Temperature dependence of the thermodynamics of helix-coil transition

The temperature-induced helix to coil transition in a series of host peptides was monitored using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). Combination of these two techniques allowed direct determination of the enthalpy of helix-coil transition for the studied peptides. It was found that the enthalpy of the helix-coil transition differs for different peptides and this difference is related to the difference in the temperature for the midpoint of helix-coil transition. The enthalpy of the helix-coil transition decreases with the increase in temperature, thus providing the first experimental estimate for the heat capacity changes upon helix-coil transition, DeltaC(p). The values for DeltaC(p) of helix-coil transition are found to be negative, which is in contrast to the positive DeltaC(p) for protein unfolding. Analysis suggests that this negative DeltaC(p) of helix-coil transition is due to the exposure of the polar peptide backbone to solvent upon helix unfolding.     

Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions.

Helix propensities of the amino acids have been measured in alanine-based peptides in the absence of helix-stabilizing side-chain interactions. Fifty-eight peptides have been studied. A modified form of the Lifson-Roig theory for the helix-coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396-3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix-capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson-Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson-Roig theory. At 0 degrees C, Ala is a strong helix former, Leu and Arg are helix-indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone ("peptide H-bonds"). The implication for protein folding is that formation of peptide H-bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side-chain interactions stabilize them.     

Unfolding of alanine-based peptides using electron spin resonance distance measurements

We describe a scheme for tagging an alanine-based peptide with a Cu(II) and a nitroxide to measure unfolding transitions. The enhancement in longitudinal relaxation rate of the nitroxide due to the presence of Cu(II) was measured at physiological temperatures by pulsed electron spin resonance (ESR). The change in relaxation rate provided the average interspin distance between the Cu(II) and the nitroxide. Control experiments on a proline-based peptide verify the robustness of the method. The change in interspin distances with temperature for the alanine-based peptide is in accord with the change in helicity measured by circular dichroism. The data provide an opportunity to examine the unfolding process in polyalanine peptides. The distance in the folded state is in concordance with molecular dynamics. However, the ESR experiment measures an average distance of 17 A in the unfolded state, whereas molecular dynamics indicates a distance of 42 A if the unfolded geometry was a polyproline type II helix. Therefore, ESR demonstrates that the unfolded state of this alanine-based peptide is not an ideal extended polyproline type II helix.     

The determination of the thermodynamic parameters of the helix-coil transition of polypeptides by means of dipole moment measurements

The theoretical change of the mean-square dipole moment of a polypeptide during the helix-coil transition is compared with the change in helix content. It is shown that, according to the theory, the determination of the helix initiation parameter σ and the enthalpy of helix formation ΔH can be determined. Experimental data on poly-benzyl-L -gluatamate in two different solvent mixtures are given.     

A theoretical model simulating the anomalous concentration dependence of the equilibrium thermal unfolding curve of noncrosslinked tropomyosin

Thermal unfolding curves of tropomyosin have so far been fit only semi-quantitatively by the statistical-mechanical theory of the helix-coil transition. The calculated values of helix content are a bit too small for the most dilute solutions and a bit too large for the most concentrated ones. The theory, as hitherto used, assumes a uniform helix-helix interaction, whereas evidence from studies on molecular segments suggests otherwise. A theoretical model incorporating such non-uniformity in helix-helix interaction is used to produce simulated thermal unfolding curves. These simulated curves, when fit to the theory using the assumption of uniformity, reveal precisely the same discrepancies seen with the experimental data. We conclude that non-uniformity in helix-helix interaction along the tropomyosin molecule is responsible for the small discrepancy between experimental data and the uniform-model theory previously employed.     

Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.     

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.     

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

The triple helix-coil transition of cyanogen-bromide peptides of the α1-chain of the calf-skin collagen

The thermal triple helix-coil transition of the CNBr peptides of the α1-chain of calf-skin collagen was studied optically and calorimetrically. Besides α1CB5, all the peptides were able to form triple-helical structures at low temperatures. The peptides with longer chain lengths showed, under the experimental conditions, hysteresis in the transition range depending on the direction of the successive temperature changes. The detailed thermodynamic analysis of the optical transition curves was only possible for the two small peptides α1CB2 and α1CB4. We observed a higher stability of α1CB2 relative to α1CB4 (α1CB2 has higher imino acid content), accompanied with increased values of both denaturation enthalpy and entropy. Further, we observed a linear relationship between the calorimetrically determined denaturation enthalpy of all the CNBr peptides and their imino acid content. Although this behavior is qualitatively in accordance with the observation of Privalov and Tiktopulo on various kinds of native collagen, the CNBr peptides showed much lower values of the thermodynamic parameters ΔH⁰ and ΔS⁰ and differed also in the rate of their change with imino acid content. These differences are interpreted as being caused by misalignment in the helical form of the CNBr peptides resulting in a rupture of the specific interactions in the native form.     

Molecular dynamics simulations of synthetic peptide folding

Because the time scale of protein folding is much greater than that of the widely used simulations of native structures, a detailed report of molecular dynamics simulations of folding has not been available. In this study, we Included the average solvent effect in the potential functions to simplify the calculation of the solvent effect and carried out long molecular dynamics simulations of the alanine-based synthetic peptides at 274 K. From either an extended or a randomly generated conformation, the simulations approached a helix-coil equilibrium in about 3 ns. The multiple minima problem did not prevent helix folding. The calculated helical ratio of Ac-AAQAAAAQAAAAQAAY-NH₂ was 47%, in good agreement with the circular dichroism measurement (about 50%). A helical segment with frayed ends was the most stable conformation, but the hydrophobic interaction favored the compact, distorted helix-turn-helix conformations. The transition between the two types of conformations occurred in a much larger time scale than helix propagation. The transient hydrogen bonds between the glutamine side chain and the backbone carbonyl group could reduce the free energy barrier of helix folding and unfolding. The substitution of a single alanine residue in the middle of the peptide with valine or glycine decreased the average helical ratio significantly, in agreement with experimental observations. © 1996 Wiley-Liss, Inc.     

Effect of a single aspartate on helix stability at different positions in a neutral alanine-based peptide.

A single aspartate residue has been placed at various positions in individual peptides for which the alanine-based reference peptide is electrically neutral, and the helix contents of the peptides have been measured by circular dichroism. The dependence of peptide helix content on aspartate position has been used to determine the helix propensity (s-value). Both the charged (Asp-) and uncharged (Asp0) forms of the aspartate residue are strong helix breakers and have identical s-values of 0.29 at 0 degree C. The interaction of Asp- with the helix dipole affects helix stability at positions throughout the helix, not only near the N-terminus, where the interaction is helix stabilizing, and the C-terminus, where it is destabilizing. Comparison of the helix contents at acidic pH (Asp0) and at neutral pH (Asp-) shows that the charge-helix dipole interaction is screened slowly with increasing NaCl concentration, and screening is not complete even at 4.8 M NaCl. Lastly, a helix-stabilizing hydrogen-bond interaction between glutamine and aspartate (spacing i, i + 4) has been found. This side-chain interaction is specific for both the orientation and spacing of the glutamine and aspartate residues and is resistant to screening by NaCl.     

Effect of phosphorylation on alpha-helix stability as a function of position

We have investigated the effect of placing phosphoserine at the N-cap, N1, N2, N3, and interior position in alanine-based alpha-helical peptides. Helix contents of each peptide were measured by CD spectroscopy and titrations performed to determine pK(a) values. Data were analyzed with modified Lifson-Roig theory to determine helix-coil parameters (n, n(1), n(2), n(3), and w) and free energy changes for phosphoserine at each helical position. Results are given for a -1 and -2 phosphoserine charge state. Results show that phosphoserine stabilizes at the N-terminal positions by as much as 2.3 kcal.mol(-1), while destabilizes in the helix interior by 1.2 kcal.mol(-1), relative to serine. The rank order of free energies relative to serine at each position is N2 > N3 > N1 > N-cap > interior. Moreover, -2 phosphoserine is the most preferred residue known at each of these N-terminal positions. Experimental pK(a) values for the -1 to -2 phosphoserine transition are in the order N2 < N-cap < N1 < N3 < interior. This order agrees well with electrostatics calculations carried out with phosphoserine at the N-terminal positions and interior positions. Combining these with calculations at the C3, C2, C1, and C-cap positions gives results for phosphoserine along the length of the helix. We see a transition from phosphoserine stabilization at the N-terminus to destabilization at the C-terminus and can explain this in terms of the balance of protein solvation, favorable interactions, and dehydration. These results give insight into the phosphorylatable control of biological systems through positive or negative changes in stability.     

Helix-coil transition of alanine peptides in water: force field dependence on the folded and unfolded structures

The force fields used in classical modeling studies are semiempirical in nature and rely on their validation by comparison of simulations with experimental data. The all-atom replica-exchange molecular dynamics (REMD) methodology allows us to calculate the thermodynamics of folding/unfolding of peptides and small proteins, and provides a way of evaluating the reliability of force fields. We apply the REMD to obtain equilibrium folding/unfolding thermodynamics of a 21-residue peptide containing only alanine residues in explicit aqueous solution. The thermodynamics of this peptide is modeled with both the OPLS/AA/L and the A94/MOD force fields. We find that the helical content and the values for the helix propagation and nucleation parameters for this alanine peptide are consistent with measurements on similar peptides and with calculations using the modified AMBER force field (A94/MOD). The nature of conformations, both folded and unfolded, that contributes to the helix-coil transition profile, however, is quite different between these two force fields.     

Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements.

Ultrasonic absorption and velocity dispersion curves have been measured in the temperature induced helix-coil transition range of poly-N5-(3-hydroxypropyl)-L-glutamine in a methanol/water mixture. The results clearly reflect an effect due to the kinetics of the conformational conversion. A practically single relaxation time is observed which passes through a maximum when plotted versus the degree of transition. This maximum occurs at definitely less than 50% helix as predicted for by the theory for the comparatively short chain length involved here. The results are discussed in relation to previous theoretical and experimental findings.     

Why water-soluble, compact, globular proteins have similar specific enthalpies of unfolding at 110 degrees C.

The changes in free energy, enthalpy, and entropy of unfolding have been measured for many water-soluble, compact, globular proteins by a number of workers. In principle, a wide range in stability could be achieved by proteins, as measured by the free energy of unfolding; in practice, evolution only allows a narrow range in this quantity. Proteins are only marginally stable at room temperature for many possible reasons, including ensuring that folding is reversible and polypeptide chains are not trapped in incorrectly folded structures. Many of these proteins have approximately the same values of enthalpy of unfolding around 110 degrees C. We show here that this arises because the change in entropy of unfolding at room temperature and the change in heat capacity on unfolding, which governs the temperature variation of the enthalpy and entropy, both vary with the magnitude of the hydrophobic effect in the protein. As all these proteins have evolved to achieve similar stabilities at room temperature, the enthalpy of unfolding will also vary with the size of the hydrophobic effect in the protein. A consequence of this is that curves of the specific unfolding enthalpy against temperature for different proteins intersect around 110 degrees C. A similar conclusion, on the basis of similar melting points rather than similar free energies of unfolding, has been reached independently by Baldwin and Muller (R. L. Baldwin, personal communication).     

Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via 13C(alpha)-NMR

Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.     

Helix-coil stability constants for the naturally occurring amino acids in water. XII. Asparagine parameters from random poly(hydroxybutylglutamine-co-L-asparagine)

The synthesis and characterization of water-soluble random copolymers containing L -asparagine with N⁵-(4-hydroxybutyl)-L -glutamine, and the thermally induced helix-coil transitions of these copolymers in water, are described. The incorporation of L -asparagine was found to decrease the helix content of the polymers in water at all temperatures. The Zimm-Bragg parameters σ and s for the helix-coil transition in poly(L -asparagine) in water were deduced from an analysis of the copolymer melting curves in the manner described in earlier papers. The computed values of s indicate that asparagine destabilizes helical sequences at all temperatures in the range 0–60°C.