Conformation of poly(L-homoarginine)

Poly(L -arginine) assumes the α-helix in the presence of the tetrahedral-type anions or some polyanions by forming the “ringed-structure bridge” between guanidinium groups and anions which is stabilized by a pair of hydrogen bonds and electrostatic interaction [Ichimura, S., Mita, K. & Zama, M. (1978) Biopolymers 17 , 2769–2782; Mita, K., Ichimura, S. & Zama, M. (1978) Biopolymers 17 , 2783–2798]. This paper describes the parallel CD studies on the conformational effects on poly (L -homoarginine) of various mono-, di-, polyvalent anions and some polyanions, as well as alcohol and sodium dodecylsulfate. The random coil to α-helix transition of poly(L -homoarginine) occurred only in NaClO₄ solution or in the presence of high content of ethanol or methanol. The divalent and polyvalent anions of the tetrahedral type (SO, HPO, and P₂O), which are strong α-helix-forming agents for poly(L -arginine), failed to induce the α-helical conformation of poly(L -homoarginine). By complexing with poly(L -glutamic acid) or with polyacrylate, which is also a strong α-helix-forming agent for poly(L -arginine), poly(L -homoarginine) only partially formed the α-helical conformation. Monovalent anions (OH^?, Cl^?, F^?, and H₂PO) did not change poly(L -homoarginine) to the α-helix, and in the range of pH 2–11, the polypeptide remained in an unordered conformation. In sodium dodecylsulfate, poly(L -homoarginine) exhibited the remarkably enlarged CD spectrum of an extended conformation, while poly(L -arginine) forms the α-helix by interacting with the agent. Thus poly(L -homoarginine), compared with poly(L -arginine), has a much lower ability to form the α-helical conformation by interacting with anions. The stronger hydrophobicity of homoarginine residue in comparison with the arginine residue would provide unfavorable conditions to maintain the α-helical conformation.     

Disorder–order transitions induced in anionic homopolypeptides by cationic detergents

CD spectra have been obtained for poly(L -glutamic acid) and poly(L -aspartic acid) as functions of temperature and concentration of cationic detergents. Dodecylammonium chloride induces a coil–helix transition in fully ionized poly(L -glutamic acid). The interaction of the monomeric detergent with the polypeptide is responsible for the conformational transition. The detergent concentration required to produce the transition is independent of temperature. The CD of fully ionized poly(L -aspartic acid) is nearly unaffected by dodecylammonium chloride, in marked contrast to the situation found with poly(L -glutamic acid). However, these results do not imply that dodecylammonium chloride interacts differently with aspartyl and glutamyl residues. The observed results can be accounted for by the well-known fact that the glutamyl residue has a higher helix-forming tendency that the aspartyl residue. Cetyltrimethylammonium chloride destabilizes the helical form of poly(L -glutamic acid). This detergent presents an exception to the usual ability of ionic detergents to promote formation of ordered structures in oppositely charged homopolypeptides.     

Optical Rotatory Dispersion of Polypeptides in the Near-Infrared Region

For the first time ORD measurements in the near-infrared region from 0.7 to 2.0 μ for well-known polypeptides, namely, poly(γ-benzyl L -glutamate), poly(L -glutamic acid), poly-L -lysine·HCl, poly-S-carbobenzoxymethyl-L -cysteine, and Bombyx mori silk fibroin, were carried out. It was found that the value of the optical activity infrared term, which is proportional to the sum of rotational strengths of vibrational transitions, depends on polypeptide conformation. The optical activity infrared term value is equal to zero for the random-coil conformation, it is small but exceeds the measurement error for the α-helical state, and finally, for the β conformation it is an order of magnitude higher than for the α-helical state. The obtained results permit one to hope that on the basis of ORD measurement in the near-infrared region it will be possible to suggest a method of determining the β-form content in polypeptides and proteins     

Rigidity of α-helical polypeptides. A new method of obtaining the persistence length from viscosimetric data

The hydrodynamic properties of α-helical poly(L -glutamic acid), (Glu)_n in aqueous solutions and in mixtures of water with organic solvents have been interpreted in terms of the persistence length of the macromolecule. A modification of the method of Vitovskaya and Tsvetkov has been proposed in order to allow a more accurate determination of this parameter. The addition of an organic solvent increases strongly the rigidity of the helical conformation of (Glu)_n. A comparison is made with some data of the literature of poly[N⁵-(3-hydroxy propyl)L -glutamine], [Gln(CH₂)₃OH]_n, and poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n.     

Vibrational analysis of peptides, polypeptides, and proteins. XXXII. alpha-Poly(L-glutamic acid)

The Raman and ir spectra of α-helical poly(L -glutamic acid) have been assigned on the basis of a normal mode calculation for this structure. The force field was based on our previously refined main-chain force constants for α-poly(L -alanine) and side-chain force constants for β-calcium–poly(L -glutamate). Despite the identical backbone α-helical structures, significantly different frequencies are calculated, and observed, in the amide III and backbone stretch regions of α-poly(L -glutamic acid), as compared with α-poly(L -alanine). This clearly demonstrates the influence of side-chain structure on mainchain vibrational modes.     

Induced CD and interaction of some porphyrin derivatives with α-helical polypeptides in aqueous solutions

Absorption spectra and induced CD have been measured on aqueous solutions of water-soluble porphyrins with α-helical poly(L -glutamic acid) or α-helical poly (L -lysine) at different mixing ratios. For the former, porphyrin is porphine-meso-tetra (4-N-methylpyridinium) (TMpyP), and for the latter, it is porphine-meso-tetra (4-benzenesulfonate) (TPPS) or porphine-meso-tetra(4-benzoate) (TPPC). All the solutions of porphyrin-polypeptide systems show hypochromism in the Soret band and induced CD in the Soret region. The CD is characterized by a positive band at a shorter wavelength and a stronger negative band at a longer wavelength. The hypochromicity and the magnitude of molar ellipticities are much larger for the TPPS– and TPPC–poly (L -lysine) systems than for the TMpyP–poly (L -glutamic acid) system. Porphyrin ions bind to the α-helix electrostatically, and the two components of the Soret transition of porphyrin are subject to dissymmetric perturbation. TMpyP ions bind to the α-helix at isolated sites, while TPPS ions and TPPC ions are in pairs on the α-helix, that is, two ions bind consecutively and dissymmetrically. In the TMpyP–poly (L -glutamic acid) system a single CD band is associated with each of the two components of the Soret transition, and these are of opposite sign. In the TPPS– and TPPC–poly (L -lysine) systems, a pair of positive and negative CD bands is associated with each of the two components, thus giving apparently a single pair of CD bands with a shoulder, owing to partial overlapping.     

Kinetic study of cooperative binding reaction of toluidine blue to poly (alpha,L-glutamic acid) by means of the electric-field pulse method.

Interaction of toluidine blue with helical and randomly coiled poly(α,L -glutamic acid) was studied with absorption spectra, titration, and electric-field pulse measurements. The obtained values of various parameters for the helical form of poly(α,L -glutamic acid)differed from those for its coiled form. The difference of parameters in these two forms of poly(α,L -glutamic acid) was attributed to differences of the binding mechanism in both forms. Furthermore, the binding of toluidine blue to poly(α,L -glutamic acid) was considered to be due to hydrogen binding in the helical conformation and ionic interaction in the coiled conformation of the polymer.     

Differentiation between the α-helical and coillike conformations of poly(L-glutamic acid) in aqueous solution through binding of pinacyanol

Pinacyanol in the presence of an excess of poly(L -glutamic acid) [polymer/dye ratio (P/D) > 10] exhibits different absorption spectra in the visible region when bound to the slightly charged polypeptide in the α-helical conformation or to the nearly completely dissociated polypeptide in the coillike conformation. These spectra reveal aggregation of the dye bound to the macromolecular chain in both conformations, although in the coillike one different kinds of aggregates may be present. Dye binding is accompanied by the appearance of CD bands in the visible region which are also different for the α-helical and the coillike conformations. The aggregates formed in the presence of the latter change slowly in time and seem to induce some conformational changes in the polypeptide chain. Furthermore, they have been found to be, at least partially, stable with respect to a subsequent reversal to the α-helical conformation. All results could be qualitatively interpreted assuming that in the coillike conformation, ordered regions exist along the chain as proposed by Krimm and Tiffany.     

Potentiometric titration of poly(L-glutamic acid) in aqueous solutions and binding of divalent cations

The potentiometric titration of poly(L -glutamic acid) was performed under conditions of varied ionic strength and concentration of added divalent cations. From these titration curves, the amount of divalent cations, especially magnesium, bound to poly(L -glutamic acid) was determined using a new method of analysis based on polyelectrolyte theory. By comparison with the polyelectrolyte, poly(acrylic acid), it was found that there are no specific interactions between metal ion and poly(L -glutamic acid) in either the helical or random coil conformation. The effect of these divalent cations on the conformation of poly(L -glutamic acid) was also discussed.     

Microcalorimetric studies of solvent-induced conformational change of sodium and cesium salts of poly(L-glutamic acid) in aqueous media

Organic solvent-induced coil → helix conformational change of poly(sodium) L -glutamate (NaPLG) and poly(cesium L -glutamate) (CsPLG) in solution in aqueous mixed solvents have been studied at 25°C. Heats of dilution of NaPLG in the water–dioxane pair have been measured as a function of polymer concentration and solvent composition. The results indicate that the overall chain conformation in the disordered form is not too different from that in the α-helical form. Heat capacity measurements by flow microcalorimetry have also been done. The apparent monomolar heat capacity at constant pressure of the polymer, C_{p, ?}, decreases with dilution similarly to other strong polyelectrolytes in aqueous media. In the water–dioxane pair, C_{p, ?} increases with the dioxane content due to partial desolvation of ionic species resulting from increasing ionic association. In the case of the water-2-chloroethanol (CE) pair, the transition takes place at low CE content and results show a fast decrease in C_{p, ?} when the α-helical conformation predominates. It is believed carboxylate groups and CE molecules associate themselves into a complex formation responsible for the transition. The size of the cation plays a significant role in the thermodynamic properties of these polyelectrolytes in solution since sodium ions are more strongly bound to the chain than cesium ions.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: Chondroitin 4-sulfate and dermatan sulfate

Circular dichroism spectroscopy has been used to study the interactions of both dermatan sulfate and chondroitin 4-sulfate with the cationic polypeptides; poly(L -arginine), poly(L -lysine), and poly(L -ornithine). The results indicate that the mucopolysaccharides have a conformation directing effect on both poly(L -arginine) and poly-(L -lysine) such that these polypeptides adopt the α-helical conformation. The extent of interaction in each polypeptide-polysaccharide system can be judged by the degree of induced helicity and the “melting temperature” at which the interaction is disrupted On comparison of these results with those previously obtained for chondroitin 6-sulfate-polypeptide mixtures, the extent of interaction can be seen to depend on the length of the amino acid side chain and the positions of the anionic groups on the mucopolysaccharide chain. Such considerations place the three mucopolysaccharides in order of increasing interaction: chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate. These results are correlated with observations that dermatan sulfate is bound more tightly to collagen in connective tissues than are the other two polysaccharides.     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

The effect of methylmercury (II) binding on the conformation of poly(L-glutamic acid) and poly(L-lysine: a Raman spectroscopic study

The binding of the methylmercury cation CH₃Hg⁺ by poly(L -glutamic acid) (PGA) and by poly(L -lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side-chain COO^? and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm^?1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α-helical and β-sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α-helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH₃Hg⁺ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.     

Azobenzene-Containing polypeptides: Photoregulation of conformation in solution

Photochromic polypeptides, with 16 to 56% azobenzene groups in the side chains, have been prepared by reaction of poly(L -glutamic acid) with p-aminozaobenzene, both in the presence of dicyclohexyl carbodiimide/N-hydroxybenzotriazole and of pivaloyl chloride. Analogous modification reactions carried out on poly(L -aspartic acid) were unsuccessful owing to the formation of N-succinimide rings. In trimethylphosphate, all the azopolypeptides exhibit the α-helix CD pattern. Irradiation produces the trans-to-cis isomerization of the azo side chains, but does not induce any variations of the backbone conformation. In water, the CD spectra indicate the presence of appreciable amounts of α helix in 16 and 21% mol azo-containing poly-(L glutamates), while a β structure is present in a 36% mol azopolypeptide. Light produces conformational changes of the polypeptide conformation which are completely reversed in the dark. The extent and kind of photobehavior depend on the azo content and the pH value at which irradiation is carried out. The light-induced effects are discussed on the basis of the pH-induced order-disorder conformational transitions. In fact, the pK values and the transition curves of the dark-adapted samples were found to be different from those of the irradiated ones.     

Conformational studies on Cu(II) polycomplexes of pyridoxal Schiff bases of polypeptides

Structures of Cu(II) complexes of pyridoxal Schiff bases with poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were investigated by absorption spectra, CD, and conformational analysis. Although the polypeptides retain their typical right-handed α-helical conformation, opposite Cotton effects were found for the poly(L -lysine) and poly(L -ornithine) polycomplexes in the whole range of wavelengths from 600 to 250 nm. As in the analogous derivatives of salicyladehyde, this effect seems to be due to a stereospecific binding of the square planar Cu(II)-bis-pyridoxylideneimine group to the α-helical matrix. Circular dichroism spectrum of poly(L -α,γ-diaminobutyric acid) polycomplex is similar to that found for poly(L -lysine) derivative, but indicates large tetrahedral distortion of the square-planar coordination of copper ion.     

Proton magnetic resonance and optical spectroscopic studies of watr-soluble polypeptides: poly-L-lysine HBr, poly(L-glutamic acid), and copoly(L-glutamic acid42, L-lysine HBr28, L-alanine)

The helix-coil transition has been studied by high-resolution NMR for three water-soluble polypeptides. Such systems are better models for protein behavior than those in TFA-CDCl₃ solvent. An upfield shift of ～7 cps is observed for the α-CH peak of poly(L -glutamic acid) and poly-L -lysine as the helix content increases over the transition. No such shift is found for copoly(L -glutamic acid⁴², L -lysine²⁸, L -alanine³⁰). The width of the α-CH peak for poly L-lysine increases rapidly as helix content rises but for poly L -glutamic acid and the copolymer, the width of this peak remains unchanged up to 60% helicity. This demonstrates a rapid rate of interconversion between helical and random conformations in partly helical polymer for the latter two polypeptides. All three polymers however, show no apparent α-CH peak at 100% helicity. Side-chain resonance lines also broaden as helix content increases and, to a greater extent, the closer the proton is to the main chain.     

Dielectric studies of aqueous solutions of poly(L-glutamic acid)

The dielectric features of poly(L -glutamic acid) are studied by the Fourier synthesized pseudorandom noise method in a time domain combined with a four-electrode cell. Polymer concentration dependence, the effect of the solvent viscosity, salt effects, and pH dependence are studied concomitantly with measurements of CD. A helix-to-coil transition occurs near pH 5.6 for a salt-free solution; at higher pH values, the polymer has an ionized random-coil conformation, and at lower pH, it has a deionized α-helical conformation. When it is in the ionized random-coil conformation, with the usual features of an electrolytic polymer, the solution shows a relaxation spectrum with a large dielectric increment at low frequencies. In the deionized α-helical state, no distinct relaxation curves are obtained, which does not deny the existence of a permanent peptide dipole. The pH dependence of the dielectric increment does not mainly correspond to the conformational change from helix to coil, but rather corresponds to the change of chain expansion on account of a charge–charge interaction under low ionic strength, which is conceived of by a viscosity measurement.     

Conformational study of poly(α-L-aspartic acid)

The conformation of poly(α-L -aspartic acid) was investigated on a sample in which β-bonds were not detected. CD and ir spectroscopy showed that poly(α-L -aspartic acid) passes through a conformational change induced by changes of the degree of ionization that is accompanied by precipitation; the precipitate is probably highly helical. The change was also detected by potentiometric titration.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: hyaluronic acid, heparitin sulfate, and keratan sulfate

Circular dichroism spectroscopy has been used to study the interactions of hyaluronic acid, heparitin sulfate, and keratan sulfate with cationic polypeptides. The results indicate that the presence of these mucopolysaccharides has an effect in the conformation of poly(L -lysine) and poly(L -arginine), such that the former adopts the “random” form and the latter takes up the α-helical conformation, rather than the “charged coil” form expected at neutral pH. The relative strengths of the interactions can be judged from the melting temperatures above which they are disrupted. Both the stoichiometry and the strength of the interactions depend on the position, number, and type of anionic groups attached to the polysaccharide backbone. Such considerations place the six common mucopolysaccharides in order of increasing strength of interaction: hyaluronic acid < chondroitin 4-sulfate < heparitin sulfate < chondroitin 6-sulfate < keratan sulfate ? dermatan sulfate. These differences should be paralleled by differences in the interaction of the mucopolysaccharides with collagen and fibrous proteins.     

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.