Experimental thermodynamics of the helix–random coil transition. II. Calorimetric measurement of transition enthalpies of poly A in acid aqueous solution

The enthalpy change accompanying the double helix–coil transition of polyriboadenylic acid (poly A) in aqueous solution has been measured optically and calorimetrically in the pH range 5.7–4.5. The course of this cooperative transition was followed optically by measuring changes in ultraviolet absorption as a function of temperature at different pH values, and calorimetrically by determining the heat capacity of the solution through the transition region. From the latter measurements, the enthalpy of transition was calculated. It is shown, that ΔH is dependent on pH as it is expected from the influence of protonation of the double helix of poly A.     

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.     

High-resolution study of the helix–coil transition of poly(L-glutamic acid): Spectroscopic data correlation and the discovery of a new transition

This article reports on both (1) the precision and capability of a computerized multidimensional spectrophotometric system recently developed in our laboratory and (2) the high-resolution study of the helix–coil transition of poly(L-glutamic acid)[poly(Glu)], especially with regard to the discovery of an overlooked transition which is attributable to order–disorder rearrangement of the poly(Glu) side chain in the α-helical conformation. This study was made possible by the high performance of the system used. The simultaneous and continuous measurement of the circular dichroism, the absorbance and light-scattering intensity, and the pH titration curve of poly(Glu) in aqueous salt solution was carried out under continuous scanning of pH ranging from 8 to 2. Besides the well-known random coil to α-helix transition that occurs at about pH 5.5, a highly cooperative transition, which is indicated as a small but definite step in several spectral dimensions, is observed for the first time at pH 4.3. The transition is ascribed to an order–disorder conversion of the side chain on the α-helix backbone.     

Mechanical properties of poly(glutamic acid) in aqueous solution

The interaction between ionizable carboxyl groups and the conformation of poly-(glutamic acid) (PGA) in aqueous solution were investigated by the mechanical method. The dynamic rigidity of the PGA solution has a maximum value at the pH corresponding to about 50% neutralization point. This may be due to establishing of a maximum attractive force by proton/charge fluctuation between ionizable carboxyl groups at that pH. The dynamic viscosity has a sharp change in the region of pH 5.5–6.5. It is suggested that this behavior is due to the helix–coil transition.     

Studies of the helix–coil transition of poly-L-lysine in film and solution

Water-insoluble films of poly-L -lysine, crosslinked with formaldehyde, were suspended in aqueous media and their relative lengths measured as a function of pH. A sharp transition of the polymer was observed in the pH range which corresponded with that observed in polylysine solutions by optical rotation or dilatometry. In NaBr and NaCl solutions the coiled form of the polylysine film shrinks with increasing salt concentration, but in NaHCO₃ solution the extent of the contraction is larger, and the coil–helix transition of polylysine occurs at lower pH when NaHCO₃ is added to the medium. If one assumes the formation of amino carbamate in this case, this phenomenon can be well explained. Urea does break up the hydrogen bonds in helical polylysine film, but not completely. This result is interesting compared with that obtained for poly(L -glutamic acid). After the coil–helix transition region was found by film experiments, the volume change associated with the coil-to-helix transition was measured and found to be about 1–l.5 ml. per amino residue after taking electrostatic interaction into consideration. This value is nearly same as that obtained for poly(L -glutamic acid). By contrast, the value for poly-γ-benzyl-L -glutamate was reported to be ?0.077 ml./mole of repeating unit. So it is still necessary to determine the magnitude and direction of the volume change for various kinds of polypeptides.     

Coupling between the helix–coil transition and nematic–isotropic transitions in polypeptide solutions

The lattice model of Flory has been extended in order to consider equilibrium between isotropic and nematic phases containing helix–coil type chains. Nearly complete exclusion of coil sequences from the lyotropic nematic phase produces an enhanced cooperativity in the helix–coil transition. In poor solvents this enhancement begins to occur at concentrations typical of some experiments.     

A nuclear magnetic resonance study of the helix–coil transition of poly(L-glutamic acid)

There have been many reports that the nuclear magnetic resonance (nmr) spectra of a large number of polypeptides exhibit peak doubling of the α-carbon and the α-carbon proton in the helix–coil transition region. One apparent exception to this generalization has been polypeptides with ionizable side chains, where the helix–coil transition is induced by changes in pH in aqueous solution. Because it is important to establish the proper theoretical reason for the peak doubling and its relation to the rate of conformational change of amino acid residues, we have reexamined the proton and carbon-13 nmr spectra, at high field, for two polydisperse samples of poly(L -glutamic acid). Doubling of the α-carbon proton resonance as well as those of the α- and β-carbon, and backbone carbonyl are observed for a low-molecular-weight sample (DP = 54), while a higher molecular weight sample (DP = 309), exhibits only single resonances. Thus, polydispersity by itself is not sufficient to observe peak doubling; low-molecular weight is also required.     

The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Studies of the helix‐to‐coil transition in dilute solutions of poly‐L ‐lysine, dissolved in mixtures of water and methanol (MeOH), have been carried under shear flow using flow birefringence and modulated polarimetry. The fraction of helical conformations in a given solution remains independent of shear rate for MeOH concentrations above and below the critical value for the helix‐coil transition (i.e., 87.5% MeOH). For the 87.5% MeOH solutions, a shear‐induced helix‐to‐“stretched” coil transition occurs above a critical shear rate. Induction times for the transition show a temperature and shear rate dependence that can be described in terms of an activated jump process. Measurements of circular birefringence on cessation of flow also show that the transition is reversible, with the stretched coil reverting to the helical state on a time scale of several seconds. The activation energy for the jump process is found to be 16.2 kJ/mole. © 1999 John Wiley & Sons, Inc. Biopoly 50: 589–594, 1999     

On the double peak of NMR in the helix—coil transition region

To attempt to resolve the controversy over “fast” and “slow” helix–coil transition rates in polypeptides, nuclear magnetic resonance spectra were measured for monodisperse poly-γ-benzyl-L -glutamate (PBLG). These results were compared with simulated line spectra which were computed by taking the molecular-weight distribution into consideration. Broad but single peaks have been observed in 220 mHz nmr for the α-CH and NH proton resonance spectra in the transition region. The shape of the line changes with the extent of polydispersity. Assuming a fast conversion rate, a molecular model of the helix–coil transition simulates these results. Consequently, the double peak which has been observed in the nmr of polypeptides at the helix–coil transition region is shown to result from the polydispersity in molecular weight.     

Effect of pressure on the helix and random coil forms of poly(D-glutamic acid)

The absorption spectrum of the aqueous solution of acridine orange (AO)-poly–(D -glutamic acid) (PDGA) complex at pH 4.5 (helix form) did not show any wavelength shift, but at pH 7.5 (coil form) changed to the absorption curve of the helix form by compression up to 4500 atm. The ionization degree of PDGA estimated from the electric conductivity of the aqueous solution of PDGA at 4500 atm. was a value of about 5.3%. The entropy of the helix formation of PDGA from the titration data at 1 atm. and 30°C. was negative ?2.98 e.u. It will be concluded in this report that the volume change for coil to helix could be positive for PDGA and negative for AO–PDGA complexes.     

Carbon-13 and proton NMR studies of helix-coil transition of poly(gamma-benzyl-L-glutamate).

The helix–coil conformational transition undergone by poly(γ-benzyl-L -glutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solvent-induced helix–coil transition, the side chain assumes a helical conformation before the backbone. In the thermally induced helix–coil transition, the results indicate the existence of an intermediate state, which is between the α-helix and random coil and is free from intramolecular hydrogen bonding.     

Helix–coil transition kinetics in aqueous poly(α,L-glutamic acid)

A polarimetric electric-field-jump relaxation apparatus is described and used to determine the relaxation spectrum for the helix–coil transition of poly(α,L -glutamic acid) in water at 24°C. A maximum relaxation time of 1.7 μc occurs at the transition midpoint (pH = 5.9) yielding a rate constant for helical growth of 6 × 10⁷ sec^?1.     

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.     

Enthalpy of helix-coil transitions from heats of solution. I. Polyglutamates

The heat of solution of a series of three polyglutamates as a function of solvent composition was measured. The abrupt increase in heat of solution at the solvent composition of the helix–coil transition (as evidenced by optical rotation data) allows the estimation of the transition enthalpy change. The difference of side chain in the three polyglutamates has no appreciable effect on the transition enthalpy, although it affects the helix stability, as judged from the solvent composition at the transition points. These facts are discussed on the basis of existing models of the transition.     

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.     

Study of helix-coil transition of DNA by dielectric constant measurement

The thermal helix–coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix–coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix–coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix–coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

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.     

15N-NMR spectroscopy. IV. Comparison of poly(L-lysine) and isopoly(L-lysine)

The natural abundance ¹⁵N-nmr spectroscopy has been used to characterize the isomeric polymers (L -Lys)_n and iso (L -Lys)_n in aqueous solution. Although the peptide nitrogens of the two polymers have nearly equivalent shifts at pH < 10, the amino nitrogens differ by 5–6 ppm at pH < 7 and provide an easy means of identification. Furthermore, the polymers are distinguishable by the pK_a of the amino group and the basicity of the peptide nitrogen. At pH 10.3 and 25°C, (Lys)_n exhibits line broadening and an upfield chemical shift of the peptide nitrogen, indicative of the coil → helix transition. The formation of 100% helix may produce a shift as large as 5 ppm, which probably makes ¹⁵N-nmr spectroscopy more suitable for studies of this transition.