The -conformation and association of low-molecular-weight poly- -benzyl-L-glutamate in solutions

Three samples of poly-γ-benzyl-L -glutamate have been prepared from γ-benzyl-N-carboxy-L -glutamate anhydride with n-hexylamine initiation at anhydride-to-initiator molar ratios, [A]/[I], of 3, 4, and 8, and their conformation and association in ethylene dichloride and dioxane have been investigated by means of infrared spectra and vapor-pressure osmometry. Two conformations, σ-and β-forms, are present in those solvents, and the content of β-form increases with increasing A/I value and concentration. At infinite dilution molecular association is absent, but the number-average molecular weight increases with cocentration, markedly in ethylene dichloride and, to a lesser extent, in dioxane. The fraction of residues involved in associated molecules have been estimated as a function of concentration. Combination of the content of β-structure with the fraction of association leads to the following results. The A/I 3 and 4 polymers form intermolecularly hydrogen-bonded aggregates, in which hydrogen-bonded residues are in the β-structure. The A/I 8 polymer has the intramolecularly hydrogen-bonded β-structure at very low concentrations, but it also forms intermolecularly hydrogen-bonded aggregates at high concentrations.     

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.     

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.     

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.     

Kinetics of coil to α-helix to β-structure transitions of poly(L-ornithine) in low concentrations of sodium dodecyl sulfate

Conformational changes of poly(L-ornithine) [(Orn)_n] were studied in a sodium dodecyl sulfate (NaDodSO₄) solution by CD. (Orn)_n adopted an unstable and a stable helical structure below and above the NaDodSO₄ concentration range where β-structure was favored, respectively. CD stopped-flow was used to monitor the transitions from coil to the unstable helix, from the helix to β-structure, and from coil to β-structure. Only the rate of the helix to β-structure transition was accelerated by an increase in NaDodSO₄ concentration, whereas the rates of the others were independent of NaDodSO₄ concentration. The fractions of coil, α-helix, and β-structure in each conformation of (Orn)_n caused by NaDodSO₄ were computed by simulating a mixed spectrum of typical CD spectra for these structures to the experimentally obtained spectrum. The contents of the unstable and stable helical structures were less than 50 and 73%, respectively.     

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.     

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.     

Hydrodynamic behavior and molecular conformation of poly(L-lysine HBr) in carbonate buffer solution

In carbonate buffer at pH 10.5, a transparent solution of poly(L -lysine HBr) was obtained up to fairly high concentration of 3 g/dl at room temperature. The hydrodynamic behavior of the solution has been studied by sedimentation analyses and viscosity measurements. A dimer form for high concentrations and a monomer form for low concentrations were inferred. The dimer and monomer forms were assigned to a β-structure and α-helix, respectively, based on the CD and optical rotary dispersion spectra. Using CD spectroscopy, a reversible transition between α-helix and β-structure was observed as a function of either poly(L -lysine HBr) concentration or temperature. An aggregated form which was assigned to the antiparallel pleated sheet appeared at 50°C on the basis of its ir spectrum.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Computer analyses of characteristic infrared bands of globular proteins.

Infrared spectra of myoglobin, ribonuclease, lysozyme, α-chymotrypsin, α-lactalbumin, and β-lactoglobulin A were obtained in deuterium oxide solution in units of absorbance versus wavenumber from 1340 to 1750 cm^?1. The spectra were resolved into Gaussian components by means of an iterative computer program. Resolved characteristic absorption peaks for the two infrared active amide I′ components of antiparallel chain-pleated sheets (β-structure) were obtained. The characteristic amide I′ peaks of α-helical regions and apparently unordered regions overlap in D₂O solution. Absorptivity values for the resolved β-structure peak around 1630 cm^?1 were estimated on the basis of the known structure of ribonuclease, lysozyme, and β-chymotrypsin. The β-structure content of β-lactoglobulin was estimated to be ca. 48% of α-lactalbumin ca. 18%, and of α_s-casein close to zero. The results are in general agreement with conclusions drawn from circular dichroism and optical rotatory dispersion studies.     

Circular dichroism of insulin a chain in water: Trifluoroethanol mixtures: Use of linear and nonlinear least squares analysis

Circular dichroic spectra obtained for S-carboxymethylated insulin A chain in water : trifluoroethanol mixtures show that a marked conformational transition occurs as the concentration of trifluoroethanol is increased to (v/v) 83% to give a polypeptide containing about 43% of the residues in the α-helical conformation. Several proposed methods of analysis, including both linear and two nonlinear least squares methods, were unable to quantitate the amount of β-structure present in the polypeptide in 83% trifluoroethanol. Examination of the methods of analysis lead to the conclusion that the current models for far UV CD analysis are not adequate for the data obtained in this study and shows that nonlinear least squares procedures may lead to erroneous conclusions.     

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.     

Molecular conformation of poly-S-carboxymethyl-L-cysteine in aqueous solutions

Poly-S-carboxymethyl-L -cysteine has been prepared by debenzylation of poly-S-carbobenzoxymethyl-L -cysteine with hydrogen bromide in acetic acid. By the infrared spectroscopic method the polymer is found to be in the extended β-conformation with an antiparallel arrangement of polypeptide chains in solid film, if it has been regenerated from dimethyl sulfoxide solution. Aqueous solutions of the polymer have been investigated by measurements of optical rotatory dispersion and viscosity. Various properties sharply change around pH 5 at different ionic strengths. By combining these with infrared studies in D₂O solutions, it has been shown that the polymer exists in the random coil conformation at higher ionization but associates into the intermolecular β-conformation at lower ionization. At the lowest pH attainable in solution, the β-form is partly converted into the random coil as the temperature is raised. The rotatory dispersion of the polymer is described by the Moffitt equation. While the random coil form has a large negative a₀ value and a zero b₀ value, the β-form is characterized by a positive a₀ value and a negative b₀ value, ?130°.     

Potentiometric titration of poly-S-carboxyethyl-L-cysteine

Potentiometric titration curves have been determined for aqueous solutions of poly-S-carboxyethyl-L -cysteine, which is subject to the β-coil transition by a change in pH. Reversibility and time dependence of the titration curves are examined by different methods in order to establish the conditions for obtaining equilibrium curves. The β-coil transition is manifest, at some region on the equilibrium titration curve, if pH – log (α/1 – α) is plotted against α. Assuming a value, 4.00, for pK_int, the free-energy change for the β-coil transition of uncharged polymer has been evaluated from the extrapolation of the observed titration curves and is found to depend on the ionic strength and polymer concentration. The Henderson-Hasselbach plot of the titration curve yields clearer distinction between the β-form and random coil, and it permits estimation of the content of β-form at, a given pH. Comparison of the conformational titration curve with the circular dichroic measurements leads to a value of ?10,000° for [θ]₂₂₃ for the pure β-structure. Precipitation which occurs at low degrees of ionization and, especially, at high ionic strength does not reveal any discontinuous change of the titration curve, which suggests that, the degree of ionization of the precipitated β-form is not very different from that in solution.     

Photoresponsive polymers: Azobenzene-containing poly(L-lysine)

Poly(L -lysine) was reacted with various azo-reagents, including p-phenylazobenzoic acid, p-phenylazobenzoyl chloride, and p-phenylazobenzoic N-hydroxy-succinimide ester, to give polypeptides containing 5–44 mol % azobenzene units in the side chains. The conformation of the azo-modified polypeptides was investigated in connection with their photochromic behavior caused by the trans ? cis photoisomerization of the azo groups present in the side chains. In methanol/water solvent mixture, the 20% azo-poly(L -lysine) adopts the α-helix conformation. The helix stability was found to be higher when the azo side chains are in cis than when they are in trans configuration. So irradiation at 340 nm (trans-to-cis isomerization), and alternately at 450 nm (cis-to-trans isomerization), produced reversible variations of the α-helix content. In hexafluoro-2-propanol/water/sodium dodecyl sulfate mixture, the 43% azo-poly(L -lysine) adopts a β-structure, as indicated by CD spectra. Irradiation at 340 nm caused the disruption of the β-structure and promoted the α-helix conformation. The effect was reversed upon irradiation at 450 nm. The photoinduced β ? helix change was explained on the basis of the different geometry and hydrophobic character of the trans and the cis azobenzene units.     

Infrared spectroscopy of human amyloid fibrils and immunoglobulin proteins

The presence of the antiparallel-β-pleated sheet coformation io isolated human amyloid protein fibrils has been confirmed by infrared spectroscopy. In most amyloid samples, this conformation was enhanced by acidic solution conditions. Infrared spectroscopy (Amide I and Amide V absorption bands) and x-ray diffraction methods were also used to examine the immunoglobulin molecule for solid state-β-structure. It was found that both heavy chains and Bence Jones proteins exhibited some β-pleated sheet content upon acid and/or heat treatment. Furthermore, pepsin digests comprising either the variable-rich region (Fd′) of the immunloglobulin heavy chain or in particular, filamentous variable segments of κ and λ Bence Jones proteins were, as isolated, very similar to amyloid in β-structure content. Data from other immunoglobulin-derived sample did not exhibit extensive β-pleated sheet content. On the other hand, most amyliod and immunoglobulin-derived samples did display some β-structure when cast from 50% HCOOH solution. Under these conditions, however, filamentous light chain-variable segments exhibited well-defined infrared patterns rich in antiparallel-β-pleated sheet structure and gave a “cross-β” x-ray diffraction pattern.     

Fourier transform IR attenuated total reflectance study on the secondary structure of poly(γ -methyl L-glutamate) surfaces treated with formic acid

Fourier transform ir attenuated total reflectance (FTIR ATR) spectra have been obtained to investigate the secondary structure of poly(γ -methyl L -glutamate) (PMLG) surfaces untreated and treated with formic acid in a quantitative manner. Curve analysis including Fourier self-deconvolution and the band fitting was applied to the ir spectra in the amide II region, revealing that the amide II band of those surfaces consists of five components. The essentially α-helical form in the PMLG surface layer transformed readily into the β-structure by the formic acid treatment, and the β-structure content increased with increasing time of the treatment. The content of random coil structure of treated PMLG was generally very little and/or negligible. The depth profile obtained by considering the depth of ir beam penetration indicated that the β-structure content also increased with approaching the outermost surface.     

Structure of Bovine αs-Casein

The secondary structure of bovine α_s-casein and chemically modified α_s-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of α_s-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-α_s-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-α_s-casein had two infrared absorption peaks centered at 1625 and 1643 cm^?1 in D₂O-CH₃OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH₃OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-α_s-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified α_s-caseins.     

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.     

Optical Rotatory Dispersion of Polypeptides in the Near-Infrared Region

For the first time ORD measurements in the near-infrared region from 0.7 to 2.0 μ for well-known polypeptides, namely, poly(γ-benzyl L -glutamate), poly(L -glutamic acid), poly-L -lysine·HCl, poly-S-carbobenzoxymethyl-L -cysteine, and Bombyx mori silk fibroin, were carried out. It was found that the value of the optical activity infrared term, which is proportional to the sum of rotational strengths of vibrational transitions, depends on polypeptide conformation. The optical activity infrared term value is equal to zero for the random-coil conformation, it is small but exceeds the measurement error for the α-helical state, and finally, for the β conformation it is an order of magnitude higher than for the α-helical state. The obtained results permit one to hope that on the basis of ORD measurement in the near-infrared region it will be possible to suggest a method of determining the β-form content in polypeptides and proteins