previously shown to adopt beta-hairpin structures in aqueous solution has been analyzed in terms of two-state (beta-hairpin left arrow over right arrow coil) equilibrium. The stability of the beta-hairpins formed by these peptides, as derived from their T(m) (midpoint transition temperature) values, parallels in general their ability to adopt those structures as deduced from independent NMR parameters: NOEs, Deltadelta(C)(alpha)(H), Deltadelta(C)(alpha), and Deltadelta(C)(beta) values. The observed T(m) values are dependent on the particular position within the beta-hairpin that is probed, indicating that their folding to a beta-hairpin conformation deviates from a "true" two-state transition. To obtain individual T(m) values for each hairpin region in each peptide, a simplified model of a successive uncoupled two-state equilibrium covering the entire process has been applied. The distribution of T(m) values obtained for the different beta-hairpin regions (turn, strands, backbone, side chains) in the six analyzed peptides reveals a similar pattern. A model for beta-hairpin folding is proposed on the basis of this pattern and the reasonable assumption that regions showing higher T(m) values are the last ones to unfold and, presumably, the first to form. With this assumption, the analysis suggests that turn formation is the first event in beta-hairpin folding. This is consistent with previous results on the essential role of the turn sequence in beta-hairpin folding.     

Is Poisson-Boltzmann theory insufficient for protein folding simulations?

The Poisson-Boltzmann theory has been widely used in the studies of energetics and conformations of biological macromolecules. Recently, introduction of the efficient generalized Born approximation has greatly extended its applicability to areas such as protein folding simulations where highly efficient computation is crucial. However, limitations have been found in the folding simulations of a well-studied beta hairpin with several generalized Born implementations and different force fields. These studies have raised the question whether the underlining Poisson-Boltzmann theory, on which the generalized Born model is calibrated, is adequate in the treatment of polar interactions for the challenging protein folding simulations. To address the question whether the Poisson-Boltzmann theory in the current formalism might be insufficient, we directly tested our efficient numerical Poisson-Boltzmann implementation in the beta-hairpin folding simulation. Good agreement between simulation and experiment was found for the beta-hairpin equilibrium structures when the numerical Poisson-Boltzmann solvent and a recently improved generalized Born solvent were used. In addition simulated thermodynamic properties also agree well with experiment in both solvents. Finally, an overall agreement on the beta-hairpin folding mechanism was found between the current and previous studies. Thus, our simulations indicate that previously observed limitations are most likely due to imperfect calibration in previous generalized Born models but not due to the limitation of the Poisson-Boltzmann theory.     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.     

How Photoisomerization Drives Peptide Folding and Unfolding: Insights from QM/MM and MM Dynamics Simulations

Photoswitchable azobenzene cross‐linkers can control the folding and unfolding of peptides by photoisomerization and can thus regulate peptide affinities and enzyme activities. Using quantum mechanics/molecular mechanics (QM/MM) methods and classical MM force fields, we report the first molecular dynamics simulations of the photoinduced folding and unfolding processes in the azobenzene cross‐linked FK‐11 peptide. We find that the interactions between the peptide and the azobenzene cross‐linker are crucial for controlling the evolution of the secondary structure of the peptide and responsible for accelerating the folding and unfolding events. They also modify the photoisomerization mechanism of the azobenzene cross‐linker compared with the situation in vacuo or in solution.     

Molecular dynamics study of peptides in implicit water: ab initio folding of beta-hairpin, beta-sheet, and beta beta alpha-motif

In this communication, we have demonstrated that molecular dynamics simulations using a GB implicit solvation model with the all-atom based force field (CHARMM19) can describe the spontaneous folding of small peptides in aqueous solution. The native structures of peptides with various structural motifs (beta-hairpin, beta-sheet, and betabetaalpha-moiety) were successfully predicted within reasonable time scales by MD simulations at moderately elevated temperatures. It is expected that the present simulations provide further insight into mechanism/pathways of the peptide folding.     

Peptide folding using multiscale coarse-grained models

The multiscale coarse-graining (MS-CG) method has been previously used to describe the equilibrium properties of peptides. The present study reveals that MS-CG models of alpha-helical polyalanine and the beta-hairpin V 5PGV 5 possess the capacity to efficiently refold in simulations initiated from unfolded configurations. The MS-CG peptides exhibit free energy landscapes that are funneled toward folded configurations and two-state folding behavior, consistent with the known characteristics of small, rapidly folding peptides. Moreover, the models demonstrate enhanced sampling capabilities when compared to systems with full atomic detail. The significance of these observations with respect to the theoretical basis of the MS-CG approach is discussed. The MS-CG peptides were used to reconstruct atomically detailed configurations in order to evaluate the extent to which MS-CG ensembles embody all-atom peptide free energy landscapes. Ensembles obtained from these reconstructed configurations display good agreement with the all-atom simulation data used to generate the MS-CG models and also corroborate the presence of features observed in the MS-CG peptide free energy landscapes. These findings suggest that MS-CG models may be of significant utility in the study of peptide folding.     

Balancing solvation and intramolecular interactions: toward a consistent generalized Born force field

The efficient and accurate characterization of solvent effects is a key element in the theoretical and computational study of biological problems. Implicit solvent models, particularly generalized Born (GB) continuum electrostatics, have emerged as an attractive tool to study the structure and dynamics of biomolecules in various environments. Despite recent advances in this methodology, there remain limitations in the parametrization of many of these models. In the present work, we demonstrate that it is possible to achieve a balanced implicit solvent force field by further optimizing the input atomic radii in combination with adjusting the protein backbone torsional energetics. This parameter optimization is guided by the potentials of mean force (PMFs) between amino acid polar groups, calculated from explicit solvent free energy simulations, and by conformational equilibria of short peptides, obtained from extensive folding and unfolding replica exchange molecular dynamics (REX-MD) simulations. Through the application of this protocol, the delicate balance between the competing solvation forces and intramolecular forces appears to be better captured, and correct conformational equilibria for a range of both helical and beta-hairpin peptides are obtained. The same optimized force field also successfully folds both beta-hairpin trpzip2 and mini-protein Trp-Cage, indicating that it is quite robust. Such a balanced, physics-based force field will be highly applicable to a range of biological problems including protein folding and protein structural dynamics.     

Mechanically untying a protein slipknot: multiple pathways revealed by force spectroscopy and steered molecular dynamics simulations

Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.     

Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.     

Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.     

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well‐defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15‐mer vascular endothelial growth factor mimicking α‐helical peptide (QK_L10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK_L10A H_α chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high‐resolution QK_L10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding–unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding–unfolding picture for the small peptide QK_L10A compatible with the nucleation–propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein–protein interactions.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

Overcoming entropic barrier with coupled sampling at dual resolutions

An enhanced sampling method is proposed for ab initio protein folding simulations. The new method couples a high-resolution model for accuracy and a low-resolution model for efficiency. It aims to overcome the entropic barrier found in the exponentially large protein conformational space when a high-resolution model, such as an all-atom molecular mechanics force field, is used. The proposed method is designed to satisfy the detailed balance condition so that the Boltzmann distribution can be generated in all sampling trajectories in both high and low resolutions. The method was tested on model analytical energy functions and ab initio folding simulations of a beta-hairpin peptide. It was found to be more efficient than replica-exchange method that is used as its building block. Analysis with the analytical energy functions shows that the number of energy calculations required to find global minima and to converge mean potential energies is much fewer with the new method. Ergodic measure shows that the new method explores the conformational space more rapidly. We also studied imperfect low-resolution energy models and found that the introduction of errors in low-resolution models does decrease its sampling efficiency. However, a reasonable increase in efficiency is still observed when the global minima of the low-resolution models are in the vicinity of the global minimum basin of the high-resolution model. Finally, our ab initio folding simulation of the tested peptide shows that the new method is able to fold the peptide in a very short simulation time. The structural distribution generated by the new method at the equilibrium portion of the trajectory resembles that in the equilibrium simulation starting from the crystal structure.