Infinite-dilution viscoelastic properties of poly-gamma-benzyl-L-glutamate in helicogenic solvents

The storage and loss shear moduli, G, and G?, have been measured for solutions of three samples of poly-γ-benzyl-L -glutamate with molecular weights from 16 to 57 × 10⁴, by use of the Birnboim-Schrag multiple-lumped resonator. The frequency range was 106 to 6060 H_z, the concentration range 0.0015–0.005 g/ml, and the temperature 25°C. Two helicogenic solvents with widely different viscosities, dimethylformamide and m-cresol, were used to provide a broader effective frequency range. The intrinsic moduli, extrapolated to infinite dilution, were compared with the predictions of the theory of Ullman for rigid rods; agreement was rather good at the lowest frequencies, but unsatisfactory at high frequencies. The data over the entire frequency range of three of logarithmic decades could be described closely by a relaxation spectrum consisting of one terminal relaxation time separated by a gap from a sequence of relaxtion times spaced as in the Zimm theory. The terminal time agrees approximately with that calculated for end-over-end rotation of a rigid rod. The additional relaxation mechanisms are tentatively attributed to modes of flexural deformation of the helix.     

Solution properties of synthetic polypeptides. XVII. Stability of the alpha-helical conformation of poly (gamma-benzyl L-glutamate) in helicogenic solvents

The Zimm–Bragg parameters s and σ were determined for poly(γ-benzyl L -glutamate) (PBLG) in m-cresol and in dimethylformamide (DMF) from ORD data as a function of molecular weight. It was found that, within the temperature range between 10 and 55°C and on the average, s = 1.6₁ ± 0.1 and √σ = 0.04 ± 0.01 in m-cresol and s = 1.6₅ ± 0.05 and √σ = 0.045 ± 0.015 in DMF. The values of s in m-cresol decreased with increasing temperature, while the values of σ in the same solvent increased. This result for s suggests that PBLG in m-cresol will undergo a thermal helix–coil transition of normal type. The parameters in DMF showed no appreciable trend to vary with temperature. Aside from the difference between the two solvents, our results are consistent with existing data for various conformation-dependent properties such as light-scattering radius, intrinsic viscosity, and dipole moment, each indicating that the polypeptide chain has some flexibility in helicogenic solvents.     

Solution structure of human beta-endorphin in helicogenic solvents: an NMR study.

Beta-endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

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.     

Solution properties of synthetic polypeptides. VI. Helix-coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine

A new procedure for evaluating u and σ characterizing σ-helix-forming polypeptides in solution was derived from Nagai's theory for the helix–coil transition of such polymers. Here u is the activity for helix formation from random coil, and σ is the helix initiation parameter. The necessary data are the helical content f_N at fixed solvent and temperature as a function of N, where N is the degree of polymerization of the polypeptide sample. Such data were obtained from ORD measurements on a number of fractionated samples of poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPG) in mixtures of water and methanol covering the complete range of composition and at various termperatures (5–40°C). When analyzed in terms of the proposed procedure, they yielded values of σ which were in the range (3.2 ± 0.6) × 10^?4, substantially independent of solvent composition and temperature. These values were much larger than those obtained recently for σ of poly(β-benzyl-L -aspartate) in m-cresol and in a mixture of chloroform and DCA. The data for [η] and s₀ (limiting sedimentation coefficient) as functions of molecular weight indicated that the molecular shape of PHPG in pure methanol is essentially rodlike, whereas that in pure water is not entirely randomly coiled but rather may be regarded as an interrupted helix. These indications were consistent with the results from ORD measurements. When plotted against the corresponding values of f_N, the values of [η] and [s₀] for PHPG in mixtures of water and methanol of various compositions and temperatures formed smooth composite curves, and we attributed these phenomena to the fact that σ of PHPG was nearly constant under these solvent conditions. Here [s₀] stands for a reduced limiting sedimentation coefficient which is equal to the inverse friction factor of the solute molecule.     

Solution properties of synthetic polypeptides. 18. Helix-coil transition of poly-N5-(2-hydroxyethyl)L-glutamine

The helix–coil transition of poly-N⁵-(2-hydroxyethyl)L -glutamine (PHEG) in aqueous isopropanol was examined by means of optical rotatory dispersion (ORD) and intrinsic viscosity [η] measurements. The Zimm–Bragg parameters σ and s for the transition were determined from the ORD data as a function of molecular weight. It was found that the transition was characterized by a relatively low cooperativity; the values of \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt \sigma $\end{document} were in the range from 0.039 to 0.066, depending on the solvent composition. These σ values are much larger than those reported for other polypeptide–solvent systems. The transition enthalpy was negative and its magnitude varied with the solvent composition, with a maximum of 620 cal/mol at 40 wt% isopropanol. The curve of [η] versus helical content for a high-molecular-weight sample exhibited a very broad minimum, and this behavior was attributed to the low cooperativity of the transition.     

Conformation study of poly(γ-methyl-L-glutamate) in helicogenic solvents by small-angle X-ray scattering

Small-angle x-ray scattering of poly(γ-methyl-L -glutamate), [Glu(OMe)]_n, in m-cresol and in pyridine was measured to determine the mass per unit length, M_q, and the radius of gyration of the cross section, 〈S〉^1/2. It was confirmed from the values of M_q that [Glu(OMe)]_n exists in an α-helical conformation in these solvents. It was elucidated from the calculations on 〈S〉^1/2 that the side chains come in moderately close contact with the main chain in these solvents. It was indicated from the analysis of the outer portion of the scattering curves that the side-chain conformation varied depending on the solvent.     

Buoyant and potentiometric titrations of synthetic polypeptides. I. Six ionizable homopolypeptides in CsCl solutions

The buoyant density and potentiometric titrations of six ionizable homopolypeptides in concentrated CsCI solutions have been studied. These six homopolypeptides were chosen as models of the behavior of ionizable residues in proteins. Their buoyant and potentiometric results will be of value in interpreting the buoyant and potentiometric results observed for proteins. The buoyant densities for all six homopolypeptides were found to increase sigmoidally as the pH is increased. These density changes are interpreted in terms of changes in the hydrations and ion binding which are associated with the titration of the residues. Preferential hydrations for the homopolypeptides are calculated. The buoyant density titrations are combined with the potentiometric titrations to determine the relationship between the buoyant density and the degree of ionization. A better method of computing buoyant densities of proteins is described. The slope of β(ρ) has been computed for CsCl using least-squares curve fitting and this is used in calculating the isoconcentration position. This method has been found to be more accurate than calculating the isoconcentration position from the normalized isoconcentration ratio, which is known only under limited conditions.     

Solvation properties of natural and synthetic ionophores. I. Stoichiometry of complexes with alkali and alkaline earth cations in aprotic organic solvents.

Ion-solvent interactions play a very important role in the studies of stoichiometry, structure, and stability of complexes of cations with natural and synthetic ionophores. These compounds are extremely useful in study of the interaction of neutral salts with macromolecules and the mechanism of cation transport across biological membranes. Knowledge of the ionophore solvation properties enables one to choose a suitable solvent for complexation studies and to obtain detailed information on the solvent effect. We would like to present in this paper a very simple method of estimating the solvation properties of ionophores. We treat the ligand as an assembly of individual noninteracting binding sites. The solvation properties of solvents can be used to represent the solvation sites in natural and synthetic ligands. The solvation properties are represented by the Gutmann donor number (DN) of the model solvent. We can define the solvation ability of a ligand binding site be "donor number of binding site" (DN binding site), which in turn can be represented by the DN of the appropriate model solvent. The average DN of the ligand (DN average) is defined as [xi ni-1 (DN binding site)i]/n, where n is the number of the ligand binding sites. Comparison of the DN average with the DN solvent, together with the knowledge of the composition of the system, characterizes remarkably well the solvation properties of the ligand. This model explains (a) the stoichiometry of many alkali and alkaline earth cation complexes with natural and synthetic ligands in aprotic organic solvents, (b) the transport of alkali and alkaline earth cations across lipid bilayers, and (c) how polypeptides and proteins interact with neutral salts in solutions.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.