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.     

Helix–coil transition in nucleoprotein—theory and applications

A general theory of helix–coil transition of irreversibly complexed nucleoproteins is presented. The equations are tested by experimental results in basic polypeptide–DNA complexes, nucleohistone I and pea bud nucleohistones. They show good agreement between theory and experiments. The theory provides direct measurement of a fraction of DNA base pairs covered by proteins, yielding a value of about 75% histone-covered base pairs in pea bud nucleohistone. It also provides a measurement of an average number of amino acid residues per nucleotide in protein-bound regions. This number varies from 1.0 to 1.4 in DNA–polylysine or DNA–polyarginine and from 2.9 to 3.3 in nucleohistone Ia, Ib, f1, and pea bud nucleohistone.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Kinetic studies of the helix–coil transition in aqueous solutions of poly(L-lysine)

The rate of conformational change of aqueous poly(α-L -lysine) solutions was measured using the electric field pulse relaxation method with conductivity detection. The relaxation time as a function of pH exhibits two maxima. One is assigned to a proton transfer reaction and the other to the helix–coil conformational transition. The helix nucleation parameter and the maximum relaxation time yield the rate constant of helix growth process (k_F) according to Schwarz's kinetic theory as k_F = 2 × 10⁷ sec^?1, which is comparable to that of the poly(glutamic acid) solution. The thermodynamic parameters of the helix growth process are compared with those of poly(glutamic acid).     

Conformational transition of poly(L-glutamic acid) in aqueous solution

Further direct evidences are given that a clear correlation exists between potentiometric and spectroscopic measurements in monitoring the poly(L-glutamic, acid) helix⁺ coil transtition. Specific Li⁺ ion poly (L -glUtamic acid) interactions have been observed, suresting that Li⁺ ions may exert a distinct destabilizing action on the helical conformation of the polyelectrolyte.     

Dielectric studies of aqueous solutions of poly(L-glutamic acid)

The dielectric features of poly(L -glutamic acid) are studied by the Fourier synthesized pseudorandom noise method in a time domain combined with a four-electrode cell. Polymer concentration dependence, the effect of the solvent viscosity, salt effects, and pH dependence are studied concomitantly with measurements of CD. A helix-to-coil transition occurs near pH 5.6 for a salt-free solution; at higher pH values, the polymer has an ionized random-coil conformation, and at lower pH, it has a deionized α-helical conformation. When it is in the ionized random-coil conformation, with the usual features of an electrolytic polymer, the solution shows a relaxation spectrum with a large dielectric increment at low frequencies. In the deionized α-helical state, no distinct relaxation curves are obtained, which does not deny the existence of a permanent peptide dipole. The pH dependence of the dielectric increment does not mainly correspond to the conformational change from helix to coil, but rather corresponds to the change of chain expansion on account of a charge–charge interaction under low ionic strength, which is conceived of by a viscosity measurement.     

Helix–coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine: Thermodynamic and statistical analysis

The thermally induced conformational changes of poly-N⁵-(3-hydroxypropyl)-L -glutamine in water and in methanol–water (3:7 v/v) have been analyzed in terms of the Lifson-Roig theory. The transitions in both solvents can be described by using v = 0.017. The thermodynamic parameters for the random coil-to-helix transition of one amino acid residue at room temperature were found to be: in water, ΔH = ? 130 cal/mole and ΔS = ? 0.45 e.u.; in methanol–water (3:7 v/v), ΔH = ? 170 cal mole and ΔS = ? 0.45 e.u. The size distribution of helical segments is broad, and the results of numerical calculations are presented for three degrees of polymerization (DP = 100, 300, and 750).     

Helix–coil transitions of compositionally heterogeneous DNA

Fragmented and mitomycin C cross-linked E. coli DNA was fractionated according to base composition by means of hydroxylapatite chromatography and density-gradient centrifugation in order to determine the effect of compositional heterogeneity on the breadth of the helix–coil transition. The transitions of some of the fractions are broader than that of the unfractionated DNA, due, presumably, to nonrandom sequences in molecules of 5 × 10⁵ daltons. Analysis of the transition breadths in terms of the known heterogeneity leads to reconsideration of current DNA helix–coil transition theory. We propose that partially denatured states include those for which the chains do not remain in strict register. Denaturation profiles are comprehensible only if this multitude of entropically favorable, degenerate states is included in the theory.     

Solvent- and salt-induced coil–helix transition of alkali metal salts of poly(L-glutamic acid) in aqueous organic solvents

The coil–helix transition has been studied for alkali metal salts of poly (L -glutamic acid) (PLG), i.e., PLGLi, -Na, -K, and -Cs, in aqueous organic solvent systems. Dependence of the transition on the solvent composition has been qualitatively discussed in terms of the solvent dielectric constant D, Gutmann's acceptor number AN, and water activity a_w. The helix formation induced by addition of alkali chlorides has also been studied. The sharpness of the transition has been interpreted as a measure of reduction of electrostatic energy of helical PLG through contact ion-pair formation between a counterion and carboxyl anion.     

Single crystals of poly(L-glutamic acid)

Lamellar single crystals of alkaline earth salts of poly(L -glutamic acid) have been grown by precipitation from dilute aqueous solution and studied by optical and electron microscopy and by x-ray and electron diffraction. The calcium, strontium and barium salts were crystallized in the β form above room temperature and could be converted to crystals of β-poly(L -glutamic acid) by washing in dilute hydrochloric acid. The magnesium salt, on the other hand, was crystallized in the α form at or below room temperature but could not be converted into crystals of α-poly(L -glutamic acid) by washing in hydrochloric acid. The crystalline lamellae are very thin (thicknesses range from 25 to 60 Å in β crystals and are about 100 Å in α crystals) and the polypeptide chains are oriented normal to the planes of the lamellae. It is clear from the disparity between crystal thickness and molecular length that the molecules crystallize by folding at the upper and lower surfaces of the crystals. Conformations of the molecules at these folds are discussed briefly.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

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.     

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.     

Helix–random chain transitions of polyamino acids in organic acid–salt solutions

Polyamino acids which are soluble and helical in acetic acid and dichloroacetic acid (DCA) have been observed to undergo a helix to random chain transition upon the addition of lithium salts of strong acids. The transition can be reversed by diluting the salt. Apparently only lithium cations are able to bring about the polycarbobenzoxy-L -lysine (PCBL) transition in acetic acid, whereas the anions display a varying degree of effectiveness; ClO₄^? > Br^? > TSA^? > Cl^? > NO₃^?. The lithium salts of carboxylate anions such as OAc^? and TFA^? do not cause polymer unwinding in acetic acid. Neither do the acids, TSA, HCl, TFA, or DCA induce the transformation in acetic acid. Poly-L -alanine (PLA) in DCA unfolds as LiBr is added, but does not unfold in the presence of 0.5M (CH₃)₄NBr, 0.25M CsBr, or 0.32M HCl. These results are explained on the basis of a direct interaction of the lithium salt with the polymer amide groups to form an ion-pair complex. The extent to which the union of the ion pair can dissociate from the complex in the low dielectric constant, environment determines the degree of unfolding of the polymer. The anion dissociation equilibrium presumably therefore would lie in the same order as given above. Acids such as HCl and TSA are considered to substantially protonate and ion-pair with the polymer, but do not readily dissociate the anion partner from the complex, and therefore do not produce an unstable positively charged helical structure.     

Pressure dependence of the helix–coil transition temperature of poly[d(G-C)]

The Pressure Dependence of the Helix-Coil Transition Temperature (T_m) of Poly[d(G-C)] was studied as a function of sodium ion concentration in phosphate buffer. The molar volume change of the transition (ΔV) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The ΔV of the transition increased from +4.80 (±0.56) to +6.03 (±0.76) mL mol^?1 as the sodium ion concentration changed from 0.052 to 1.0M. The van't Hoff enthalpy of the transition calculated from the half-width of the differentiated transition displayed negligible pressure dependence: however, the value of this parameter decreased with increasing sodium ion concentration, indicating a decrease in the size of the cooperative unit. The volume change of the transition exhibits the largest magnitude of any double-stranded DNA polymer measured using this technique. For poly[d(G-C)] the magnitude of the change in ΔV with sodium ion concentration (0.94 ± 0.05 mL mol^?1) is approximately one-half that observed for either poly[d(A-T)] or poly (dA)·poly(dT). The ΔV values are interpreted as arising from changes in the hydration of the polymer due to the release of counterions and changes in the stacking of the bases of the coil form. As a consequence of solvent electrostriction, the release of counterions makes a net negative contribution to the total ΔV, implying that disruption of the slacking interactions contributes a positive volume change to the total ΔV. The larger magnitude of the ΔV compared with that of other double-stranded polymers may be due in part to the high helix-coil transition temperature of poly[d(G-C)], which will attenuate the contribution of electrostriction to the total volume change. The data in addition show that in the absence of other cellular components, the covalent structure of DNA is stabile under conditions of temperature and pressure more extreme than those experienced by any known organism. © 1995 John Wiley & Sons, Inc.     

Isodichroic point and the β–random coil transition of poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine) in the absence of added salt

The β-coil transition of poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂CO₂H)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys((CH₂)₂CO₂H)]) was followed by CD, potentiometric titration, and viscosity in the absence of added salt. These different properties give consistent results for poly[Cys((CH₂)₂CO₂H)]. The CD spectra of poly[Cys(CH₂CO₂H)] change considerably with the degree of neutralization α even for a low-molecular-weight sample incapable of forming the β-structure. Because of the superposition of this additional effect, the dependence of CD on α is inconsistent with titration data for the case of poly[Cys(CH₂CO₂H)], particularly when the nπ transition is used to follow the β-coil transition. The change of CD inherent to the β-coil transition is characterized by an isodichroic point: 215 nm for poly[Cys((CH₂)₂CO₂H)] and 218 nm for poly[Cys(CH₂CO₂H)]. A criterion supporting the stacking of the pleated sheet is suggested based on the isodichroic point.     

Helix — coil transition of poly(γ-bensyl L-glutamate) in dioxan — dichloracetic acid mixtures

Measurements have been made on solutions of poly(γ-benzyl-l-glutamate) in solvents comprising dichloracetic acid (volume fraction φ₁) and dioxan over the whole range of φ₁. The transition point at φ₁ = 0.91 found from intrinsic viscosity is close to that obtained by others via dielectric measurements. However, the specific refractive index increments at constant composition and constant chemical potential as well as the selective adsorption coefficient of dichloracetic acid all exhibit sharp changes at about φ₁ = 0.75, and the partial specific volume of the polymer increases when φ₁ >0.75. An unusual minimum in the dependence of the refractive index increment on solvent composition is attributed to a small change in the refractive index of the polymer. This corresponds to a decrease of about 2% in the molecular polarizability of the polypeptide during the helix → coil transition.