Relative stability of the α-helix of deuterated poly(γ-benzyl-L-glutamate)

The coil-to-helix transition temperatures of hydrogen bearing and deuterated poly(γ-benzyl-L -glutamate) in 1,3-dichlorotetrafluoroacetone/H₂O and/D₂O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α-helical form of poly(γ-benzyl-L -glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ-benzyl-L -glutamate), while the polypeptide-active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β-benzyl-L -aspartate) reveals an anomaly with respect to the present analysis.     

Experimental theromodynamics of the helix-random coil transition. I. Influence of polymer concentration and solvent composition in the PBG-DCA-EDC system

The course of the reversible helix formation of poly(γ-benzyl L -glutamate) (PBG) dissolved in a mixture of dichloroacetic acid (DCA) and 1,2-dichloroethane (EDC) was followed by measuring the heat capacity and the optical rotation of the system through the transition region. The results of these measurements indicate that the transition enthalpy ΔH the transition temperature T_c, and the Zimm-Bragg parameter σ depend considerably on the PBG concentration as well as on the composition of the solvent. For the standard state of infinite dilution, however, a linear extrapolation of the measured ΔH if values results in a standard value ΔH° = 950 cal./mole, independent of the solvent composition. The results of the calorimetric measurements are discussed in relationship to changes in optical rotation. Some peculiarities in the measured thermodynamic and optical properties in solutions with relatively high content of dichloroacetic acid are reported.     

Helix–coil transition in poly(γ-benzyl-L-glutamate) and poly-ε-carbobenzoxy-L-lysine

In this paper two points are considered: the methods of evaluating the helical content θ and the calculation of the parameters of the transition from experimental data and its interpretation. The parameter ΔH obtained is in good agreement with the calorimetric one and v is found to be independent of temperature and solvent and in agreement with the ordinarily accepted value for poly(γ-benzyl-L -glutamate). The different methods of estimating θ are discussed for both polypeptides.     

Deuteration and solvent composition effects in the helix–coil transition of poly-γ-beiizyl-L-glutamate

The effects of deuteration and of changes in solvent composition on the thermo dynamics of the helix–coil transition have been studied by calorimetric and optical measurements in the poly-γ-benzyl-L -glutamate–dichloroacetic acid–1,2-dichloro-ethanc system. For a given solvent composition, deuteration of the polypeptide and of the acid lowers the transition temperature T_c, while an increase in the volume fraction of acid in the solvent raises T_c. A rise in T_c is accompanied by a decrease in both the van't Hoff and the calorimetric heats of transition, but at different rates. The result is a temperature dependency in the Zimm-Bragg cooperation parameter σ. Possible causes of this result and its implications are discussed.     

Nuclear magnetic resonance and optical spectroscopic studies of block copolymers of polypeptides. I. Block copoly((benzyl-L-glutamate) n : (benzyl-L-aspartate) m )

In a study of A–B type block copolymers of γ benzyl-L -gultamate and β-benzyl-L -aspartate use has been made of the observations: (1) that for poly aspartate esters the chemical shifts of the α-CH and NH resonances are sensitive to the helix sense, (2) that in both helical and random coil conformations the same resonances of poly-γ-benzyl-L -glutamate are well separated from those of poly aspartates. Since the sense of poly-β-benzyl-L -aspartate is very sensitive to the inclusion of γ-benzyl-L -glutamate residues, the degree of overlap between the blocks can be studied by monitoring the helix sense of the aspartate. The ability of the NMR method to make separate observation of the two blocks removes the necessity of relying on an overall ORD parameter such as b₀. The copolymers studied include those having lefthanded, righthanded, and mixed-sense aspartate, corresponding to differing degrees of overlap.     

Further evidence of the protonation of the C-terminal of the poly-γ-benzyl-L-glutamate helix by acids

The change of the specific Kerr constant upon the addition of several acids to poly-γ-benzyl-L -glutamate solution in ethylene dichloride has been measured by the rectangular pulse method. The addition of a small amount of strong acid (trifluoroacetic acid, trichloroacetic acid, monochloroacetic acid, or hydrogen chloride) caused a rapid decrease of the specific Kerr constant. On the other hand, the effect of weak acids (formic acid, acetic acid, and propionic acid) was small. These facts show that the apparent dipole moment of a helical poly-γ-benzyl-L -glutamate molecule is considerably diminished by the protonation of terminal peptide groups. The electric conductivity of poly-γ-benzyl-L -glutamate solution in ethylene dichloride–dichloroacetic acid mixtures has been measured. It was found that the specific conductivity per unit concentration of poly-γ-benzyl-L -glutamate increased considerably at small fractions of dichloroacetic acid. This shows that poly-γ-benzyl-L -glutamate can react as a base (proton acceptor) with dichloroacetic acid. This result also confirms the previous conclusion.     

Thermodynamics of the helix-coil transition in polypeptides in mixed organic solvents: the influence of inert solvent and side chain

The thermally induced coil–helix transition of poly(γ-benzyl-L -glutamate) (PBLG) and poly(γ-methyl-L -glutamate) (PMLG) in binary solvent mixtures was investigated by calorimetric and optical rotatory dispersion (ORD) measurements. Dichloroacetic acid was the common active solvent, and the inert solvent was one of the chlorinated hydrocarbons, such as chloroform, 1,3-dichloropropane, 1-chlorobutane, or 1-chlorooctane. The thermodynamic parameters characterizing the intramolecular polypeptide and polypeptide–solvent interactions were calculated using the Karasz and Gajnos theoretical model [(1973) J. Phys. Chem. 77 , 1139–1145]. It was found that the enthalpy (ΔH₁) and entropy (ΔS₁) of helix stabilization in the absence of the active solvent depend on the inert solvent, but only in the case of PBLG. This is explained by the additional helix stabilization achieved by the stacking of the benzyl groups. The stacking is more pronounced in less polar chlorinated hydrocarbons with longer aliphatic chains. The results obtained indicate that the maximum helix stability is reached in chlorinated hydrocarbons with 12 C atoms. In the case PMLG, with an aliphatic ester side group, ΔH₁ and ΔS₁ are independent of the inert solvent. The ORD measurements were used to determine the maximum fraction of helicity attained at constant solvent composition and the transition temperature, T_c, at the point where f_H = 0.5. It was found that, for the same solvent composition, T_c was higher than the temperature of the midpoint of the calorimetric peak. This is explained by the fact that the maximum fraction of helicity is less than unity. The finite transition width was taken into account by calculating the phase boundaries for different fractions of helicity using the value of σ estimated from the calorimetric and van't Hoff enthalpies in the usual manner.     

Etude par spectroscopie infrarouge de la transformation hélice–chaîne statistique des polypeptides. II. Etude de l'interaction de quelques polypeptides à chaînes latérales polaires avec l'acide trifluoracétique

The various polymer–acid solvation possibilities occuring in the helix–coil transition process of polypeptides with polar side chains were systematically analyzed by infrared spectroscopy. The following samples have been considered: poly-γ-benzyl-L -glutamate (PBLG), alternating poly-γ-benzyl-D ,L -glutamate (PBD-LG), and poly-β-benzyl-L -as-partate (PBLA). The behavior of the amide A, I, II, and νC?O ester absorptions of each polymer dissolved in trifluoroacetic acid–chloroform mixtures was studied in depth. The classical assumptions concerning the interaction between a polypeptide and a proton donor solvent are discussed. This interaction was previously proposed in a theoretical model of helix–coil transition. For PBLG, the spectral characteristics of the cooperative transition are evidenced by the amide bands. These bands also show main chain–acid hydrogen bonding (I) Quantitative analysis of phenomenon (I) was performed in order to localize the “binding sites” of the polymer. In agreement with the theory, only the complexation of peptide units belonging to random coil and terminal helical regions were observed. However, in contrast to the theory in which the association constants KCO and KNH of these residues are generally kept equal, the present results have shown that the main binding site is the carbonyl group (KNH ? 0 or « KCO ). The behavior of the polar side chains of these polypeptides were analyzed during the transition. Similarly to the peptide backbone, they bind the acid by hydrogen bonding (II) Furthermore, this association is more important when the side chains are localized in the coiled regions than in the helical ones. This result suggests, by analogy with the main chain behavior, that the helix–coil transition theory should take into account two more association constants for polar side chains, namely k₁ for the helical regions and k₂ > k₁ for the coiled ones.     

Intermolecular association of tetra-(γ-benzyl-L-glutamate)s in ethylene dichloride solutions

Light scattering from ethylene dichloride solutions of tetra-(γ-benzyl-L -glutamate)s has been measured and their association in solution is examined. One of the peptides is monodisperse o-nitrophenylthio-tetra-(γ-benzyl-L -glutamate) ethylamide prepared by a stepwise condensation method, and the other is low-molecular-weight poly(γ-benzyl-L -glutamate) prepared by the N-carboxyanhydride method with n-hexilamine initiation at [A]/[I] = 4 and factionated by dosslution in formic acid. Concentration-dependent association of both peptides occurs noncooperatively, without giving critical micelle concentrations. The aggregate size is small: about 23 for the former tetrapeptide and about 7 for the latter polypeptide. While angular dissymmetry is close to unity, light scattering shows anomalous angular dependence, the intensity being symmetrically low with respect to the scattering angle of 90°. The observed angular dependence is interpreted in terms of the effect of optical anisotropy of peptide units. Formation of the anisotropic phase in concentrated solutions of these peptides is also examined briefy.     

Solution properties of synthetic polypeptides. II. Sedimentation and viscosity of poly-γ-benzyl-L-glutamate in dimethylformamide

Nine samples of poly-γ-benzyl-L -glutamate (PBLG), ranging in M?_w from 19,000 to 410,000, were examined viscomctrically and by ultracentrifugation with dimethylforma-mide (DMF) at 25°C. as helicogenic solvent. The data for [η] and s₀ (limiting sedimentation coefficient) as functions of M?_w were fitted well by the theories for a rigid prolate ellipsoid of revolution whose major axis increases linearly with M?_w, but whose minor axis is independent of M?_w. This implies that the overall shape of the PBLG molecule in DMF is represented by a straight cylinder whose cross section is independent of its length. The length per monomeric residue h evaluated from [η] is about 1.3 A., whereas that from s₀ is about 1.6 A. No adequate explanation for this difference in h can be found at present. More serious is the fact that these hydrodynamically evaluated values of h are appreciably larger than, the value obtained from our light-scattering measurements reported previously. All these values of h from our studies are not consistent with the value characteristic of the α-helix, for which h is 1.5 A. The concentration dependence of s₀ was found to agree well with the recent theoretical prediction of Peterson for cylindrical macromolecules.     

Enthalpy changes in the helix-coil transition of poly(gamma-benzyl L-glutamate)

The helix-coil transition temperature T_c of poly(γ-benzyl L -glutamate) in binary solvent mixtures of dichloroacetic acid and 1,4-dichlorobutane, 1-chlorooctane, or 1-chlorododecane have been measured. A treatment is presented with which the transition enthalpy can be calculated from the observed dependence of T_c on solvent composition. Results are compared with previously obtained calorimetric data. The underlying assumptions of the calculation are discussed.     

Poly( -p-nitrobenzyl-L-glutamate): synthetic aspects, sedimentation, and viscosity studies

A series of poly(γ-p-nitrobenzyl-L -glutamates), PNBG, has been synthesized by the polymerization of N-carboxyanhydride (NCA) derivatives of γ-p-nitrobenzyl-L -glutamate, NBG, using triethylamine as an initiator. We studied the influence of (a) the solvents dioxane, nitrobenzene, dimethylformamide (DMF), and DMF–1,2-dichloroethane mixture and (b) the anhydride–initiator ratio (A/I) for the polymerization in nitrobenzene (A/I varying from 50 to 750) on the properties of the polymers obtained. In order to improve its synthesis, NBG, was prepared by three different methods. Ten samples of PNBG, ranging in M_w from 10,000 to 50,000, were examined viscometrically in DMF and dichloroacetic acid (DCA) and by ultracentrifugation in DMF. The data for [η] and S_o (limiting sedimentation coefficient) as functions of M_w for PNBG in DMF were utilized, applying theories of Kuhn and Kuhn,¹³ Schachman,¹⁴ and Perrin, ¹⁵ for the estimation of the length per monomeric residue h. Viscosity data gave a h value of about 2.3 Å, Whereas sedimentation yielded 1.5 Å. Treating viscosity and sedimentation data for poly(γ-benzyl-L -glutamate), PBLG, in the same way leads to somewhat higher hvalues (2.4 Å and 1.7 Å, respectively). Although a nitroaromatic effect was shown to be absent for PNBG in DMF, it can be concluded that in this medium PNBG has a somewhat more compact structure than PBLG.     

Rigidity of α-helical polypeptides. A new method of obtaining the persistence length from viscosimetric data

The hydrodynamic properties of α-helical poly(L -glutamic acid), (Glu)_n in aqueous solutions and in mixtures of water with organic solvents have been interpreted in terms of the persistence length of the macromolecule. A modification of the method of Vitovskaya and Tsvetkov has been proposed in order to allow a more accurate determination of this parameter. The addition of an organic solvent increases strongly the rigidity of the helical conformation of (Glu)_n. A comparison is made with some data of the literature of poly[N⁵-(3-hydroxy propyl)L -glutamine], [Gln(CH₂)₃OH]_n, and poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n.     

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.     

Syntheses of poly[γ-(l)-menthyl L- and D-glutamates] and their secondary structures

γ-(l)-Menthyl L - and D -glutamates were prepared by a fusion reaction of N-phthalyl-L - and D -glutamic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were polymerized to poly[γ-(l)-menthyl L - and D -glutamates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as ethyl ether, chloroform, tetrahydrofuran, and n-hexane. From the results obtained by a study of the infrared absorption spectra, the x-ray photographs, the optical rotatory dispersions and the circular dichroisms, poly[γ-(l)-menthyl L -glutamate] was found to be a right-handed α-helix in the solid state and in solution. Similarly, poly[γ-(l)-menthyl D -glutamate] was a left-handed α-helix. The helix-coil transition of these polymers was observed in the vicinity of 40% dichloroacetic acid in a chloroform–dichloroacetic acid mixture.     

On the amino-acid-sequence distribution in benzylglutamate-methionine copolymers prepared from N-carboxyanhydrides

The amino-acid-sequence distribution in poly(γ-benzyl-L -glutamate, L -methionine) prepared by polymerization of the respective N-carboxyanhydrides has been investigated. This copolymer was converted first to poly(L -glutamic acid, L -methionine), which was subsequently cleaved by treatment with cyanogen bromide. The resulting material was fractionated into oligomers of (glutamic acid)_n-homoserine whose relative molar amounts were determined quantitatively. The results have been compared with those for a random incorporation of the methionine in a γ-benzylglutamate host polymer. Fairly close agreement has been found.     

Helix-coil transition of poly(gamma-benzyl-l-glutamate) in the dioxane-dichloracetic acid mixture.

The thermal helix–coil transition of four samples of poly(γ-benzyl-L -glutamate) in the dioxane–dichloroacetic acid (DCA) mixture was studied by optical rotatory dispersion method. The transition occurred at very high DCA content (97.5% by weight) in this system. The transition parameters have been evaluated by two methods based on the theories of Zimm and Bragg and of Nagai. The values of the helix initiation parameter and of the enthalpy of transition were found to be (4.4 ± 0.4) × 10^?5 and 340 cal/mole, respectively. The range of validity and equivalence of the two methods is discussed.     

Helix-coil transition of poly-gamma-N-carbobenzoxy-L-alpha, gamma-diaminobutyrate and poly-delta-N-carbobenzoxy-L-ornithine

The helix–coil transition of poly-γ-N-carbobenzoxy-L -α,γ-diaminobutyrate (PCLB) and poly-δ-N-carbobenzoxy-L -ornithine (PCLO) in chloroform–dichloroacetic acid mixtures was followed by optical rotatory dispersion. PCLB displays a “normal” temperature-induced transition, but PCLO an “inverse” one. The thermodynamic parameters for helix formation of the two polymers were determined using the Zimm-Bragg theory. The enthalpy for adding an amide residue to a helical region, ΔH, and the initiation factor σ were ΔH = ?180 cal/mole and σ = 9.2 × 10^?5 for PCLB and ΔH = +490 cal/mole and σ = 1.9 × 10^?5 for PCLO.     

NMR studies of poly-gamma-benzyl-l-glutamate in trifuloroacetic-deuterochloroform solutions: temperature dependence

The helix–random-coil transition process for poly-γ-benzyl-L -glutamate (PBG) in solvent mixtures trifluoroacetic acid/deuterochloroform (TFA/CDCl₃) at different temperatures has been studied by nmr. The chemical shift behavior of the α-CH resonances of the peptide chain and of the TFA carboxylic protons is reported.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.