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.     

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.     

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.     

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.     

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.     

Towards an understanding of the heat capacity of liquids. A simple two-state model for molecular association

A model for the temperature dependence of the isobaric heat capacity of associated pure liquids C(p,m)(o)(T) is proposed. Taking the ideal gas as a reference state, the residual heat capacity is divided into nonspecific C(p) (res,ns) and associational C(p) (res,ass) contributions. Statistical mechanics is used to obtain C(p)(res,ass) by means of a two-state model. All the experimentally observed C(p,m)(o)(T) types of curves in the literature are qualitatively described from the combination of the ideal gas heat capacity C(p)(id)(T) and C(p)(res,ass)(T). The existence of C(p,m)(o)(T) curves with a maximum is predicted and experimentally observed, for the first time, through the measurement of C(p,m)(o)(T) for highly sterically hindered alcohols. A detailed quantitative analysis of C(p,m)(o)(T) for several series of substances (n-alkanes, linear and branched alcohols, and thiols) is made. All the basic features of C(p,m)(o)(T) at atmospheric and high pressures are successfully described, the model parameters being physically meaningful. In particular, the molecular association energies and the C(p)(res,ns) values from the proposed model are found to be in agreement with those obtained through quantum mechanical ab initio calculations and the Flory model, respectively. It is concluded that C(p,m)(o)(T) is governed by the association energy between molecules, their self-association capability and molecular size.     

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.     

The protein folding transition state: insights from kinetics and thermodynamics

We perform extensive lattice Monte Carlo simulations of protein folding to construct and compare the equilibrium and the kinetic transition state ensembles of a model protein that folds to the native state with two-state kinetics. The kinetic definition of the transition state is based on the folding probability analysis method, and therefore on the selection of conformations with 0.4相似文献   

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.     

Temperature- and length-dependent energetics of formation for polyalanine helices in water: assignment of w(Ala)(n,T) and temperature-dependent CD ellipticity standards

Length-dependent helical propensities w(Ala)(n,T) at T = 10, 25, and 60 degrees C are assigned from t/c values and NMR 13C chemical shifts for series 1 peptides TrpLys(m)Inp2(t)Leu-Ala(n)(t)LeuInp2Lys(m)NH2, n = 15, 19, and 25, m = 5, in water. Van't Hoff analysis of w(Ala)(n,T) show that alpha-helix formation is primarily enthalpy-driven. For series 2 peptides Ac-Trp Lys5Inp2(t)Leu-(beta)AspHel-Ala(n)-beta-(t)LeuInp2Lys5NH2, n = 12 and 22, which contain exceptionally helical Ala(n) cores, protection factor-derived fractional helicities FH are assigned in the range 10-30 degrees C in water and used to calibrate temperature-dependent CD ellipticities [theta](lambda,H,n,T). These are applied to CD data for series 1 peptides, 12 < or = n < or = 45, to confirm the w(Ala)(n,T) assignments at T = 25 and 60 degrees C. The [theta](lambda,H,n,T) are temperature dependent within the wavelength region, 222 +/- 12 nm, and yield a temperature correction for calculation of FH from experimental values of [theta](222,n,T,Exp).     

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is pre-organized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T 7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.     

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.     

Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling

Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.     

Understanding the folding rates and folding nuclei of globular proteins

The first part of this paper contains an overview of protein structures, their spontaneous formation ("folding"), and the thermodynamic and kinetic aspects of this phenomenon, as revealed by in vitro experiments. It is stressed that universal features of folding are observed near the point of thermodynamic equilibrium between the native and denatured states of the protein. Here the "two-state" ("denatured state" <--> "native state") transition proceeds without accumulation of metastable intermediates, but includes only the unstable "transition state". This state, which is the most unstable in the folding pathway, and its structured core (a "nucleus") are distinguished by their essential influence on the folding/unfolding kinetics. In the second part of the paper, a theory of protein folding rates and related phenomena is presented. First, it is shown that the protein size determines the range of a protein's folding rates in the vicinity of the point of thermodynamic equilibrium between the native and denatured states of the protein. Then, we present methods for calculating folding and unfolding rates of globular proteins from their sizes, stabilities and either 3D structures or amino acid sequences. Finally, we show that the same theory outlines the location of the protein folding nucleus (i.e., the structured part of the transition state) in reasonable agreement with experimental data.     

Primary and secondary structure dependence of peptide flexibility assessed by fluorescence-based measurement of end-to-end collision rates

The intrachain fluorescence quenching of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is measured in short peptide fragments, namely the two strands and the turn of the N-terminal beta-hairpin of ubiquitin. The investigated peptides adopt a random-coil conformation in aqueous solution according to CD and NMR experiments. The combination of quenchers with different quenching efficiencies, namely tryptophan and tyrosine, allows the extrapolation of the rate constants for end-to-end collision rates as well as the dissociation of the end-to-end encounter complex. The measured activation energies for fluorescence quenching demonstrate that the end-to-end collision process in peptides is partially controlled by internal friction within the backbone, while measurements in solvents of different viscosities (H2O, D2O, and 7.0 M guanidinium chloride) suggest that solvent friction is an additional important factor in determining the collision rate. The extrapolated end-to-end collision rates, which are only slightly larger than the experimental rates for the DBO/Trp probe/quencher system, provide a measure of the conformational flexibility of the peptide backbone. The chain flexibility is found to be strongly dependent on the type of secondary structure that the peptides represent. The collision rates for peptides derived from the beta-strand motifs (ca. 1 x 10(7) s(-1)) are ca. 4 times slower than that derived from the beta-turn. The results provide further support for the hypothesis that chain flexibility is an important factor in the preorganization of protein fragments during protein folding. Mutations to the beta-turn peptide show that subtle sequence changes strongly affect the flexibility of peptides as well. The protonation and charge status of the peptides, however, are shown to have no significant effect on the flexibility of the investigated peptides. The meaning and definition of end-to-end collision rates in the context of protein folding are critically discussed.     

Stability of cyclic beta-hairpins: asymmetric contributions from side chains of a hydrogen-bonded cross-strand residue pair

Amino acid structural propensities measured in "host-guest" model studies are often used in protein structure prediction or to choose appropriate residues in de novo protein design. While this concept has proven useful for helical structures, it is more difficult to apply successfully to beta-sheets. We have developed a cyclic beta-hairpin scaffold as a host for measurement of individual residue contributions to hairpin structural stability. Previously, we have characterized substitutions in non-backbone-hydrogen-bonded strand sites; relative stability differences measured in the cyclic host are highly predictive of changes in folding free energy for linear beta-hairpin peptides. Here, we examine the hydrogen-bonded strand positions of our host. Surprisingly, we find a large favorable contribution to stability from a valine (or isoleucine) substitution immediately preceding the C-terminal cysteine of the host peptide, but not at the cross-strand position of the host or in either strand of a folded linear beta-hairpin (trpzip peptide). Further substitutions in the peptides and NMR structural analysis indicate that the stabilizing effect of valine is general for CX(8)C cyclic hairpins and cannot be explained by particular side-chain-side-chain interactions. Instead, a localized decrease in twist of the peptide backbone on the N-terminal side of the cysteine allows the valine side chain to adopt a unique conformation that decreases the solvent accessibility of the peptide backbone. The conformation differs from the highly twisted (coiled) conformation of the trpzip hairpins and is more typical of conformations present in multistranded beta-sheets. This unexpected structural fine-tuning may explain why cyclic hairpins selected from phage-displayed libraries often have valine in the same position, preceding the C-terminal cysteine. It also emphasizes the diversity of structures accessible to beta-strands and the importance of considering not only "beta-propensity", but also hydrogen-bonding pattern and strand twist, when designing beta structures. Finally, we observe correlated, cooperative stabilization from side-chain substitutions on opposite faces of the hairpin. This suggests that cooperative folding in beta-hairpins and other small beta-structures is driven by cooperative strand-strand association.     

Folding mechanisms of individual beta-hairpins in a Go model of Pin1 WW domain by all-atom molecular dynamics simulations

This paper examines the folding mechanism of an individual beta-hairpin in the presence of other hairpins by using an off-lattice model of a small triple-stranded antiparallel beta-sheet protein, Pin1 WW domain. The turn zipper model and the hydrophobic collapse model originally developed for a single beta-hairpin in literature is confirmed to be useful in describing beta-hairpins in model Pin1 WW domain. We find that the mechanism for folding a specific hairpin is independent of whether it folds first or second, but the formation process are significantly dependent on temperature. More specifically, beta1-beta2 hairpin folds via the turn zipper model at a low temperature and the hydrophobic collapse model at a high temperature, while the folding of beta2-beta3 hairpin follows the turn zipper model at both temperatures. The change in folding mechanisms is interpreted by the interplay between contact stability (enthalpy) and loop lengths (entropy), the effect of which is temperature dependent.     

Determination of critical indices by "slow" spectroscopy: NMR shifts by statistical thermodynamics and density functional theory calculations

The temperature dependencies of NMR shifts in the critical region of two coexisting phases have been simulated using statistical thermodynamics and graph-theory consideration of equilibrium processes of molecular association. Microparameters of magnetic screening of various water and water/pyridine structures used in the statistical averaging have been evaluated by density functional theory calculations (PBE1PBE and B3PW91 functionals in the 6-311++G** basis set). The gauge-including atomic orbital (GIAO) approach has been applied to ensure gauge invariance of the results. Solvent effects were taken into account by a polarized continuum model (PCM). NMR shifts "order parameters" (Deltadelta = |delta+ - delta-|) and "diameters" (phidelta = |(delta+ + delta-)/2 - deltaC|, where delta+, delta-, and deltaC are the chemical shifts of coexisting phases and at the critical point respectively) have been calculated in each case close to the lower critical solution point (TL) and processed using linear regression analysis of Deltadelta approximately |T - TL| and phidelta approximately |T - TL| in the log-log plot. It has been shown that the critical index beta can be evaluated with high precision from the slope of Deltadelta = f(T - TL) at any realistic set of model input parameters. The slope of diameter has been found to depend on both input beta and alpha values. The obtained phidelta slopes (0.58-0.63) are very close to 2beta values. The results are discussed within the concept of complete scaling. Results of simulation are compared and supported by experimental NMR data for water/2,6-lutidine, acetic anhydride/n-heptane, and acetic anhydride/cyclohexane systems.     

Cross-strand coupling of a beta-hairpin peptide stabilized with an Aib-Gly turn studied using isotope-edited IR spectroscopy

Isotope-edited IR spectroscopy was used to study a series of singly and doubly 13C=O-labeled beta-hairpin peptides stabilized by an Aib-Gly turn sequence. The double-labeled peptides have amide I' IR spectra that show different degrees of vibrational coupling between the 13C-labeled amides due to variations in the local geometry of the peptide structure. The single-labeled peptides provide controls to determine frequencies characteristic of the diagonal force field (FF) contributions at each position for the uncoupled 13C=O modes. Separation of diagonal FF and coupling effects on the spectra are used to explain the cross-strand labeled spectral patterns. DFT calculations based on an idealized model beta-hairpin peptide correctly predict the vibrational coupling patterns. Extending these model results by consideration of frayed ends and the hairpin conformational flexibility yields an alternate interpretation of details of the spectra. Temperature-dependent isotopically labeled IR spectra reveal differences in the thermal stabilities of the individual isotopically labeled sites. This is the first example of using an IR-based isotopic labeling technique to differentiate structural transitions at specific sites along the peptide backbone in model beta-hairpin peptides.