Electric field-induced conformational change of poly(L-lysine) studied by transient electric birefringence

Transient electric birefringence measurements on poly(L -lysine hydrobromide) in methanol–water mixtures have been carried out at various solvent compositions in the vicinity of the helix–coil transition region (from 87 to 98 vol % methanol). Anomalous birefringence transients were observed between 90 and 95 vol % methanol above a threshold field strength. A distinct difference between the responses to weak and strong electric fields was noticed over a narrow range of the solvent composition. The effects of polymer concentration and temperature on the field-strength dependence of the birefringence were studied at a solvent composition of 90 vol % methanol where the anomalous transients appeared most clearly. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different concentrations and temperatures could be superimposed by shifting them horizontally along the abscissa. The threshold field strength which was determined from the shift factor decreased with decreasing concentration. The results provide further evidence that strong electric fields can cause a helix–coil transition in this system under favorable conditions.     

Electric birefringence of ionized polypeptides in solution and the effect of high electric fields on the helix-coil transition

Electric birefringence and circular dichroism measurements have been made on solutions of two po!y (L-lysine) homologs. The specific Kerr constant and the molar ellipticity at 222 nm of poly (L-alpha, gamma-diaminobutyric acid hydrochloride) in methanol/water mixtures underwent an abrupt change between 75 and 80 vol% methanol at 25 degrees C, corresponding to a solvent-induced helix-coil transition. On the helix side of the transition region, i.e., between 78 and 80 vol% methanol, anomalous birefringence transients indicative of field-induced helix-to-coil transition were observed at high fields. In the case of poly (L-ornithine hydrobromide) in methanol/water mixtures, a helix-coil transition was induced between 93 and 98 vol% methanol and anomalous birefringence transients were observed between 96 and 98 vol% methanol. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for various solvent compositions and polymer concentrations could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transients were observed. This enabled us to estimate the threshold field strength.     

Electro-optical studies on the electric field-induced helix-to-coil transition of poly(l-ornithine hydrobromide) in methanol/water mixtures

Transient electric birefringence and circular dichroism measurements have been made on poly(L-ornithine hydrobromide) in methanol/water mixtures of various compositions. The specific Kerr constant and the molar ellipticity at 222 nm underwent an abrupt change between 93 and 98% (v/v) methanol at 25°C corresponding to a solvent-induced helix-coil transition. Anomalous birefringence transients were observed at high electric fields between 96 and 98% (v/v) methanol, i.e. on the helix side of the transition region. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transient were observed. This behavior served to determine the threshold field strength. The results indicate that a conformational change from the charged helix to the charged coils is induced by high electric fields in this system, as in the cases of poly(L-lysine hydrobromide) and poly(L-αγ-diaminobutyric acid hydrochloride) in methanol/water mixtures.     

Helix–coil transition in copolypeptides. II. Poly(γ-benzyl L-glutamate-co-ε-carbobenzoxy-L-lysine)

The thermal helix–coil transition of poly(γ-benzyl L -glutamate-co-ε-carbobenzoxy-L -lysine) copolypeptides was studied in solvent mixtures of different compositions. The cooperativity parameter v changes linearly with polymer (and solvent) composition, whereas the heat of the transition shows a very pronounced minimum as a function of polymer composition. This minimum cannot be due only or mainly to the solvent changes and must be attributed to the effect on the transition of the side chains of the polypeptides.     

Electric birefringence of poly-L-lysine hydrobromide in methanol-water mixtures and helix-coil transition induced by high electric fields

The electric birefringence of poly-L -lysine hydrobromide in methanol–water mixtures has been measured at 25 °C over a wide range of field strengths by use of the rectangular pulse technique. An abrupt change in the specific Kerr constant was observed between 87 and 90 vol % methanol, corresponding to the solvent-induced helix–coil transition. The specific Kerr constant increased rapidly with dilution in the random coil form, and more slowly in the helical conformation. The field strength dependence of the bire fringence at various concentrations, for both the helical and coil conformations, can be described by a common orientation function, which resembles the theoretical one for the case of permanent dipole moment orientation. This is interpreted in terms of the saturation of ion–atmosphere polarization. The optical anisotropy for the helical conformation was much larger than that for the coil form. Anomalous birefringence signals were observed above a critical field strength (about 5 kV/cm) in 90 vol % methanol. The birefringence passed through a maximum and began to decrease slowly before the pulse terminated, reaching a steady-state value. This steady-state value was closer to that of the coil in the coil in the limit of very high fields. The results indicate that a transition from the charged helix to the charged coil is induced by high electric fields in the transition region. This effect can be explained on the basis of the polarization mechanism proposed by Neumann and Katchalasky.     

A Study of stepwise conformational changes in poly(β-benzyl-L-aspartate) with the helix–coil transition by means of proton and carbon-13 NMR

The helix–coil transition for poly(β-benzyl-L -aspartate) [poly(Asp[OBzl])] in solvent mixtures of trifluoroacetic acid/deuterated chloroform (F₃AcOH/CDCl₃) was studied by means of proton and carbon-13 nmr. Conformational fixation of the side chain occurs before the coil–helix transition of the backbone, when neighboring phenyl rings face each other. Another type of conformational fixation occurs in the side chain after the coil–helix transition of the backbone. These conformational changes of the side chain are due to the changes of the strength of the interaction between the side-chain ester group and the F₃AcOH molecule. In the absence of F₃AcOH (coil-forming solvent), the polymer has a rather rigid structure in which the side chain may wrap around the backbone. These conformational changes of the polymer are closely related to the changes of the interaction between the polymer and F₃AcOH molecules.     

The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Studies of the helix‐to‐coil transition in dilute solutions of poly‐L ‐lysine, dissolved in mixtures of water and methanol (MeOH), have been carried under shear flow using flow birefringence and modulated polarimetry. The fraction of helical conformations in a given solution remains independent of shear rate for MeOH concentrations above and below the critical value for the helix‐coil transition (i.e., 87.5% MeOH). For the 87.5% MeOH solutions, a shear‐induced helix‐to‐“stretched” coil transition occurs above a critical shear rate. Induction times for the transition show a temperature and shear rate dependence that can be described in terms of an activated jump process. Measurements of circular birefringence on cessation of flow also show that the transition is reversible, with the stretched coil reverting to the helical state on a time scale of several seconds. The activation energy for the jump process is found to be 16.2 kJ/mole. © 1999 John Wiley & Sons, Inc. Biopoly 50: 589–594, 1999     

Electric and hydrodynamic properties of polypeptides in solution. IV. A new conformational change of poly(L-glutamic acid) in dimethylsulfoxide–methanol mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in dimethylsulfoxide (DMSO)–methanol mixtures has been measured by use of the rectangular pulse technique. The length distribution curve, the mean molecular length, and the mean apparent permanent dipole moment of PLGA in solution have been obtained from the decaycurve and field strength dependence of the steady-state birefringence according to the method developed for analyzing the electric birefringence of a polydisperse system. The length distribution curve exhibits one or two peaks. The length corresponding to a high peak and the mean length of PLGA undergo an abrupt change in the vicinity of 50 to 60 vol % DMSO at 30°C. Moreover, a sharp change of the Moffitt b₀ parameter with the solvent composition is observed. These results provide evidence for the existence of a solvent-induced transition from a helical conformation (presumably α-helix) to another helical conformation with shorter length per amino acid residue. Further, the temperature dependence of the length distribution of PLGA in 50 vol % DMSO suggests the existence of a temperature-induced helix ? helix transition.     

Threshold effects observed in conformation changes induced by electric fields

Single-stranded polynucleotides are used as model systems for the investigation of conformational changes induced by electric fields. It is demonstrated that the single-strand helix–coil transition in poly(A), poly(dA), and poly(C) can be induced by application of high electric fields. The transition is measured by UV absorbance using polarized light at an angle of 54.8° with respect to the vector of the electric field and by electrodichroism. A linear increase of the absorbance, reflecting the helix-to-coil transition, is observed at increasing field strength. When ions are added to the polymer, electric fields do not induce conformation changes, unless a threshold value of the electric field strength E₀ is exceeded. At field strengths above this threshold, the degree of transition is a linear function of the increase in field strength. The threshold values E₀ show a linear increase with the logarithm of the ion concentration. Bivalent ions cause thresholds at much lower ion concentrations than mo-novalent ions. The shielding efficiency of ions is correlated to the binding affinity of these ions to the polymer. The conformation changes induced by the field and the existence of thresholds can be explained on the basis of dissociation field effects. Similar threshold effects may be expected for other macromolecules as well as for membrane structures and may be important in the regulation of bioelectricity.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

Electric-field orientation of sodium poly(l-glutamate) in methanol/water and ethylene glycol/water mixtures

Transient electric birefrinqence and circular dichroism measurements have been made on sodium poly(l-glutamate) in methanol/waer and ethylene glycol/water mixtures of various compositions. The specific Kerr constant increased upon the transition from coil to helix, but decreased with further increase in methanol or ethylene glycol content on the helix side.     

Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Influence of isopropanol-water solvent mixtures on the conformation of poly-L-lysine

The helix–coil transition of poly-L -lysine (PLL) in water–isopropanol solvent mixtures has been investigated at room temperature by circular dichroism measurements. Within the range of 70%–80% isopropanol concentration (by volume), the polymer undergoes a sharp transition, characterized by the formation of a fully charged α-helical structure. On the basis of some experimental evidence the role of the organic component in solution appears more complicated than that of strengthening the intramolecular hydrogen bonds in the polymer. By analogy with the distribution of the components of alcohol–water mixtures in simple ionic systems, it is thought that only an high co-solvent concentration brings about an extensive and possible cooperative depletion of the clusters of firmly-bound water molecules in the domain of the polylelectrolyte, favoring the transition to the α-helical structure. On the other hand, CD spectral patterns show that the addition of NaCl in the alcohol-rich–water mixtures of charged poly-L -lysine gives rise to a transition from the α-helical to a β-structures conversion obeys a first-order rate law at all times, with a rate constant dependent on solvent composition and ionic strength. In these conditions, the rate of the process is close to that found for the thermally induced α–β transition. Higher polymer concentration and/or ionic strength cause a phase separation of β-PLL, suggesting that in this case interchain reactions (where hydrogen bonding should play the major role) predominate. Titration experiments on charged α-helical poly-L -lysine in 85% or 90% isopropanol mixtures confirm the occurrence of a conformational transition, which takes place within a degree of dissociation α of 0.2–0.75. The transition is accompanied by a visible turbidity, which increases as the titration proceeds. Implications of the solvent distribution around the macroion on the observed conformational phenomena are also discussed.     

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.     

Determination of the relaxation time of the helix–coil transition of poly(γ-benzyl-L-glutamate) by the nmr technique

NMR measurements of poly(γ-benzyl-L -glutamate) are reported in several different strengths of magnetic field to determine the relaxation time of the helix–coil transition. Nmr spectra of various samples had line shapes varying from the double to single, depending on the extent of the polydispersity of the sample. This result indicated that the correct line shape of a polypeptide is obscured in the overlapping of multipeaks, which are due to the heterogeneity of the molecular weight in the sample. Thus, the conventional line-shape analysis could not be applied to the kinetic study of the helix–coil transition of polypeptides without consideration of this polydispersity effect on the line shape. To overcome this difficulty, we measured linewidths of nmr spectra for fairly monodisperse samples, using various nmr spectrometers, having field strengths from 60 to 220 MHz. The results were analyzed by a quadratic equation, which involves an additional term proportional to the frequency difference of two sites. The equation differs from the conventional quadratic equation, usually utilized in the case of the fast-exchange limit, only in this additional term. This modification is required to evaluate correctly the unusual broadening of the linewidth resulting from the polydispersity effect and to determine the relaxation time reflected in nmr. Nmr spectra of three samples (DP-35, 85, and 250) were measured by 220-, 100-, and 60-MHz spectrometers in trifluoroacetic acid/chloroform at 28°C and linewidths were analyzed. Relaxation times of the helix–coil transition obtained at the transition midpoint are 2.5 × 10^?4, 7 × 10^?4, and 1.1 × 10^?3 sec, for DP-35, 85, and 250, respectively.     

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.     

Reversible helix/coil transitions of left-handed Z-DNA structures. Comparison of the thermodynamic properties of poly(dG).poly(dC), poly[d(G-C)].poly[d(G-C)], and poly(dG-m5dC).poly(dG-m5dC)

In contrast to poly(dG).poly(dC), which remains in the B-DNA conformation under all experimental conditions the polynucleotides with the strictly alternating guanine/cytosine or guanine/5'-methylcytosine sequences can change from the classical right-handed B-DNA structure to the left-handed Z-DNA structure when certain experimental conditions such as ionic strength or solvent composition are fulfilled. Up to now the investigation of the helix/coil transition of left-handed DNA structures was not possible because the transition temperature exceeds 98 degrees C. By applying moderate external pressure to the surface of the aqueous polymer solution in the sample cell the boiling point of the solvent water is shifted up the temperature scale without shifting the transition temperature, so that we can measure the helix/coil transition of the polynucleotides at all experimental conditions applied. It can thus be shown that the Z-DNA/coil transition is cooperative and reversible. The Tm is 125 degrees C for poly(dG-m5dC).poly(dG-m5dC) in 2mM Mg2+, 50mM Na+, pH 7.2 and 115 degrees c for poly[d(G-C)].poly[d(G-C)] in 3.04M Na+. The transition enthalpy per base pair was determined by the help of an adiabatic scanning microcalorimeter.     

Interaction of poly(α-L-glutamic acid) and sodium poly(α-L-glutamate) with solvent components in water–2-chloroethanol mixtures

The preferential interaction of sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) with the solvent components in water/2-chloroethanol mixtures has been determined using density-increment measurements. The degree of preferential interaction was deduced from the density increments at constant molality of 2-chloroethanol and at constant chemical potential of 2-chloroethanol. Sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) are both preferentially hydrated in the whole range of solvent composition. A dehydration process occurs during the 2-chloroethanol-induced coil-to-helix transition of sodium poly(α-L -glutamate). This dehydration process was attributed to the release of some moles of water from the neighborhood of the peptide bond during the nucleation of the helix. After the conformational transition, sodium poly(α-L -glutamate) is solvated by one 2-chloroethanol molecule. The location of water and 2-chloroethanol molecules in the different parts of the residue (more polar and less polar portions) is also discussed.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

13C nmr study of the helix–coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine

The helix–coil transition of poly-N⁵-(3-hydroxypropyl)-L -glutamine in methanol–water solutions has been observed using ¹³C nuclear magnetic resonance. Two signals appear in the α-CH region in the course of the transition; this is in contrast with previous proton magnetic resonance experiments carried out on this polymer in the same solvent system.