NMR and optical activity observations on the helix-coil transition in poly(gamma-benzyl L-glutamate) solutions

The helix–coil transition of poly(γ-benzyl L -glutamate) was studied by comparing proton magnetic relaxation behavior with optical activity studies. The transition temperature as determined by magnetic relaxation was lower than that obtained by optical activity. The concentration dependence of the transition was also studied. The relationship of these experiments with previous NMR studies and also with calorimetric investigation of the transition is developed.     

Enthalpy change of the coil-helix transition of poly(gamma-benzyl L-glutamate) in dichloroacetic acid-1,2-dichloroethane mixtures

By combining the heat of dilution of a poly(γ-benzyl L -glutamate)–dichloroacetic acid–1, 2-dichloroethane solution with the corresponding heat of mixing of two solvents, the integrated heat of the coil–helix transition of poly(γ-benzyl L -glutamate) in the solution was estimated to be about 750 cal/mole.     

Kinetic properties and the electric field effect of the helix-coil transition of poly(gamma-benzyl L-glutamate) determined from dielectric relaxation measurements

Dielectric relaxation of poly(γ-benzyl L -glutamate) in solution has been studied in the 5 kcps-10 Mcps range for various values of the helix content. The results give first experimental evidence for three effects of major significance. (1) The system exhibits dielectric relaxation due to a chemical rate process (namely helix formation). This confirms recent theoretical predictions. (2) The mean relaxation time τ* of the helix–coil transition could be evaluated as a function of the degree of transition. The results are in excellent agreement with a previously developed theory. At the midpoint of transition it is found τ*_max = 5 × 10^?7 sec. The elementary process of helical growth turns out to be practically diffusion-controlled (with a rate constant of hydrogen bond formation of 1.3 × 10¹⁰ sec^?1). (3) There is a considerable electric field effect of the helix–coil transition. This indicates that conformation changes in biological systems could be potentially caused by direct action of an electric field.     

Stability of the alpha-helix in poly(gamma-benzyl L-glutamate) and related polymers

A series of poly(L -glutamic acid) esters have been synthesized and studied by optical rotatory dispersion, x-ray diffraction, and infrared spectrometry. The results obtained emphasize the importance of the outer portions of the side chains in determining both the stability and precise conformation of the α-helix.     

Conformational studies on concentrated solutions of poly(gamma-benzyl L-glutamate)

We have studied the effect of polypeptide concentration on the helix–coil transition of poly(γ-benzyl L -glutamate) (PBLG) in both dichloroacetic acid (DCA) and DCA–chloroform (CHF) mixtures. In agreement with other reports, we find the van't Hoff transition enthalpy to be strongly dependent on PBLG concentration. Also, an apparent effect of polypeptide concentration was noted on the transition temperature; however, corrections for finite PBLG concentration on the mole fraction of DCA seem to remove this effect. In order to explain our data, as well as some calorimetric data in the literature, we consider the transition free energy and enthalpy as a sum of three partial terms. These represent the thermodynamic parameters associated with: (1) conformational changes of the polypeptide, e.g. formation or disruption of intramolecular hydrogen bonds; (2) binding by the strong acid to the nonhelical segments of the polypeptide; (3) an overall (weak) interaction of the polypeptide with the nonbound solvent giving rise to dilution parameters that are dependent on the polypeptide conformation. The latter effect is generally ignored, since it is assumed that solvent interactions, other than specific binding, are similar for both the helical and the nonhelical conformation. Striking effects of water (small amounts) and solution aging on the formation of PBLG helices was observed. Water, as expected, acts as a helicogenic solvent when combined with DCA. The processes occurring during solution aging are not known, although the net effect is to stabilize the helical conformation. Finally, we present some rather unique thermally induced transitions of concentrated PBLG (about 200 mg/ml) in DCA. At low temperatures the soluble randomly coiled conformation is present. Heating produces first an isotropic gel, followed at higher temperatures by an isotropic solution consisting of about 70% α-helicity.     

Epitaxial crystallization of poly(gamma-benzyl L-glutamate) on alkali halide single crystals

Single crystals of poly(γ-benzyl L -glutamate) were formed by epitaxial crystallization from solution in mesitylene on NaCl, KI, and KCl (001) cleavage faces. From electron microscopy and diffraction studies, the structure of these overgrowths was determined to be that of lamellae containing chain-folded α-helical macromolecules. The usual type of crystal perfection, that of ordered helix axes and disordered side groups, was exhibited by this synthetic polypeptide. Unique orientation regimes were observed with each substrate.     

Electric dichroism studies: interaction of trifluoroacetic acid with poly (gamma-benzyl L-glutamate)

Electric dichroism studies on poly(γ-benxyl L -glutamate) show that the addition of small amounts (up to 5%) of trifluoroacetic acid causes complete disappearance of dichroism in contrast to the electric birefringence, which drops to an observable plateau at 20% of its initial value. This loss of dichroism cannot be explained simply by a decrease in the effective dipole moment of the benzyl ester side chains, and the nature of this interaction is explored by nuclear magnetic resonance and infrared spectral studies. Theories of the helix-coil transition which require an initial protonation of the helix backbone to form a more flexible rod consisting of helical segments interrupted by regions of random coil are shown to be inadequate to describe the changes in poly(γ-benzyl L -glutamate) effected by small amounts of trifluoroacetic acid. Rather, trifluoroacetic acid in small amounts interacts with the ester carbonyl oxygen in the side chain, either by hydrogen bonding or protonation, before there is evidence of any effect on the backbone or of loss of helix content.     

Osmotically induced helix-coil transition in poly(glutamic acid)

Protein folding and conformational changes are influenced by protein-water interactions and, as such, the energetics of protein function are necessarily linked to water activity. Here, we have chosen the helix-coil transition in poly(glutamic acid) as a model system to investigate the importance of hydration to protein structure by using the osmotic stress method combined with circular dichroism spectroscopy. Osmotic stress is applied using poly(ethylene glycol), molecular weight of 400, as the osmolyte. The energetics of the helix-coil transition under applied osmotic stress allows us to calculate the change in the number of preferentially included water molecules per residue accompanying the thermally induced conformational change. We find that osmotic stress raises the helix-coil transition temperature by favoring the more compact α-helical state over the more hydrated coil state. The contribution of other forces to α-helix stability also are explored by varying pH and studying a random copolymer, poly(glutamic acid-r-alanine). In this article, we clearly show the influence of osmotic pressure on the peptide folding equilibrium. Our results suggest that to study protein folding in vitro, the osmotic pressure, in addition to pH and salt concentration, should be controlled to better approximate the crowded environment inside cells.     

Synthesis and ITC characterization of novel nanoparticles constituted by poly(gamma-benzyl L-glutamate)-beta-cyclodextrin

Imparting desired technological characteristics to polymeric nanoparticles requires the development of original polymers. In the present work, the synthesis and characterization of a novel PBLG-derivative, the poly(gamma-benzyl L-glutamate)-beta-cyclodextrin (PBLG-beta-CD-50), have been carried out. Nanoparticles from either PBLG-beta-CD-50 polymer or from mixtures with PBLG have been prepared using a modified nanoprecipitation method. Spherically shaped nanoparticles with diameter in the range of 50-70 nm were obtained, as determined by dynamic laser light scattering and transmission electron microscopy. The presence of a surfactant in the suspension medium had almost no influence on these parameters and was not necessary to the shelf-stability of the suspension. Further, isothermal titration microcalorimetry (ITC) experiments have been used to show unambiguously that about 20% of the cyclodextrins remain functional within the particles. Consequently, this system may be of interest when association of large amounts of hydrophobic drugs to nanoparticles is required.     

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. 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.     

Raman scattering of some synthetic polypeptides: poly(gamma-benzyl L-glutamate), poly-L-leucine, poly-L-valine, and poly-L-serine

The Raman spectra of poly-γ-benzyl-L -glutamate, poly-L -leucine, poly-L -valine, and poly-L -serine are reported. For the α-helical polymers, the conformationally sensitive amide I, II, and III modes are observed in the Raman as, well as the infrared. For the β form, the Raman effect, supplies the infrared inactive inphase motion which is useful for the determination of a parallel or antiparallel chain alignment. Modes characteristic of the specific polypeptide are also observed which are insensitive to conformation.     

Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique

Fluorescent dansyl labels were covalently attached to poly (L-lysine) (poly(Lys)) with a degree of polymerization of 300 to 600. The degree of labeling was 0.01 to 0.085 (mol label to mol amino acid residues). From the decay of the anisotropy of fluorescence it was concluded that the labels were highly mobile both in the coiled and helical state. A decrease of fluorescence intensity accompanied the helix-coil transition. Identical pH induced transition curves were measured by circular dichroism and fluorescence. The midpoint of the transition was at pH 10.2. The kinetics of the transition were studied by temperature-jump relaxation using fluorescence detection. A single relaxation phase was observed. The relaxation time tau exhibited a distorted bell shaped dependence on the degree of helicity f with a maximum value tau(max) = 15 micros at f = 0.3 and 20 degrees C. It was independent of polymer concentration and of the degree of labeling. A rate constant of helix propagation kF = 10(7) s(-1) was calculated from tau(max) and published values of the nucleation parameter sigma. The activation energy was 16 kJ mol . The observed rate constant is comparable to that of poly(L-glutamic acid) but two orders of magnitude smaller than that found for polyamino acids with nonionizable side chains.     

Potentiometric titrations and the helix-coil transition of poly(L-glutamic acid) and poly-L-lysine in aqueous salt solutions

The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L -lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L -glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK₀), slope of pK_app versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°_hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN–PLL and with LiCl–PLGA. In the presence of KSCN, the poly-L -lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pK_app versus α plots is greatly reduced. However, ΔG°_hel (for the uncharged molecule) is not affected, and pK₀ is only slightly higher. We interpret these data in terms of binding of SCN^? primarily to the side-chain amino groups (both to R? NH₃⁺ and to R? NH₂) solutions. (L -glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pK_app versus α plots and the absolute values of ΔG°_hel are lower than in KCl solution. We interpret these results in terms of binding of Li⁺ to side chains as well as to the peptide bond.     

Enthalpy of helix-coil transition of DNA: dependence on Na+ concentration and GC-content

The enthalpy of helix-coil transition of DNA (delta H) is determined from the experiments on DNA melting with ligands by means of absolutely general formula, which contains only values directly known from the experiment (M.D. Frank-Kamenetskii, and A.T. Karapetian, Mol. Biol. USSR 6, 621 (1972)) with the combination of the "area" method (P.O. Vardevanian, et al., Biophysica 28, 130 (1983)). The experimentally obtained data show that delta H depends on both concentration of Na+ in solution and GC-content of DNA and is of high accuracy.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

Radiation effects of poly(l-glutamic acid) and poly(l-lysine) in the helix-coil transitional state

Summary The degradation of poly(l-glutamic acid) and poly(l-lysine) by X-rays and ultraviolet radiation has been studied at variouspH values, including the helix-coil transition region. It was found that X-rays cause maximum degradation in the transition region for both polypeptides, indicating that the efficiency of degradation is affected not only by the secondary structure but also by some other factors related to the transition. On the contrary, the degradation by monochromatic ultraviolet 2537 radiation showed a strong dependence on helix content of PLGA, suggesting that degradation occurs more effectively in the helical form than in the coil form. Theoretical calculations were made to approximate thepH dependence of degradation, assuming different cross sections for the peptide bonds in helix, coil, and junctions between helical and coil regions.     

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.