Conformational studies on poly-L-tryptophan: circular dichroism and x-ray diffraction studies

Circular dichroism (CD) measurements were carried out on various copolymers of L -tryptophan and γ-ethyl L -glutamate in ethylene glycol monomethyl ether as the solvent. On increasing the L -tryptophan content of the copolymers a gradual change in the CD spectra was observed. The typical spectrum of the right-handedα-helix becomes more and more evident as the L -tryptophan content decreases. On the basis of these results we assumed that no conformational transition occurs on proceeding from pure poly (γ-ethyl L -glutamate) to pure poly-L -tryptophan in ethylene glycol monomethyl ether: therefore the conformation of poly-L -tryptophan should be that of a right-handed α-helix. Moreover we observed that the change in the CD spectra of the copolymers is gradual but not linear on increasing the tryptophan content. The deviations from linearity were attributed to interactions among side-chain chromophores whose contributions to the optical activity are not simply additive. An x-ray analysis carried out on oriented films of poly-L -tryptophan casted from solutions of the polymer in dimethylformamide shows conclusively that the solid-state conformation of the polymer is that, of an α-helix.     

Conformational studies of poly-beta-1-naphthylmethyl-L-aspartate.

Poly-β-1-naphthylmethyl-L -aspartate and copolymers of β-1-naphthylmethyl-L -aspartate and γ-benzyl-L -glutamate were prepared. From the results obtained by a study of infrared and circular dichroism spectra, poly-β-1-naphthylmethyl-L -aspartate was found to be a left-handed α-helix both in the solid state and in solution. The fluorescence spectra of these polymers showed excimer emission of the naphthyl chromophores and gave some information about the arrangement of the side-chain chromophores. By optical titration experiments, it was found that an increasing amount of β-1-naphthylmethyl-L -aspartate residues in the copolymers induces a progressive instability of the helical structure.     

Nuclear magnetic resonance and optical spectroscopic studies of copolymers of polypeptides. II. Random copoly(benzyl-l-glutamate:benzyl-L-aspartate) and (benzyl-D-glutamate:benzyl-L-aspartate)

High-resolution NMR is used to study two series of benzyl-L - or D -glutamate: benzyl-L -aspartate random copolymers. The helix sense of the L -aspartate residues determined from the αCH and NH chemical shifts agrees with that obtained from ORD. The stability of the helical copolymers to TFA addition shows a minimum at the composition of helix-sense inversion for the L -glutamate:L -aspartate copolymers but no minimum for the lefthanded D -glutamate: L -aspartate series. The helix-coil transition of the glutamate and aspartate residues in each polymer is compared. For the D -glutamate: L -aspartate series no differences are found, indicating random copolymerization. In the L -glutamate:L -aspartate series significant differences between the transition midpoints are interpreted as resulting from irregular distribution of component residues along the chain.     

Effect of glycyl residues on the stability of the alpha-helix

A series of polypeptides containing ordered sequences of glyeyl and γ-ethyl L -glutamyl residues has been synthesized. The properties of the polymers were investigated by x-ray diffraction, infrared spectrophotometry, and optical rotator dispersion, and the results indicate that glycine appreciably reduces the stability of the γ-ethyl L -glutamate helix.     

The random copolymerization of γ-benzyl-L-glutamate and L-valine N-carboxyanhydrides: Reactivity ratio and heteogeneity studies

The random copolymerization of the N-carboxyhydrides of γ-benzyl-L -glutamate and L -valine using triethylamine as the initiator in low dielectric media reults in high-molecular-weight copolymers at low convenrson. This behavior makes it possible to apply the monomer reactivity ration theory, which was dervied for addition polymerizations, and from the use of the copolymer composition equation, the respective monomer reactivity ratios, the average and incremental copolymer compositions, and the monomer feed ratio at any conversion can be determined. A comparison of the reactivity ratios for the copolymerization of γ-benzyl-L -glutamate NCA and L -valine NCA in benzene/methylene chloride (r_G = 2.1, r_V = 0.6) with those obtained using dioxane (r_G = 2.7, r_V = 0.3) indicates that the interchain compositional heterogeneity is greater for copolymers prepared in the dioxane. For Example, at 100% conversion of the monomeric NCAs, Poly[Glu(OBzl)⁵⁰Val⁵⁰] prepared in dioxance has an interchain composition ranging from 74 to 0 mol % γ-benzyl-L -glutamate, whereas in benzene/methylene chloride the interchain composition of γ-benzyl-L -glutamae ranges from 65 to 0 mol %. Once the reactivity ratios are obtained for any pair of α-amino and N-carboxyanhydrides, the use of the aforementioned parameters relating to interchain composition can give insight into the compositional heterogeneity between chains as a function of conversion and provide a basis for the preparation of random α-amino acid copolymers that are homogeneous.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

Occurrence of beta-chain conformation in poly-gamma-methyl glutamate membranes

Molecular chain conformations of poly-γ-methyl-L -glutamate, poly-γ-methyl-D -glutamate, and poly-γ-methyl-D ,L -glutamate in membranes prepared by using mainly trifluoroacetic acid and formic acid as solvents were investigated by infrared, X-ray diffraction, and optical rotatory dispersion measurements. It was pointed that these polymers exist in the α-helix form in membranes cast from trifluoroacetic acid solutions, but in the β-chain form in membrances swollen in formic acid. The β-chain structure was also observed in crystals precipitated from dilute solutions including formic acid. The formation of the β-chain structure was discussed.     

Conformational studies of polymers and copolymers of L-aspartate ester. II. Infrared studies and the factors involved in the formation of the omega-helix

It has already been show that the helix senses of poly(β-benzyl L -aspartate) and poly(β-methyl L -aspartate) are left-handed, while the poly esters of n-propyl, isopropyl, n-butyl, and phenethyl L -asparate are all right-handed. The effect of changes in helix sense from the left-handed to the right-handed α-helical form on the infrared spectra of copolymers of benzyl L -aspartate with ethyl, n-butyl, isopropyl, n-propyl, and phenethyl L -aspartate have been studied. Those show that for the right-handed helical form the amide band frequencies fall within the range given by Elliott,⁷ while for the left-handed form the frequencies are higher. The frequency ranges for the two helix senses are given and have been used to show that poly (β-n-propyl L -aspartate) in chloroform solution undergoes a transition from the right-handed to the left-handed helix form on heating. Polarized infrared studies of the different copolymers show that the disposition of the side chain ester groups is different for the two forms. Although methyl L -aspartate forms a left-handed α-helix similar to benzyl L -aspartate, the introduction of methyl L -aspartate residues into poly (β-benzyl L -aspartate) prevents the formation of the ω-helix. The factors involved in the formation of this helix form are discussed.     

13C-nmr chemical shift and conformation of L-alanine residues incorporated into poly(β-benzyl L-aspartate) in the solid state

¹³C-nmr spectra of poly(β-benzyl L-aspartate) containing ¹³C-enriched [3-¹³C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent ¹³C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine C_β carbon show significant ¹³C chemical shift displacement depending on conformational change, and gave the ¹³C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane.     

Syntheses of poly[γ-(l)-menthyl L- and D-glutamates] and their secondary structures

γ-(l)-Menthyl L - and D -glutamates were prepared by a fusion reaction of N-phthalyl-L - and D -glutamic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were polymerized to poly[γ-(l)-menthyl L - and D -glutamates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as ethyl ether, chloroform, tetrahydrofuran, and n-hexane. From the results obtained by a study of the infrared absorption spectra, the x-ray photographs, the optical rotatory dispersions and the circular dichroisms, poly[γ-(l)-menthyl L -glutamate] was found to be a right-handed α-helix in the solid state and in solution. Similarly, poly[γ-(l)-menthyl D -glutamate] was a left-handed α-helix. The helix-coil transition of these polymers was observed in the vicinity of 40% dichloroacetic acid in a chloroform–dichloroacetic acid mixture.     

Poly(L-lysyl-L-alanyl-alpha-L-glutamic acid). II. Conformational studies.

The solution characterization of poly(Lys-Ala-Glu) is described. This polytripeptide is zwitterionic at neutral pH and is shown to take on a conformation which is dictated by the state of ionization, molecular weight, temperature, and solvent. The polypeptide is almost entirely α-helical at low pH and temperature for polymers of greater than 25,000 molecular weight. Melting profiles for these conditions show t_m ～ 20°C. Analysis of circular dichroism curves shows the α-helical content to vary in a linear manner with molecular weight in the range 3000–30,000. At neutral pH the charged polypeptide is essentially random, but substantial α-helix could be induced by addition of methanol or trifluoroethanol. At temperatures where the sequential polypeptide is a random coil, addition of trifluoroethanol produces a polymer which is mostly α-helical but also contains an appreciable ammount of β-structure. The infrared spectrum of a low-molecular-weight fraction assumed to be cyclo(Lys-Ala-Glu)₂ was tentatively assigned a β-pleated sheet structure. A comparison of this polytripeptide in various ionization states with other polytripeptides containing L -alanine and L -glutamate or L -lysine shows the α-helix directing properties for the (uncharged) residues to lie in the order Ala > Glu > Lys.     

On the flexibility of poly(γ-benzyl L-glutamate) in helicogenic solvents

The molecular-weight dependence of the rms radius of gyration of poly(γ-benzyl L -glutamate) (PBLG) in helicogenic solvents shows negative and positive deviations from expectations for an intact and rigid α-helix in the higher and lower molecular-weight ranges, respectively. In order to study the reason for both deviations, we compare the extant experimental data of with those computed for wormlike chain, freely jointed rod, and a rigid rod having random-coil portions at both ends. The computation for the freely jointed rod and the rigid rod having frayed ends is carried out by a simulation method of Muroga. From the Zimm and Bragg theory and the above comparisons, it is concluded that both deviations can be self-consistently explained if PBLG in helicogenic solvents has an essentially intact α-helical structure with some flexibility arising from random fluctuations in hydrogen bond length. This flexibility explains the negative deviations in the high molecular weight region. The positive deviations in the low molecular weight region result from the tendency of helices to unwind at the ends. © 1998 John Wiley & Sons, Inc. Biopoly 45: 281–288, 1998     

Thermal and charge-induced coil to -helix transition of poly-L-glutamic acid and random L-glutamic acid-L-alanine copolymers

Thermal and charge induced random coil to α-helix transitions of poly-L -glutamic acid (PGA) were measured by optical rotatory dispersion in various solvents. The data of PGA in 0.1M Nacl were analyzed by the Zimm-Rice theory. Enthalpy and entropy changes for the coil-to-helix transition in the unionized state were obtained: ΔH° = ?1020 ± 100 cal/residue mole; ΔH° = ?3.0 ± 0.4 e.u./residue mole. The initiation parameter, σ, of the Zimm-Rice theory was given by a value of 5 ± 1 × 10^-3. Random copolymers of L -glutamic acid and L -alanine containing 10, 30, and 40 molar percents of alanyl residue were synthesized. Stabilities of α-helix of the copolymers were compared to that of PGA. In water and water-ethanol solutions, stabilities of the polymers were almost equal after the simple correction about the ionized charge density of the polymers. In 0.1 M NaCl solution these copolymers showed some deviations from the transition curve of PGA, which would suggest the hydrophobic contribution of the alanyl residues.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Circular dichroism measurements of benzyl L-aspartate–nitrobenzyl L-aspartate copolymers and their use in detecting and characterizing preferred polymer conformations

We have examined the nature of the circular dichroism band at 330 mμ for a series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate. The circular dichroism band arises from an electronic transition in the nitroaromatic group. In order to interpret the effect quantitatively, we employed a simplified statistical treatment and curve fitting for six copolymers. Both approaches gave consistent results, which indicates that the dichroism comes from pairwise interactions between two nitrobenzyl groups. We constructed a molecular model that meets the constraints and requirements of the analyses developed in this paper. In this model, it is proposed that the main chain forms a right-handed α-helix and that nitrobenzyl groups separated by four residues interact with each other.     

Conformational studies of alternating copoly(Leu-Lys) and copoly(Leu-Orn) in alcohol-water solvent mixtures

Conformational transitions of alternating copoly(l-leucyl-l-lysine) and copoly(l-leucyl-l-ornithine) in organic solvents and in alcohol-water mixtures were determined by c.d. measurements and the results compared with those from random copoly(Leu^48.3, Lys^51.7). As reported previously^16,17, in salt-free water these alternating copolymers undergo a conformational transition from a disordered to β-structure when the pH is raised or when various salts are added, whereas random copolymers adopt an α-helix conformation under similar conditions. However, both alternating copolymers reveal a tendency to form α-helix in 2,2,2-trifluoroethanol and in alcohol-water mixtures at neutral pH, as does the random copolymer. The alcohol concentration at which the α-helix can be induced is dependent on the kind of alcohol, the α-helix promoting power follows the the series: 2,2,2-trifluoroethanol > isopropanol > ethanol > methanol. In addition, these alternating copolymers in methanol-water mixtures below 50% (by volume) methanol form the β-structure when the pH is raised. On the other hand, above 60% methanol the fraction of α-helix already formed at neutral pH is enhanced at higher pH-values.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Interchain heterogeneity in 2-hydroxyethylglutamine-l-valine random copolymers

Solid state circular dichroism (c.d.) and infrared (i.r.) studies of water soluble and insoluble fractions of poly(hydroxyethylglutamine-valine) random copolymers, prepared from parent γ-benzyl l-glutamate valine copolymers, show that interchain conformational heterogeneity with interchain compositional heterogeneity is present when the respective N-carboxyanhydrides are copolymerized in dioxan or benzene/methylene chloride. Use of previously determined reactivity ratios for the aforementioned copolymer systems permits the determination of the variation of the average copolymer composition, $\text{f}$_G, with conversion. The experimentally determined average copolymer composition.$\text{f}$_G for the use of the respective reactivity ratios and the copolymer hydroxyethylglutamine, valine are predicted by the use of the respective reactivity ratios and the copolymer composition equation. As the valine content of the copolymer chains in the fractions increases, the expected increase in β-sheet contribution is seen. Comparison of the experimentally determined solid state c.d. spectra with Greenfield and Fasman's computer generated c.d. spectra for varying amounts of α-helix, β-sheet and random structures, shows that the water insoluble fractions with their increased valine contents have a greater contribution of β-sheet structure than the respective soluble fractions.     

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.     

Correlation between the sequence-length distribution and structure of statistical copolymers of L-valine and γ-benzyl-L-glutamate

Statistical copolymers were prepared from N-carboxyanhydrides of L -valine and γ-benzyl-L -glutamate in dioxan with triethylamine as an initiator. The copolymerization conversion was determined by ir spectroscopy, the copolymer composition by amino acid analysis, and the molecular weights by light scattering. The monomer reactivity ratios were found to be r_Val = 0.14 and r_Glu(OBzl) = 6.4. High-molecular-weight copolymers are formed even at low conversions. The content of β-structure in the copolymers was estimated from the ir spectra in copolymerization mixtures. The sequence-length distribution of L -valine and γ-benzyl-L -glutamate copolymers was calculated and its dependence on copolymerization conversion is discussed. Relations between the sequence-length distribution and the content of β-structure were studied. It was found that the content of β-structure in samples with the same composition is different for low- and high-conversion copolymers. The formation of β-structure in copolymers in the copolymerization mixture requires a certain minimal sequence length, which has been found to be about 6 valine units.