The viscosity behavior and conformations of low-molecular-weight poly-γ-benzyl-L-glutamate in various solvents

Reduced viscosity and infrared spectra of low-molecular-weight poly-γ-benzyl-L -glutamate (which was prepared by polymerization of the N-carboxyanhydride with n-hexylamine initiation at [A]/[I] 3, 4, and 8) have been measured in various organic solvents. Infrared spectra indicate that the polypeptide molecules consist of a series of residues of two forms, the solvated σ-form and the hydrogen-bonded β-form, and relative abundance of the two forms depends on solvent species and polypeptide concentration. An approximate method is developed for estimating the content of β-structure from a single spectrum of dissolved polypeptide. The reduced viscosity of some solutions is scarcely dependent in polypeptide concentration, in which a single conformation is predominantly kept over the concentration range. In the other solutions the reduced viscosity displays a strong concentration dependence or some anomalous behavior. The observed viscosity behavior has been attributed to the changes in size and shape of aggregates, which are determined by the number of hydrogen bonds in the aggregate. This unusual behavior is exhibited by solutions of the polypeptides which have a moderate content of β-structure at a finite concentration. Both the content of β-structure and the extent of association increase in the following solvents, ranked in order of effectiveness: dimethylformamide, trifluoroethanol < trimethyl phosphate < chloroform < dioxane < ethylene dichloride < ethylene dibromide. Infrared spectra suggest that the conformation of the polypeptide in dichloroacetic acid differs from either the σ- or the β-conformation.     

Rotatory dispersion and circular dichroism of low-molecular-weight poly- -benzyl-L-glutamate

Rotatory dispersion and circular dichroism of low-molecular-weight poly-γ-benzyl-L -glutamate, which was prepared from the N-carboxyanhydride by n-hexylamine initiation at [A]/[I] 3, 4, and 8, have been measured in ethylene dichloride and dioxane at different concentrations. The rotatory properties of the polypeptides are all characterized by a trough at 233 mμ of a negative Cotton effect or by a negative circular dichroic band at 223 mμ. With increasing A/I value or concentration, dextrorotation increases and the negative dichroic band becomes deeper. Both the trough magnitude and the negative ellipticity are linearly dependent on the content of β-structure, and the rotatory parameters for the pure β-structure are estimated by extrapolation of the linear relations. Circular dichroism and infrared spectra of the polypeptides have also been measured in trifluoroethanol, and the effect of solvents on the polypeptide conformation is discussed.     

Structure of micelles formed by monodisperse hexa-(γ-benzyl-L-glutamate) in solution

The angular dependence of light scattering and the concentration dependence of the relative viscosity have been measured in solutions of o-nitrophenylthio-hexa-(γ-benzyl-L -glutamate) ethylamide in ethylene dichloride. Both the reduced intensity of scattered light and the reduced viscosity of the solution suddenly increase above a certain critical concentration, below which both of them remain low and constant. The Debye plot of light scattering indicates that primary micelles having an aggregation number 48 are formed at the critical micelle concentration and that secondary micelles, each consisting of 294 molecules, then appear in increasing amounts with increasing concentration beyond the critical micelle concentration. The secondary micelle is rodlike and has a length of 1170 Å, if it is rigid. An analysis of the reduced viscosity leads to the intrinsic viscosity for the primary micelle, 0.360 dL g^?1, and to that of the secondary micelle, 1.28 dL g^?1. If the secondary micelle is represented by a prolate ellipsoid, it should have an axial ratio of 47. If the polypeptide chains are extended in the micelle, the observed aggregation number and axial ratio of the secondary micelle can well accommodate the intermolecularly hydrogen-bonded in-register β-structure of anti-parallel chains. In the primary micelle, some folded polypeptide chains are involved, and an intermolecularly hydrogen-bonded out-of-register structure would form a rather open network.     

Conformations of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,and its critical concentration of β-form formation in solution

Nps-[Glu(OBzl)]₆-NHEt has been prepared by coupling Nps-[Glu(OBzl)]₂-OH with HCl,H-[Glu(OBzl)]₄-NHEt by means of dicyclohexylcarbodiimide. The ir spectra of its nujol mull show that the hexapeptide has the β-structure of antiparallel chains. When it is dissolved in dioxane or ethylene dichloride, the hexapeptide consists of a mixture of the β-form and the solvated σ-form, but the β-form can exist only above a certain critical concentration. The critical concentration is about 0.4g dl^?1 in dioxane and 0.08g dl^?1 in ethylene dichloride, and the content of β-form increases with increasing concentration above it. The CD of the dioxane and ethylene dichloride solutions shows concentration dependence in visible and uv regions.     

Intermolecular association and molecular weight of aggregates of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,in solution

Light scattering from the solutions of Nps-[Glu(OBzl)]₆-NHEt in dioxane or ethylene dichloride has been measured at different concentrations, and a critical concentration of intermolecular association is found to exist, which is equal to the critical concentration of β-form formation. The Debye plot of light scattering leads to the molecular weight of aggregates at the critical concentration, which corresponds to an aggregation number 15 in dioxane and 53 in ethylene dichloride. In the latter solvent the aggregates further associate into a larger aggregate consisting of 330 molecules when the concentration is increased beyond the critical concentration. The content of β-form, which is a measure of number of hydrogen bonds, is derived from the ir data previously obtained. The results on the modes of intermolecular association and hydrogen bonding lead to possible structures of aggregates formed by both hydrogen bonds and other nonbonding side-chain interactions.     

Infrared spectra and conformation of monodisperse Oligo-γ-Benzyl-L-Glutamates in solution

The ir spectra of σ–nitrophenylthio-tetra- and hexa-γ-benzyl-L -glutamate ethylamides were measured at different concentrations in chloroform and ethylene dichloride. The molar extinction coefficients of two bands, each for the amide I and A modes, were observed as indicating the content of β structure and the fraction of hydrogen bonds; they were analyzed for elucidating the hydrogen-bonded state of peptide residues in the σ and β conformations of oligopeptides. While the content of β structure of the tetra-peptide increases with increasing concentration, the hexapeptide is in the β conformation above the critical concentration only. The fraction of hydrogen bonds remains finite even at infinite dilution or below the critical concentration, indicating the intramolecular hydrogen-bonding in the σ—form. As the fundamental structure of folded forms having only intramolecular hydrogen bonds, the 2₇ ribbon is most likely. With increasing concentration or above the critical concentration, the extended forms are stabilized by the intermolecular hydrogen bonds between residues of the β-form. The β-form is present only when intermolecular hydrogen bonds link two residues in an antiparallel way. Possible structures of the oligopeptides in the σ– and β conformations in the two solvents are described briefly.     

The infuence of interchain compositional heterogeneity on the conformation in random copolymers of γ-benzyl-L-glutamate and L-valine

Variation in the solvent used for the copolymerization of γ-benzyl-L -glutamate and L -valine N-carboxyanhydrides provides copolymers which have variable interchain compositions, and this variation in interchain compositional heterogeneity is reflected in the solid-state conformations of the respective copolymers. Poly[Glu(OBzl)²⁹Val⁷¹] prepared in dioxane exhibits a β-structure, whereas a copolymer of the same average composition prepared in benzene/methylene chloride shows predominantly an β-helix conformation with a small amount of β-structure. The use of the monomer reactivity ratio permits the calculation of the average and incremental copolymer compositions at any conversion; thus, correlations between conformation and interchain compositional heterogeneity can be made. In general, copolymers prepared in dioxane show a greater distribution of chain composition and therefore permit a wider variety of conformation than copolymers prepared in benzene/methylene chloride under identical conditions.     

Aggregation of poly(γ-benzyl-L-glutamate) in mixed solvent systems

The aggregation of poly(γ-benzyl L -glutamate) has been studied by measuring the specific Kerr constants (B/c) over a tenfold range of concentration and the intrinsic viscosities of solutions of a low molecular weight sample of the polymer in four solvent mixtures, viz., benzene–dimethylformamide (DMF), benzene–ethylene dichloride (EDC), dioxane–DMF and dixoane–EDC. Sharp changes are found in the experimentally determined quantities on the addition of small amounts of polar solvent to solutions of the polymer in either benzene or dioxane; this implies that lyotropic phase changes are occuring. The aggregation in benzene produces a birefringent, viscous solution which is probably a smeetic liquid crystal. This changes on addition of polar solvent to an aggregation involving only a few molecules; the second aggregate is most likely antiparallel. Aggregation in dioxane is antiparallel; the existence of a smectic phase is ruled out by the low intrinsic viscosities.     

Correlation between the sequence-length distribution and structure of statistical copolymers of L-valine and γ-benzyl-L-glutamate

Statistical copolymers were prepared from N-carboxyanhydrides of L -valine and γ-benzyl-L -glutamate in dioxan with triethylamine as an initiator. The copolymerization conversion was determined by ir spectroscopy, the copolymer composition by amino acid analysis, and the molecular weights by light scattering. The monomer reactivity ratios were found to be r_Val = 0.14 and r_Glu(OBzl) = 6.4. High-molecular-weight copolymers are formed even at low conversions. The content of β-structure in the copolymers was estimated from the ir spectra in copolymerization mixtures. The sequence-length distribution of L -valine and γ-benzyl-L -glutamate copolymers was calculated and its dependence on copolymerization conversion is discussed. Relations between the sequence-length distribution and the content of β-structure were studied. It was found that the content of β-structure in samples with the same composition is different for low- and high-conversion copolymers. The formation of β-structure in copolymers in the copolymerization mixture requires a certain minimal sequence length, which has been found to be about 6 valine units.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Poly(L-lysyl-L-alanyl-alpha-L-glutamic acid). II. Conformational studies.

The solution characterization of poly(Lys-Ala-Glu) is described. This polytripeptide is zwitterionic at neutral pH and is shown to take on a conformation which is dictated by the state of ionization, molecular weight, temperature, and solvent. The polypeptide is almost entirely α-helical at low pH and temperature for polymers of greater than 25,000 molecular weight. Melting profiles for these conditions show t_m ～ 20°C. Analysis of circular dichroism curves shows the α-helical content to vary in a linear manner with molecular weight in the range 3000–30,000. At neutral pH the charged polypeptide is essentially random, but substantial α-helix could be induced by addition of methanol or trifluoroethanol. At temperatures where the sequential polypeptide is a random coil, addition of trifluoroethanol produces a polymer which is mostly α-helical but also contains an appreciable ammount of β-structure. The infrared spectrum of a low-molecular-weight fraction assumed to be cyclo(Lys-Ala-Glu)₂ was tentatively assigned a β-pleated sheet structure. A comparison of this polytripeptide in various ionization states with other polytripeptides containing L -alanine and L -glutamate or L -lysine shows the α-helix directing properties for the (uncharged) residues to lie in the order Ala > Glu > Lys.     

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.     

Vibrational spectroscopic analysis of LD-sequential, bacterial cell wall peptides: an IR and Raman study

The synthetic, zwitterionic bacterial cell wall peptides—D -Glu^γ-L-Lys, D -Glu^γ-L-Lys-D -Ala, D -Glu^γ-L-Lys-D -Ala-D -Ala, and L-Ala-D -Glu^γ-L-Lys-D -Ala-D -Ala—have been investigated in the crystalline and aqueous solution state applying ir and Raman spectroscopy. Additionally, aqueous solutions of the tetra- and pentapeptide have been investigated by CD spectroscopic techniques. Apart from the dipeptide, whose spectral features were dominated by end-group vibrations, the corresponding ir and Raman active bands of the crystalline peptides in the amide and skeletal regions were found at similar wave numbers, thus suggesting an analogous three-dimensional structure of these compounds. Dominant amide A, I, II, and III bands near 3275, 1630, 1540, and 1220–1250 cm^?1, respectively, in the ir are interpreted in favor of an intermolecularly hydrogen-bonded, β-like structure. The absence of any amide components near 1680–1690 cm^?1, together with the presence of strong amide bands near 1630 cm^?1, and weak bands near 1660 cm^?1 in the ir, which, conversely, were found in the Raman spectra as weak and strong bands, but at corresponding wave numbers, is taken as strong evidence for the presence of the unusual, parallel-arranged β-structure. On the basis of comparative theoretical considerations, a parallel-arranged, “β-type ring” conformation [P. De Santis, S. Morosetti, and R. Rizzo (1974) Macromolecules 7 , 52–58] is hypothesized. The solubilized peptides exhibited distinct similarities with their crystalline counterparts in respect to frequency values and relative intensities of the corresponding ir and Raman-active amide I/I′ components, and of some Raman bands in the skeletal region. This is interpreted in terms of residual short-range order, persisting even in aqueous solution. We concluded that the peptides show a strong propensity to form hydrated, strongly associated aggregates in water. On the basis of amide I/I′ band positions, stable, intramolecular interactions via the amide groups are discarded for the solubilized peptides. Complementarily, the CD data obtained suggest the presence of weakly bent, “open-turn”-like structures for the tetra- and pentapeptide in aqueous solution.     

Structure of aggregates and helix-coil transition of polypeptides by dielectric measurements. I. Poly-gamma-benzyl-L-glutamate in dioxane and dioxane-dichloroacetic acid

Dielectric dispersion measurements on poly-γ-benzyl-L -glutamate (PBLG) in dioxane and dioxane–dichloroacetic acid (DCA) mixtures in the frequency range 200 Hz–2 MHz were made in order to study the structure of molecular aggregates. The structure of aggregates is explained on the basis of the variation of dipole moment and relaxation time with degree of aggregation. PBLG was found to form linear head-to-tail-type aggregates in dioxane. These aggregates gradually reduce in size without loosing their α-helical structure during the process of disaggregation obtained by either adding DCA to the solution in dioxane or by heating. It was confirmed that the addition of 30 wt % DCA completely destroys the aggregation of PBLG in dioxane at 30°C. Thermal disaggregation, however, was not complete even at a temperature approaching the boiling point of the solvent. A reaction scheme for aggregation is proposed and equilibrium constants are calculated at various stages of aggregation. The enthalpy of aggregate formation is found to be ?3 kcal/mol. Results of optical rotatory dispersion measurements of the helix–coil transition in this system are also presented.     

Microcalorimetric studies of solvent-induced conformational change of sodium and cesium salts of poly(L-glutamic acid) in aqueous media

Organic solvent-induced coil → helix conformational change of poly(sodium) L -glutamate (NaPLG) and poly(cesium L -glutamate) (CsPLG) in solution in aqueous mixed solvents have been studied at 25°C. Heats of dilution of NaPLG in the water–dioxane pair have been measured as a function of polymer concentration and solvent composition. The results indicate that the overall chain conformation in the disordered form is not too different from that in the α-helical form. Heat capacity measurements by flow microcalorimetry have also been done. The apparent monomolar heat capacity at constant pressure of the polymer, C_{p, ?}, decreases with dilution similarly to other strong polyelectrolytes in aqueous media. In the water–dioxane pair, C_{p, ?} increases with the dioxane content due to partial desolvation of ionic species resulting from increasing ionic association. In the case of the water-2-chloroethanol (CE) pair, the transition takes place at low CE content and results show a fast decrease in C_{p, ?} when the α-helical conformation predominates. It is believed carboxylate groups and CE molecules associate themselves into a complex formation responsible for the transition. The size of the cation plays a significant role in the thermodynamic properties of these polyelectrolytes in solution since sodium ions are more strongly bound to the chain than cesium ions.     

Reversible and irreversible conversion between the intermolecular β-structure and the disordered state of poly(s-carboxymethyl-l-cysteine) in aqueous media

Conversion between the intermolecular β-structure and the disordered state of a fractionated low molecular weight sample of poly(S-carboxyniethyl-L-cysteine) was examined mainly by the measurements of circular dichroism in the absence of salt as well as in the presence of 20 mM NaClO₄, or NaCl. In 20 mM NaClO₄ or NaCl solutions, the conversion was reversible. Under this condition, it was confirmed by direct and unambiguous evidence provided from the viscosity and the reduced scattering intensity that the β-structure was formed by intermolecular association. At low degrees of neutralization, the pH increased on dilution while it remained constant over a wide range of concentration at a high degree of neutralization. In the absence of salt, the conversion was often irreversible with respect to a concentration change at a constant degree of neutralization or to a change in the degree of neutralization at a constant concentration. The extent of the irreversible conversion decreased with the amount of β-structure in the solution. The dissociation of aggregates was very slow at low ionic strengths. It was inferred that the irreversible nature of the conversion arose from this slow dissociation of aggregates.     

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.     

Infrared and Raman study in the solid state of fully protected,monodispersed homooligopeptides of L-valine,L-isoleucine,and L-phenylalanine

An ir-absorption and Raman-scattering study, in the solid state, has been carried out on monodispersed, N- and C-protected homooligopeptides (number of residues, n, from 2 to 7) of L -valine, L -isoleucine, and L -phenylalanine. The amide I, II, III, V, and vNH regions have been examined. Some deuterated (ND) samples have been examined to complete the assignments. L -Phenylalanine dipeptide displays spectral characteristics compatible with the parallel β-structure; L -isoleucine and L -valine dipeptides are probably in a distorted structure. A mixture of parallel and antiparallel extended chains cannot be excluded for the peptides with n = 3. In the amide I region the spectra of peptides with n ≥ 4 show the existence of the β-conformation. The problem of chain orientation within the pleated-sheet structure is discussed on the basis of a recent theoretical treatment of vibrational interactions of the amide I mode.     

Laser Raman scattering of cobramine B, a basic protein from cobra venom

Cobramine B, a small basic protein from cobra venom, is selected as a model for studying the scattering intensity of tyrosyl ring vibrations in the Raman spectra of proteins. All three tyrosines in this protein appear to be “buried” in the interior of the molecule and probably involved in interactions which are similar to those of the three “buried” tyrosines in RNase A when it is dissolved in water. Spectral evidence is presented and discussed. The Raman spectra in the 300–1800 cm^?1 region of cobramine B in the solid and solution are compared quantitatively. Several differences exist between the two spectra and may be interpreted in terms of difference in conformation. In the amide I region, a strong single line was observed at 1672 cm^?1 both in the solid and solution spectra, suggesting that this protein may contain a large fraction of antiparallel-β structure. This is supported by the presence of a line at 1235 cm^?1 in the amide III region, which is also characteristic of β-structure. The resolved peaks at 1254 and 1270 cm^?1 indicate the coexistence of some hydrogen-bonded random-coil and some α-helix with the β-structure.     

Vibrational circular dichroism of polypeptides. IV. Film studies of L-alanine homo-oligopeptides

Vibrational CD (VCD) and ir absorption data are reported for a series of films of Boc-(L -Ala)_n-OMe homo-oligopeptides (n = 3–7) in the amide I and A regions. The data evidenced a sharp change between n = 3 and n = 4, which parallels the onset of β-structure formation, and another between n = 5 and n = 6, which parallels the full development of β-structure. This represents the first report of the application of VCD to oligopeptide conformation. The data resembled earlier reported film VCD studies of higher-molecular-weight polypeptides of known β-structure.