Helix–coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(γ-benzyl-L-glutamate-co-L-alanine) and poly(γ-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host–guest technique has been applied to the determination of the helix–coil stability constants of two naturally occurring amino acids, L -alanine and L -leucine, in a nonaqueous solvent system. Random copolymers containing L -alanine and L -leucine, respectively, as guest residues and γ-benzyl-L -glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix–coil transition behavior in a dichloroacetic acid (DCA)–1,2-dichloroethane (DCE) mixture. Two types of helix–coil transitions were carried out on the copolymers: solvent-induced transitions in DCA–DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA–DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, σ and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L -alanine residue stabilizes the α-helical conformation more than the L -leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.     

Simulation of α-helix–coil transitions in simplified polyvaline: Equilibrium properties and brownian dynamics

A quantitative understanding of helix–coil dynamics will help explain their role in protein folding and in folded proteins. As a contribution to the understanding, the equilibrium and dynamical aspects of the helix–coil transition in polyvaline have been studied by computer simulation using a simplified model of the polypeptide chain. Each amino acid residue is treated as a single quasiparticle in an effective potential that approximates the potential of mean force in solution. The equilibrium properties examined include the helix–coil transition and its dependence on chain position and well depth at the coil–helix interface. A stochastic simulation of the Brownian motion of the chain in its solvent surroundings has been used to investigate dynamical properties. Time histories of the dihedral angles have been used to study the behavior of the helical structure. Auto and cross-correlation functions have been calculated from the time histories and from the state (helix or coil) functions of the residues with relaxation times of tens to hundreds of picoseconds. Helix–coil rate constants of tens of ns^?1 were found for both directions of the transition. © 1993 John Wiley & Sons, Inc.     

Behavior of amphipathic helices on analysis via matrix methods, with application to glucagon, secretin, and vasoactive intestinal peptide

A configuration partition function, which incorporates concepts embodied in the amphipathic helix hypothesis, has been formulated for a polypeptide in the presence of zwitterionic phospholipid. An enhanced probability is assigned to helix formation in any region of the polypeptide chain where side chains bearing charges of opposite sign will be situated on the same side of the α-helix but displaced from one another by one turn. This situation will arise when residues i ? 4 (or i ? 3) and i bear charges of opposite sign and residue i ? 4 (or i ? 3) through i are in a helical state. Illustrative calculations are performed for polypeptide chains in which the generalized nonionic amino acid residue serving as host has Zimm-Bragg parameters of σ = 10^?4, s = 1. These calculations define conditions under which two interacting charged pairs can cooperate in a synergistic helix augmentation even when the two pairs are separated by significantly more than four generalized nonionic amino acid residues. Furthermore, the two interacting charged pairs, as well as the intervening amino acid residues, may become helical as one unit. Significant augmentation in helicity is observed with plausible values for the enhanced probablity assigned to helix formation for an interacting pair. This model predicts correctly that glucagon and secretin, but not vasoactive intestinal peptide, undergo a coil-to-helix trnsition in the presence of zwitterionic phospholipid. This prediction is made with plausible values for the parameter used to express the helicity enhancement. The experimental observation with zwitterionic phospholipids is the direct opposite of that seen for these three peptides in the presence of anionic lipids and detergents. In anionic lipids the amount of induced helicity is in the following order: glucagon < secretin < vasoactive intestinal peptide. Results obtained with these three peptides demonstrate that the nature of the head group of the lipid is important for lipid–protein interaction and that the resulting conformational changes can be rationalized by matrix methods.     

Dichroic statistical model for prediction and analysis of peptide helicity

Traditional statistical models for the prediction of peptide helicity are written in terms of the mean fractional helicity of the peptide residues. Far ultraviolet circular dichroic measurements of peptide solutions are converted to mean fractional helicity by partitioning the observed ellipticity between that of a perfect helix and a random coil. This partition does not adequately represent the ensemble of peptide molecules present in solution that populate imperfect helical conformations of quite variable lengths. A new dichroic statistical model has been written in terms of ellipticity rather than fractional helical content that recognizes (1) the source of ellipticity, peptide bond adsorption; (2) the differential ellipticity of peptide bonds in the terminal and interior helical turns; and (3) the contributions of each participant in a conformational ensemble to the observed ellipticity. Comparative analyses of host/guest peptides indicates that significant differences are obtained between residue w and n weights and ellipticity values using the traditional and dichroic statistical models. Proteins 28:467–480, 1997. © 1997 Wiley-Liss, Inc.     

O-Linked glycopeptides retain helicity in water.

A 17-residue O-linked glycopeptide model incorporating a central alpha-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.     

Effect of the N3 residue on the stability of the alpha-helix

N3 is the third position from the N terminus in the alpha-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH(3)CO-[AAX AAAAKAAAAKAGY]-NH(2)) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the alpha-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.     

Helix–coil transitions in a simple polypeptide model

A simplified model of a polypeptide chain is described. Each residue is represented by a single interaction center. The energy of the chain and the force acting on each residue are given as a function of the residue coordinates. Terms to approximate the effect of solvent and the stabilization energy of helix formation are included. The model is used to study equilibrium and dynamical aspects of the helix–coil transition. The equilibrium properties examined include helix–coil equilibrium constants and their dependence on chain position. Dynamical properties are examined by a stochastic simulation of the Brownian motion of the chain in its solvent surroundings. Correlations in the motions of the residues are found to have an important influence on the helix–coil transition rates.     

Positional independence and additivity of amino acid replacements on helix stability in monomeric peptides

The 17-residue peptide acetylAEAAAKEAAAKEAAAKAamide, described as an autonomous folding unit (Marqusee & Baldwin, 1987), has been used to examine the effect of amino acid replacements on helix stability. Alanine residues(s) at positions 4, 9, and 14 in the peptide sequence were replaced either singly or multiply by either serine or methionine residues with solid-phase peptide synthesis. The thermal dependence of the helix/coil transition of each peptide was observed by far-ultraviolet circular dichroism. Within experimental variation, all three single replacements exhibit a common thermal transition, and all three double replacements exhibit a different common thermal transition. These results suggest that replacement of the central alanine residue in the repeat EAAAK located in the N-terminus, in the middle, or in the C-terminus of the peptide helix has the same effect on helix stability. The melting temperature of each thermal transition was estimated by assuming a linear van't Hoff plot and a change in molar ellipticity of 33,500 deg cm2 dmol-1. Such analysis indicates that each replacement of an alanine residue by a serine residue diminishes the melting temperature by 11 +/- 1 degrees C and that each replacement of an alanine residue by a methionine residue diminishes the melting temperature by 6 +/- 1 degrees C. These results suggest that the effect of these replacements on helix stability is additive.     

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.     

Effect of the substitution Ala----Gly at each of five residue positions in the C-peptide helix

The substitution Ala----Gly has been studied in a unique-sequence peptide (related in sequence to the C-peptide of ribonuclease A) to determine its effect on C-peptide helicity at different residue positions. There is a substantial decrease in helicity for Ala----Gly at residue position 4, 5, or 6 but only a small decrease in helicity for Ala----Gly at end residue 1 and no decrease at end residue 13. The change for Ala----Gly is similar at position 4, 5, or 6; the change is caused chiefly by the difference in s, the helix growth parameter in the Zimm-Bragg model for alpha-helix formation, between Ala and Gly. Thus, the helicity of C-peptide depends sensitively on s at interior positions. The small change in helicity found for Ala----Gly at either end position suggests that the end residues are largely excluded from the helix, with the result that helicity is relatively unaffected by replacement of an end residue. Another possibility is that some helix-stabilizing effect is exerted by Gly only at an end position. Exclusion of an end residue from the helix might be caused either by fraying of the helix ends or by helix termination at an interior residue, resulting from a helix stop signal such as the Glu-2- -Arg-10+ salt bridge or the Phe-8-His-12+ ring interaction.     

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii

Gas vesicles are proteinaceous, gas‐filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in‐silico 3D‐model of GvpA of the predicted coil‐α1‐β1‐β2‐α2‐coil structure is available and implies that the two β‐chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + A_mut transformants for their ability to form gas vesicles (Vac⁺ phenotype). In most cases, an alanine substitution of a non‐polar residue did not abolish gas vesicle formation, but the replacement of single non‐polar by charged residues in β1 or β2 resulted in Vac^– transformants. A replacement of residues near the β‐turn altered the spindle‐shape to a cylindrical morphology of the gas vesicles. Vac^– transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt‐bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac^– transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid‐state NMR.     

Analysis of peptides for helical prediction

Two terminally blocked peptides, acetylAETAAAKFLRQHMamide and acetylAETSSSRYLRQHMamide, were obtained by solid-phase synthesis, purified by reversed-phase chromatography, and characterized by fast atom bombardment mass spectrometry. Both peptides were soluble in aqueous solutions and remained monomeric over the concentration range examined. Changes in the temperature, pH, and trifluoroethanol concentration of solutions of each peptide produced changes in the far-ultraviolet circular dichroic spectrum characteristic of a two-state helix/coil transition. The limiting mean residue ellipticity of the coil and helix form of each peptide was estimated by addition of the denaturant guanidinium chloride at elevated temperature and by addition of trifluoroethanol at subzero temperatures, respectively. The midpoint for the thermal transition of the peptide SSSRY is lowered by about 30 degrees C relative to that of peptide AAAKF, in qualitative agreement from predictions based on helix probabilities of amino acid residues. The magnitude of the change observed in the midpoint of the thermal transitions suggests that the effect of single amino acid replacements on helix formation should be experimentally measurable.     

Prediction of the stability of coiled coils using molecular dynamics simulations

We have performed 40–80 ns-long molecular dynamics (MD) simulations of the GCN4 leucine zipper and synthetic coiled coils using the GROMOS96 (43a2) and OPLS-AA force fields, with the aim of predicting coiled coil stability. Starting with an initial configuration of two peptides placed in an ideal coiled coil configuration, we find that changing the amino acid sequence modestly or decreasing peptide length can lead to a decrease in the final α-helicity of coiled coils, although for peptides as long or longer than 16 residues, the values of helicity do not decrease to the low values seen in the experimental results of Lumb et al. (Biochemistry. 1994, 33, 7361–7367) or of Su et al. (Biochemistry. 1994, 33, 15501–15510), presumably because the simulations are not long enough. We find, however, that helicity correlates positively with the number of close hydrophobic interactions between the two peptides, showing that stable coiled coils in the simulations are tightly packed. The minimum interhelical distances are 0.50–0.66 nm for charged groups, indicating that favorable charge interactions are also important for the stability of the coiled coil.     

Effect of central-residue replacements on the helical stability of a monomeric peptide

The peptide acetylYEAAAKEARAKEAAAKAamide exhibits the dichroic features characteristic of a monomeric helix/coil transition in aqueous solution. Nineteen variants of this peptide each containing a different residue at position 9 were prepared by solid-phase peptide synthesis and purified by reversed-phase chromatography. The thermal dependence of the far-ultraviolet dichroic spectrum of each of these peptides except that containing proline is characteristic for an alpha-helix/coil transition. The relative stability of the helical forms of these peptides does not correlate with the preference of the variable amino acid to occupy a middle position in a protein helix. It is likely that the specific interactions of the variable residue with its local environment obscure any inherent preference of the residue to reside in an alpha-helix.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. IV. Local substitutions in the collagen triple helix

The properties of collagen are affected by the replacement of Pro by imino acid analogues. The structural effect of the low-level local substitution of L -azetidine-2-carboxylic acid (Aze) has been analyzed by computing the energy of CH₃CO-(Gly-Pro-Pro)₄-NHCH₃ triple helices in which a single residue of one strand has been replaced by Aze. When Aze is in position Y of a (Gly-X-Y) unit, low-energy local deformations are introduced in the triple helix, i.e., it becomes more flexible. On the other hand, the flexibility of the triple helix is not increased with Aze in position X. The energy of the triple helix to coil transition is not changed significantly by this amount of substitution. In an earlier study, we have demonstrated that the regular substitution of Aze in every tripeptide distorts or destabilizes the triple helix to a large extent [A. Zagari, G. Némethy, & H. A. Scheraga (1990) Biopolymers, Vol. 30, pp. 967–974 ]. Thus, it appears that a high level of substitution is required to cause the observed chemical and biological effects of Aze on collagen. © 1994 John Wiley & Sons, Inc.     

The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states

It has long been understood that the proline residue has lower configurational entropy than any other amino acid residue due to pyrrolidine ring hindrance. The peptide bond between proline and its preceding amino acid (Xaa-Pro) typically exists as a mixture of cis- and trans-isomers in the unfolded protein. Cis–trans isomerization of Xaa-Pro peptide bonds are infrequent, but still occur in folded proteins. Therefore, the effects of the cis–trans isomerization equilibrium in both unfolded and folded states should be taken into account when estimating the stability contribution of a specific proline residue. In order to study the stability contribution of the four proline residues to the hyperthermophilic protein Ssh10b, in this work, we expressed and purified a series of Pro→Ala mutants of Ssh10b, and performed correlative unfolding experiments in detail. We proposed a new unfolding model including proline isomerization. The model predicts that the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between cis- and trans-isomers both in the unfolded and folded states, agreeing well with the experimental results.     

Ligand-induced melting reaction of specific-sequence DNA molecules

An algorithm for the calculation of ligand-induced helix–coil transitions of macromolecules of any specific sequence was derived. The probabilities of each residue to be in helix and ligand-free, helix and ligand-bound, coil and ligand-free, and coil and ligand-bound states can be calculated by extending the recursion relation formula proposed by D. Poland [(1974) Biopolymers 13 , 1859–1871]. Calculations using hypothetical DNAs having block heterogeneities for melting reactions showed that cooperative binding specific to single strands weakens the effect of the heterogeneity. Fine structures in the melting profiles or cooperatively melting regions, which have so far been found in thermal melting experiments, were predicted to exist in ligand-induced melting reactions for natural DNA fragments.     

Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Theory of protein molecule self-organization. II. A comparison of calculated thermodynamic parameters of local secondary structures with experiments

Constants of the helix–coil transition for all natural amino acid residues are evaluated on the basis of thermodynamic parameters obtained in paper I of this series. The specific effects at the termini of the helices are also considered as well as the parameters controlling the formation of β-bends in the unfolded protein chain. Evaluated s constants of the helix–coil transition agree with the experimental data on helix–coil transitions of synthetic polypeptides in water. Only a very qualitative correlation exists between s constants (both experimental and theoretical) and the occurrence of corresponding residues in internal turns of α-helices in globular proteins: residues with s > 1 occur in helices as a rule more often than residues with s < 1. At the same time a direct correlation is demonstrated between theoretical parameters of residue incorporation into α-helical termini and β-bends in an unfolded polypeptide chain and the occurrence of residues in corresponding positions of the globular protein secondary structures.     

Helix–coil stability constants for the naturally occurring amino acids in water. XXI. Glutamine parameters from random poly(hydroxypropylglutamine-co-L-glutamine) and poly(hydroxybutylglutamine-co-L-glutamine)

Water-soluble, random copolymers containing L -glutamine and either N⁵-(3-hydroxypropyl)-L -glutamine or N⁵-(4-hydroxybutyl)-L -glutamine were synthesized, fractionated, and characterized. The thermally induced helix–coil transitions of these copolymers were studied in water. A short-range interaction theory was used to deduce the Zimm-Bragg parameters σ and s for the helix–coil transition in poly(L -glutamine) in water from an analysis of the melting curves of the copolymers in the manner described in earlier papers. The computed values of s indicate that L -glutamine is helix-indifferent at low temperature and a helix-destabilizing residue at high temperature in water. At all temperatures in the range of 0–70°C, the glutamine residue promotes helix–coil boundaries since the computed value of σ is large.