Nuclear magnetic resonance investigation of the helix in random coil transformation in poly-alpha-amino acids. I

High-resolution nuclear magnetic resonance spectra at 100 MHz and 220 MHz have been obtained on two samples of poly-L -alanine of differing molecular weights (2500 and 42 500) in the chloroform–trifluoroacetic acid system under various conditions of solvent composition, temperature, and polypeptide concentration. Separate helix and random coil peaks are observed for the α-CH and peptide NH backbone proton resonances, thereby permitting the determination of helix content. This observation of separate peaks demonstrates that the lifetimes of the helix and random coil portions of poly-L -alanine have lower limits of about 10^?1 sec. It is suggested that solvent–peptide versus peptide–peptide hydrogen bond competition, coupled with a destabilizing effect of the trifluoroacetic acid on the helix, is responsible for the helix–random coil transformation.     

Optical and other properties of a hydrocarbon-soluble polypeptide, poly-gamma-(N-dodecyl)-L-glutamate

The polypeptide poly-γ-(n-dodecyl)-L -glutamate (PDLG) is soluble in hydrocarbon solvents such as hexane, cyclohexane, and dodecane. The CD spectra of PLDG in these solvents are reported here. These spectra are typical α-helix spectra and show none of the wavelength shifts and magnitude changes displayed by partially helical proteins in membrane preparations. This observation rules out the possibility that the membrane protein CD spectra result from solvent effects. The PDLG helix is stable in dodecane up to at least 70 °C. However, it is easily disrupted by trifluoroacetic acid, with the helix–coil transition centered at 3% TFA in hexane. Viscosity measurements of PDLG in dry dichloroacetic acid exhibit polyelectrolyte effects which can be suppressed by addition of several percent water.     

Helix-coil transition in heterogeneous DNA.

The broadening of a helix–coil transition due to base pair heterogeneity is calculated on the basis of a cumulant perturbation expansion in the quasi-grand ensemble. In this ensemble the fictitious, homogeneous chain, to which the perturbation is referred, automatically decreases its correlation length as the heterogeneity increases. This “renormalization” seems to stabilize the perturbation expansion, in view of the good agreement between the present results and the exact theory of a heterogeneous polypeptide helix–coil transition. For the DNA model in which ring entropy is included, the transitions is found to be extremely narrow for an infinite random chain with conventional parameters. A tentative reconciliation of this result with contradictory calculations of some other workers is offered on the basis of end effects, coarse graining, or approximation to the ring entropy. An application of the new method to DNA with a non-random base pair distribution requires evaluation of the correlation function between molecular states (helix or coil), at different sites of the reference chain. The evaluation is reduced to quadrature, but numerical calculations have been made only for the random chain.     

Carbon-13 and proton NMR studies of helix-coil transition of poly(gamma-benzyl-L-glutamate).

The helix–coil conformational transition undergone by poly(γ-benzyl-L -glutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solvent-induced helix–coil transition, the side chain assumes a helical conformation before the backbone. In the thermally induced helix–coil transition, the results indicate the existence of an intermediate state, which is between the α-helix and random coil and is free from intramolecular hydrogen bonding.     

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.     

Comparison of several calculations of helix-coil transitions in heterogeneous polymers

Random number methods are used to calculate helix—coil transition curves for the model of a heterogeneous polypeptide of random sequence. These curves are compared with several other calculations. The random number computations confirm the exact calculation of Lehman; among the several approximate calculations examined only that of Fixman and Zeroka agrees closely with results of the random number method over the whole range of conditions considered. Calculations are also reported of the average length of helix and coil sections in a heterogeneous molecule of random sequence which is undergoing the helix-coil transition.     

Study by circular dichroism and optical rotatory dispersion of polypeptides with aromatic side-chain chromophores

Poly(ortho-, meta-, and para-γ-nitrobenzyl-L -glutamates) were studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in two helicogenic solvents, hexafluoroisopropanol (HFIP) and dichloroethane (EDC), and two non-helicogenic solvents, dichloracetic acid (DCA) and trifluoroacetic acid (TFA). The corresponding glutamates were also studied in DCA and TFA. The symmetric nitrobenzylic chromophore is optically active when the polymers are in solution in DCA and TFA. The corresponding glutamates are also optically active under the same conditions. Thus, it was not possible to explain the origin of the optical activity of the side-chain chromophore when the polymer is in solution in a helicogenic solvent. Nevertheless, from a side-chain dichroic band, a helix–coil transition curve was determined and the stability of each poly(γ-nitrobenzyl-L -glutamate) given; this stability depends on the position of the nitro substituent on the aromatic ring.     

Helix–coil transitions in a simple polypeptide model

A simplified model of a polypeptide chain is described. Each residue is represented by a single interaction center. The energy of the chain and the force acting on each residue are given as a function of the residue coordinates. Terms to approximate the effect of solvent and the stabilization energy of helix formation are included. The model is used to study equilibrium and dynamical aspects of the helix–coil transition. The equilibrium properties examined include helix–coil equilibrium constants and their dependence on chain position. Dynamical properties are examined by a stochastic simulation of the Brownian motion of the chain in its solvent surroundings. Correlations in the motions of the residues are found to have an important influence on the helix–coil transition rates.     

Simulation of α-helix–coil transitions in simplified polyvaline: Equilibrium properties and brownian dynamics

A quantitative understanding of helix–coil dynamics will help explain their role in protein folding and in folded proteins. As a contribution to the understanding, the equilibrium and dynamical aspects of the helix–coil transition in polyvaline have been studied by computer simulation using a simplified model of the polypeptide chain. Each amino acid residue is treated as a single quasiparticle in an effective potential that approximates the potential of mean force in solution. The equilibrium properties examined include the helix–coil transition and its dependence on chain position and well depth at the coil–helix interface. A stochastic simulation of the Brownian motion of the chain in its solvent surroundings has been used to investigate dynamical properties. Time histories of the dihedral angles have been used to study the behavior of the helical structure. Auto and cross-correlation functions have been calculated from the time histories and from the state (helix or coil) functions of the residues with relaxation times of tens to hundreds of picoseconds. Helix–coil rate constants of tens of ns^?1 were found for both directions of the transition. © 1993 John Wiley & Sons, Inc.     

Helix–coil transition of poly(α,L-glutamic acid) at an interface: Correlation with static and dynamic membrane properties

When adsorbed from an aqueous dilute solution at high pH into the pores of an inert cellulose acetate filter, poly(α,L -glutamic acid) remains strongly anchored to the pore walls. The existence of the helix–coil transition for the adsorbed polypeptide in a certain pH range is evidenced by static and dynamic membrane properties displayed by the “activated” filter, such as excess cation uptake, membrane potential, and hyraulic permeability. In particular, the variations of the hydrodynamic thickeness present a sigmoidal shape characteristic of the helix–coil transition at the interface, a transition apparently less sharp than in solution.     

Degradation of N5-(2-hydroxyethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain

The rate of degradation of poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG), poly(L-glutamic acid) (PGA) and poly[HEG-co-GA] random copolymers by papain was measured in the pH range 4.0-7.5, employing the gel permeation chromatography method. The effect of the degree of ionization on the polymer conformation was measured by circular dichroism (c.d.). PHEG, which is uncharged, had a random coil conformation and an almost constant degradation rate within the whole pH interval. The ionization of PGA increased with increasing pH and was accompanied by conformational transition from helix to random coil. The hydrolysis of PGA by papain depended on pH with the optimum at about pH 5, indicating that both the high content of helix (at pH less than 5) and increasing charge density (at pH greater than 5), decreased the degradation rate. Contrary to PGA, pH profiles of the degradation rate of poly[HEG-co-GA] copolymers are monotonous and do not decrease at pH less than 5. In the copolymers the HEG residues act as a helix breaker and limit the formation of helical conformation. The role of structural features of a macromolecular substrate, i.e. the charge, helical conformation and the nature of amino acid residues, in the interaction between enzyme and polymer is discussed.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been 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.     

Solvent effects on the helix–coil transition in polypeptides

A simple way to incorporate the solvent–peptide interaction in any available theory of the helix–coil transition is developed. The competition between the intramolecular hydrogen bonding and the solvent–polymer hydrogen bonding is considered in multi-component solvents where some of the components have hydrogen-bonding capacity. Molecular averages are computed by using the theory of Lifson and Roig. The experimental data of Yang are analyzed, and the range of acceptable values of the equilibrium constants of hydrogen bond formation is deduced. The enthalpy of the transition in multicomponent solvents is calculated.     

The effect of polydispersity on the nuclear magnetic resonance spectra of poly-gamma-benzyl-L-glutamate

The effect of poly dispersity on the nuclear magnetic resonance spectra of samples of poly-γ-benzyl-L -glutamateein the helix–random coil transition is studied. In the transitionregion the α-CH proton resonance shows two peaks whose behavior does not change appreciably upon fractionation by gel permeation chromatography. Theoretical spectra were computed with both a polydispersity model of the transition and a model for slow nucleationof helix from completely random coil molecules. The results suggest that the double peak behavior in the nmr spectra results from a slow rate of helix nucleation rather than polydispersity.     

Adsorption of polypeptides on the solid surface. II. Effect of secondary chain structure and helix–coil transition

The theory of adsorption of semistiff chains on a planar surface developed by the authors previously has been used to consider the helix–coil transition in single-stranded macromolecule interacting with an adsorbent plane. The cases of nonselective interaction when the adsorption energy is independent of the unit conformation (a) and selective interaction with only helical (b) or coiled (c) sequences active in adsorption were investigated. In case (b) the existence of secondary structure favors chain bonding to the surface. This leads to the increase in the stability of the helical state and complete polypeptide chain spiralization. The profile of the conformational helix–coil transition acquires an asymmetrical shape inherent to the second-order phase transition. In case (c) the bonding of a partially helical chain to the surface is similar to the adsorption of Gaussian coils and is accompanied by the destruction of secondary structure, this destruction being appreciable even if the helical state in space was favorable.     

Influence of magnesium ions on helix-coil transition of DNA determined by modified differential scanning calorimeter

The thermal effect of magnesium ions on the helix–coil transition of DNA was studied calorimetrically by a modified differential scanning calorimeter (DSC). It was found that the transition temperature of DNA depends on both the DNA and magnesium ion concentrations. The dependence of the helix–coil transition of DNA on the mole ratio of magnesium ions to DNA(P) can be classified into two groups. When this mole ratio is less than 1, magnesium ions tend to stabilize the double-helix DNA, so that the transition temperature increases linearly and the heat of transition increases significantly with increasing mole ratio. When the mole ratio is more than 1, magnesium ions tend to destabilize the double-helix DNA, so that DNA precipitates when the temperature is raised above the transition temperature. In this case, both the transition temperature and the heat of transition decrease with increasing mole ratio.     

The strand-separation transition of T2 bacteriophage DNA

Strand separation of T2 DNA has been investigated in a helix-destabilizing solvent. Temperature-shift experiments in which the conformation of the DNA is monitored by its viscosity, sedimentation behavior, and kinetics of helix formation show that a well-defined strand-separation transition follows the helix–coil transition usually observed by changes in absorbance. For T2 DNA, this strand-separation transition is 70% as broad as the helix–coil transition, and is characterized by extremely slow kinetics of conformational change in the population. Strand separation requires the expansion of the two-stranded coil observed at the end of the helix–coil transition. This expansion is apparently coupled with the disurption of the last remaining base pairs in the molecule. The expansion process increases the viscosity, and can be readily followed as a function of time and/or temperature. Subsequent separation of the expanded form into complementary strands results in a viscosity decrease, the net result of a reduction in hydrodynamic volume and the halving of the molecular weight. Only under conditions where the driving force for strand separation is large are these events at all synchronous in the population. When the kinetics of conformational change are complete in the strand-separation transition, a mixture of expanded forms and separate strands is observed; the breadth of the transition reflects differences in stability with respect to strand separation among the molecules in the population. The transition exhibits hysteresis and is not a reversible equilibrium between double-stranded and single-stranded forms. It appears that renucleation is kinetically forbidden within the strand-separation region.     

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.     

Study of helix-coil transition of DNA by dielectric constant measurement

The thermal helix–coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix–coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix–coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix–coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.