An infrared study on the conformation of poly-L-proline in concentrated aqueous salt solutions

The infrared absorption of poly-L -proline in concentrated aqueous salt solutions was measured in the fundamental region. Of primary interest were the carbonyl absorption of the peptide linkage and the methylene C–H bending absorption of the pyrrolidine ring. These spectral regions each show an additional component in the concentrated salt solutions. Using the position of the absorptions of poly-L -proline I (cis) and II (trans) as models, we conclude that both cis–trans linkages are present in the peptide in salt solutions. Increasing the temperature shifts the equilibrium slightly in favor of cis.     

Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA

The optical density–temperature profile of double-stranded poly(A + U), triple stranded poly(A + 2U), and double-stranded RNA from rice dwarf virus in solutions with and without poly-L -lysine has been examined. When poly-L -lysine is added, more than one melting temperature T_m is observed for poly(A + U) and poly(A + 2U). One of them is considered to correspond to the melting of the polynucleotide molecule free from poly-L -lysine, and another to the melting of a polynucleotide–poly-L -lysine complex. For rice dwarf virus RNA, the T_m assignable to the complex is not found to be lower than 99°C. In every case, however, the hyperchromicity observed at the T_m of the free poly-nucleotide molecule is lowered linearly as the amount of poly-L -lysine added to the solution increases. This fact is taken as indicating that there is a stoichiometric complex formed. The stoichiometric ratio lysine/nucleotide in each complex is determined by examining the relation between the amount of poly-L -lysine added to the solution and the percentage of hyperchromicity remaining at T_m of the free polynucleotide molecule. The ratio is found to be 2/3 for all of the three complexes. A discussion is given on the molecular conformations of four types of polynucleotide–polylysine complex hitherto found: (A) double-stranded DNA plus poly-L -lysine in which the lyslne/nucleotide ratio is 1, (B) three-stranded RNA [poly(A + 2U)] plus poly-L -lysine in which the ratio is 2/3, (C) double-stranded RNA [poly (A + U) or rice dwarf virus RNA] plus poly-L -lysine in which the ratio is 2/3, and (D) double-stranded RNA [poly(I + C)] plus poly-L -lysine in which the ratio is 1/2.     

Cooperative transition between two helical conformations in a linear system: Poly-L-proline I ⇌ II. I. Equilibrium studies

The solvent-induced conformational transition between the two helical forms of poly-L -proline is studied as a model for cooperative order ? order transitions. The chain length dependent equilibrium data in two solvent systems are described by Schwarz's theory, which is based upon the most general formulation of the linear Ising model with nearest neighbor interactions. The parameter σ which describes the difficulty of nucleation of a I (II) residue in an uninterrupted II (I)-helix is 10^?5 in both solvent systems. The ratios of the nucleation difficulties of states I and II at the ends of the chains β′ and β″ are very different in the two systems. Nucleation difficulty within the chain is interpreted as being due to unfavorable excess interaction energies at the I–II and II–I junctions, which add up to 7 kcal/mole of nuclei as calculated from the σ value. A similar value is computed from the atomic interactions at the junctions. In contrast to this intrinsic properly of poly-L -proline, the energies of I and II residues at the ends are heavily influenced by interactions of the endgroups with the solvent. The above values of the nucleation parameters are determined by a new least-square fitting procedure which does not necessitate the assumption of the dependence of the equilibrium constant s for propagation upon the external parameters, but yields this function from the experimental transition data. A quantitative explanation of this experimental s function through the binding of solvent is attempted. In the transition region a very small free energy change (about 0.1 kcal/mole), arising from a preferential binding of solvent molecules to one of the conformational states, is sufficient for a complete conversion from one helical form to the other.     

The circular dichroism spectrum of poly-L-acetoxyproline

We report the circular dichroism (CD) spectrum of poly-L acetoxyproline in trifluoroethanol, a solvent in which the form I to form II conformational isomerization occurs slowly enough to permit observation of the spectral changes. A comparison is made to poly-L proline. As judged by the similarity of the CD spectra, the conformations of the corresponding forms of the two polymers must be nearly the same. Transition assignments are proposed; these are shown to agree with the theoretical calculations of Pysh. There is a serious unexplained discrepancy between our solution data and those of Fasman for poly-L acetoxyproline.     

The solution behavior of poly-L-proline: II. Fluorescence behavior of labeled polyproline in water and conentrated aqueous neutral salt solytions

Fluorescence depolarization experiments performed on labaled poly-L -proline Forme II suggest the occurrence of aggrgation in water while 6M guanidinium-HCl induces dissociation. The solvent 4M CaCl₂ results in a reduction of polymer structural orgganization. These findings corroborate suggestion of polyproline aggregation and solution behavior in aqueous neutral salt solytion (see preceding article).     

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.     

Morphological evidence for the folding of polyproline II helices

Spherulites of poly-L -proline II were grown by film casting from a dilute formic acid solution. Electron diffraction indicated that the molecular axis was parallel to the surface and essentially normal to the face of the lamellae. The molecular length was five times greater than the width of the lamellae. This is considered to he substantial evidence for the molecular folding of poly-L -proline II. A correlation between the molecular conformation of poly-L -proline in solution and the observed morphological form in the solid state has been determined. As the solution mutarotates from form I to form II, the solid morphology varies from an indistinetly shaped spherulite with little inner detail to a well formed crystalline spherulite complete with lamellae.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

Synthesis of poly-L-arginine

Poly-L -arginine was prepared by the guanidization of poly-L -ornithine with 1-guanyl-3,5-dimethylpyrazole. The poly-L -ornithine was derived from poly-δ,N-trifluroacetyl-L -ornithine by removal of the blocking groups under mild basic conditions (1M piper-idine).     

Diamide model for the optical activity of collagen and polyproline I and II

A diamide, N-acetyl-L -proline-N,N-dimethylamide (AcProDMA), in water solution has optical rotatory dispersion (ORD) and circular dichroism (CD) spectra very similar to those of poly-L -proline II and the fibrous protein collagen. In contrast, AcProDMA in cyclohexane solution has optical activity resembling that of poly-L -proline I. Conformational analysis shows that AcProDMA is confined by steric constraints to either of two narrow regions of conformational space. The trans isomer of AcProDMA assumes conformations near those of polyproline II and collagen nearest neighbors, while cis-AcProDMA assumes conformations near that of polyproline I nearest neighbors. Nuclear magnetic resonance (NMR) experiments show that an equilibrium mixture of the cis and trans isomers of AcProDMA is present in solution. The trans isomer predominates in aqueous solution, but the equilibrium shifts to favor the cis isomer in nonpolar organic solvents such as cyclohexane. Analysis of the ORD spectra in terms of two basic spectra reveals a solvent dependent isomerization which parallels that observed by NMR. The optical activity of the pure isomers of AcProDMA can be derived from the ORD, CD and NMR data. A comparison of component cotton effects confirms the similarity in optical activity of trans-AcProDMA, polyproline II, and collagen on the one hand, and of cis-AcProDMA and Polyproline I on the other.     

Steric structure of L-proline oligopeptides. II. Far-ultraviolet absorption spectra and optical rotations of L-proline oligopeptides

The results of the measurement of the far-ultraviolet absorption spectra of L -proline oligomers in water and acetonitrile are summarized as follows. The monomer has an absorption maximum at 182.5 mμ in acetonitrile. The absorption maximum of the dimer is found at 185 mμ and a shoulder appears around 200 mμ, that is, splitting of the absorption spectrum is observed in the dimer. As the degree of polymerization increases, the position of the shoulder shifts toward the wavelength of the absorption maximum of poly-L -proline II, with an accompanying increase in intensity. We may describe the absorption peak around 203 mμ of poly-L -proline II as identical with the shoulder with an increased intensity. By measurements of optical rotatory dispersion and circular dichroic spectra, it was also confirmed that the appearance of the helical conformation commences at the tetramer. When the number of residues is five or greater, the conformation of the helical structure of poly-L -proline II seems to be completed.     

An X-Ray diffraction study of poly-L-ornithine hydrobromide

An X-ray study has been made of the synthetic polypeptide poly-L -ornithine hydrobromide to investigate whether, like the chemically related polypeptides poly-L -lysine and poly-L -arginine hydrochlorides, it can undergo conformational changes merely from variations in its degree of hydration. X-ray powder and fiber photographs of specimens with from half up to about three molecules of water per ornithine residue show features that suggest a “cross-β-pleated-sheet” structure. Each pleated sheet is formed from parallel chains and the sheets are piled up along the b axis. The spacings, which do not vary appreciably with hydration, can be satisfactorily indexed in terms of an orthogonal unit cell with a = 4.60 Å, b = 30.2 Å, and c = 6.64 Å. These dimensions are shown by models to be compatible with the proposed structure. Removal of the last half molecule of water results in a very diffuse pattern but on rehydration the sharp pattern reappears. Specimens containing four to nine molecules of water per residue show a quite different pattern. Reflections other than equatorial are absent in oriented diagrams except for a 5.4 Å diffuse streak across the meridian which is suggestive of an α-helical structure. Increasing the relative humidity from 86% to about 100% causes the a axis of the hexagonal unit cell to increase from 14.7 Å to 15.3 Å. On drying, the β structure reappears once again. These conformational changes are very similar to those observed in poly-L -lysine hydrochloride except that the latter shows a more stable α-helical form. This difference may be explained in terms of stabilizing hydrophobic interactions between side chains, since ornithine has a shorter side chain than lysine.     

Lysine oligopeptides. Preparation by ion-exchange chromatography

The preparation of L -lysine peptides (Lys_n, n = 2–14) from polyL -lysine is described. Fractionation by ion-exchange column chromatography of poly-L -lysine hydrolysates on a preparative scale resulted in 0.2–1.0 g quantities of individual members of the poly-L -lysine series. The peptides isolated proved to be analytically pure and the optical configuration was fully retained, as demonstrated by complete enzymic digestion. Peptides higher than n = 14 were also prepared. They consisted of oligolysine groups of narrow and accurately determined size distribution. Potentiometric titrations were used both to characterize the products and to demonstrate the characteristic dependence of the dissociation constants on size of the peptide.     

Steric structure of L-proline oligopeptides. I. Infrared absorption spectra of the oligopeptides and poly-L-proline

Poly-L -prolines I and II were differentiated by the characteristic bands in the far infrared region. Form I showed two broad bands at about 280 and 160 cm^?1 and form II two bands at, 400 and 670 cm.^?1. Furthermore, three broad bands at about 250, 200, and 100 cm.^?1 were observed in the spectrum for form II. Infrared absorption bands of the pentamer, hexamer, and octamer of tert-amyloxycarbonyl-L -proline were almost similar to those of poly-L -proline II in the 1800–75 cm.^?1 region. In the far-infrared region, especially, the absorption bands of these three oligopeptides were in good agreement with that of poly–L –proline II. Accordingly we concluded that the molecules of pentamer, hexamer, and octamer had a helical structure of a left-handed threefold screw axis. The tetrapeptide of tert-amyloxycarbonyl-L -proline might also have a left-handed helix, probably one turn, since the tetramer clearly showed an absorption band at about 400 cm.^?-1 characteristic of poly–L –proline II.     

Vibrational spectra and dispersion curves of poly-L-proline II chain

Using Wilson's GF-matrix method as modified by Higgs for infinite helical polymers, dispersion curves and the frequency distribution function have been calculated for poly-L -proline II chain. Infrared spectrum is obtained and a Urey-Bradley force field, which provides best fit with the observed frequencies, is evaluated. The result are discussed from the viewpoint of the conformational characteristics of forms I and II.     

Synthesis of poly-L-asparagine and poly-L-glutamine

Poly-β-N-diphenylmethyl-L -asparagine and poly-γ-N-diphenylmethyl-L -glutamine were prepared from the corresponding N-carboxyanhydrides. Poly-L -aspuragine and poly-L -glutamine were obtained by removal of the diphenylmethyl protecting groups with liquid anhydrous hydrofluoric acid.     

Effect of side-chain hydrophobic bonding on the stability of homopolyamino acid alpha-helices: conformational studies of poly-l-leucine in water

The conformational properties of block copolymers of poly-L -leucine in water have been examined. The degree of polymerization of the poly-L -leucine block was 11 and 21, respectively, for samples prepared by the Merrifield procedure, and 56 for a sample prepared by the polymerization of leucine N-carboxyanhydride. The optical rotatory dispersion parameter b₀ was used to obtain the helix content θ_h at various temperatures. Application of the Lifson-Roig theory gave the following parameters for the transition of a residue from a coil to a helical state: v = 0.05–0.011, ΔH = +100 cal/mole, ΔS = +0.70–1.00 e. u. These parameters, as well as those for other polyamino acids, are accounted for by hydrophobic bonds involving the nonpolar side chains in the helical and randomly coiled forms. From the data for poly-L -alanine and theoretical values of the thermodynamic parameters for hydrophobic bond formation, the parameters for formation of a polyglycine helix are computed. By separating the contributions of the backbone, it is possible to obtain a set of thermodynamic parameters for the side-chain contributions of a number of polyamino acids. Increased size of the nonpolar side chain (with a larger contribution from hydrophobic bonding) makes a larger contribution to the stability of the α-helix which is reflected, among other ways, in a higher helix content at given temperature.     

Conformational aspects of polypeptide structure. XLI. Crystal structure of S-thiazolidine-4-carboxylic acid and helical structure of poly((S)-thiazolidine-4-carboxylic acid)

The helical structures of poly[(S)-thiazolidine-4-carboxylic acid], in the cis and trans forms, were redetermined by using the new sets of bond angles and bond lengths established by X-ray diffraction analysis of L -thioproline. Calculations of the helical structures of poly-L -proline and poly[(S)-oxazolidine-4-carboxylic acid] were also repeated. As a result of these energy calculations, it is suggested that, in contrast to poly-L -proline and poly[(S)-oxazolidine-4-carboxylic acid], poly[(S)-thiazolidine-4-carboxylic acid] should not mutarotate from the trans to the cis form. This result is due to the fact that the energy barrier for the conversion is most likely too high. Previous experimental work is consistent with this finding.     

Calorimetric measurement of enthalpy change in the isothermal helix--coil transition of poly-L-lysine in aqueous solution

The heat ΔH° for converting an uncharged lysine residue from a coil to an α-helical state in poly-L -lysine in 0.1N KCl has been determined calorimetrically to be ?1200 cal/mole at both 15°C and 25°C. Essentially the same value has been obtained for the conversion of an uncharged residue from a coil to a β-pleated sheet state. 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°, the observed Calorimetric heat was corrected for the heat of breaking the sample cell, the heal of dilution of HCl, the heat of neutralization of OH^? ion, and the heat of ionization of the ε-amino group in the random coil. The latter was obtained from similar Calorimetric measurements on poly-D ,L -lysine, which was shown to be a good model for the random coil form of poly-L -lysine. The measured transition heat was ～0.7 cal., which is only 7% of the total heat liberated when a 40 ml solution of 0.25% w/v poly-L -lysine is brought, from pH 11 to pH 7; nevertheless it could be determined with a precision of ±8%. The conformation of poly-L -lysine at pH 11 appears to be completely helical at 15°C, but a mixture of 90% α-helix, 5% β form, and 5% coil at 25°C. Since ΔH° ～ 0 for the α ? β conversion, the polymer behaves like one of 95% α-helix and 5% coil in the calorimeter at 25°C. At neutral pH, poly-L -lysine is an extended coil, like poly-D ,L -lysine.     

Binding of alcohols to the peptide CO group of poly-L-proline in the I and II conformation. II. Binding constants from infrared measurements in solution

Trifluoroethanol, benzyl alcohol, and n-butanol bind to the peptide and acelyl CO groups of poly-O-acetyl-L -hydroxyproline in dichloromethane via hydrogen bonds. The binding aflinity decreases from trifhioroelhanol to n-buitanol. For the acelyl CO groups the binding does not depend on the conformation of the polymer but for the peptide CO groups the binding constants are larger by a factor of two to five time when it is in the helix II conformation (all peptide bonds trans) than when it assumes the helix I conformation (all peptide bonds cis). This preference is explained by the higher accessibility of the peptide CO groups in the II helix. The small additional energy which results from the preferential binding is sufficient, to induce a complete I → II transition because of the very high cooperativily of the system. The quantitative dependence of the equilibrium constant s for the propagation step of the transition on solvent composition (ratio of trifluoroethanol or benzyl alcohol to n-butanol) is derived from the binding data. It agrees satisfactorily with the empirical relation obtained from a best fit to transition curves of Ganseret al. The I ? II conversion of poly-L -proline is therefore an example of a conformational transition whose solvent dependence can be explained by a binding mechanism.