Local conformations of ends of α-helical poly[Glu(OBzl)] studied by circular dichroism of terminal chromophores

Conformations of terminal peptide units of α-helical poly(γ-benzyl-L -glutamate), poly-[Glu(OBzl)], were examined by an induced circular dichroism (CD) of chromophores which were covalently attached to both ends of the chain. In chloroform, where the helical poly-[Glu(OBzl)] exists as a head-to-tail-type dimeric associate, the chromophores showed a strong CD induced by an asymmetric perturbation from the helical structure. The induced CD almost disappeared by an addition of a few percent of dichloroacetic acid, which has been reported as a powerful breaker of the associate. These results are explained by an incorporation of terminal peptide units into a helical conformation in the head-to-tail associate and a local unfolding of the terminal portions by the addition of acid. An induced CD of a charge-transfer complex between the two terminal chromophores was also observed and the structure of the helix–helix junction of the head-to-tail dimer is discussed.     

Reversing-pulse electric birefringence of poly (γ-benzyl-L-glutamate). II. Field-strength dependence of steady-state and decay signals of well-fractionated samples

The electric field dependence (up to 21 kV/cm) of the steady-state and decay signals has been examined on the four well-fractionated samples of poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n, in N,N-dimethylformamide at 535 nm and 20°C. Together with the data previously obtained from the reversing-pulse electric birefringence [Ueda, K., Nomura, M. & Yamaoka, K. (1983) Biopolymers 22 , 2077–2090], the steady-state birefringence and field-free relaxation time were analyzed by a method that takes into account the polydispersity of the chain length. The weight-average chain length, (l_w), permanent dipole moment, (μ_w), electric polarizability anisotropy, (Δα_w), and the length-independent optical anisotropy factor were evaluated. The axial translation per residue was calculated for the [Glu(OBzl)]_n helix, but the uncertainly involved in the weight-average molecular weights, determined from light scattering by different investigators, makes the determination of the exact conformation of [Glu(OBzl)]_n difficult. The contribution of Δα_w to electric field orientation was found to be significant, since Δα_w was approximately proportional to l_w. A linear relationship also exists between μ_w and l_w, when the [Glu(OBzl)]_n helix is shorter than about 1200 Å.     

Conformations of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,and its critical concentration of β-form formation in solution

Nps-[Glu(OBzl)]₆-NHEt has been prepared by coupling Nps-[Glu(OBzl)]₂-OH with HCl,H-[Glu(OBzl)]₄-NHEt by means of dicyclohexylcarbodiimide. The ir spectra of its nujol mull show that the hexapeptide has the β-structure of antiparallel chains. When it is dissolved in dioxane or ethylene dichloride, the hexapeptide consists of a mixture of the β-form and the solvated σ-form, but the β-form can exist only above a certain critical concentration. The critical concentration is about 0.4g dl^?1 in dioxane and 0.08g dl^?1 in ethylene dichloride, and the content of β-form increases with increasing concentration above it. The CD of the dioxane and ethylene dichloride solutions shows concentration dependence in visible and uv regions.     

Monte Carlo simulation on the intrachain end-to-end charge-transfer interaction on oligosarcosines

Monte Carlo conformational calculations for oligosarcosines having terminal electron-donor (D) and -acceptor (A) chromophores [A-(Sar)_n-D, n = 2–8] were carried out. The bulkiness of the terminal chromophores, the conformational energies, and the charge-transfer (CT) interaction energy were taken into account in the calculation. The equilibrium constant for the intramolecular end-to-end CT interaction was evaluated, and the result was compared with the experimental data. The alternating chain-length dependence of the equilibrium constant observed experimentally was reproduced by the simulation and the solvent dependence of the equilibrium constant was also reasonably explained.     

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.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Preferred conformations of protected homo-oligo-L-glutamate peptides in CDCl3 and CDCl3/TFA mixtures

A conformational analysis of protected glutamate homo-oligopeptides Z-[Glu(OEt)]_n-OEt (n = 2–7) was carried out in chloroform solution using high-resolution ¹H-nmr spectroscopy. At dilute peptide concentrations, the backbone NH and α-CH resonances are well resolved and can be assigned by combining extensive homonuclear decoupling experiments with data for co-oligopeptide derivatives. The structure of these peptides in solution was then assessed using information from chemical shifts, coupling constants, temperature coefficients, and titration of each oligomer with trifluoroacetic acid (TFA). The di- and tripeptides are found to be in disordered forms in deuterochloroform (CDCl₃) and CDCl₃/TFA mixtures. The tetrapeptide exhibits a folded structure with intramolecular hydrogen bonding at Glu² in CDCl₃ and undergoes a transition to increasingly disordered forms as TFA is added. The pentamer to heptamer show a folded structure with a strong intramolecular hydrogen bond at Glu² and a weaker hydrogen bond at Glu³, which are disrupted as these peptides go to random coils at high TFA/CDCl₃ ratios. In addition, the N-terminal portions of these glutamate peptides appear to be involved in side chain–main chain interactions. The results support the hypothesis that protected linear homo-oligopeptides may possess two or more segments of conformation with intramolecular folding preferred near the N-terminal portion.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo[Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)2, and cyclo(Glu-Pro)2, were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)-Pro]2 and cyclo(Glu-Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). I. Transient behavior of fractionated samples in the low electric field region

Reversing-pulse electric birefringence (RPEB) was measured for the first time for four fractionated poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n, samples in N,N-dimethylformamide (DMF) at 20°C and at 535 nm. The RPEB signal showed a deep minimum for each sample on reversal of an applied electric field. The profiles of the reverse-transient signal were analyzed by taking into account the polydispersity for the continuous distribution of molecular lengths. The best set of three quantities (l_w, l_w/l_n, (β_w)²/2_γw), which determine a signal profile, was evaluated for each sample. By combining the experimental data of intrinsic viscosity and RPEB, the diameter of a cylinder, which is assumed for the [Glu(OBzl)]_n helix, was found to be 17 Å. The value (β_w)²/2_γw, which is related to the ratio of weight-average permanent dipole moment μ_w, and electric polarizability anisotropy Δα_w, was found to be in the range of 20–45. This indicates that the former contributes predominantly to electric field orientation, but the latter also should not be ignored. With the three parameters from the reverse portion, the rise and decay curves were regenerated theoretically in excellent agreement with experimental signals.     

Conformational characteristics of polyglutamic acid esters: Persistence of orientational correlation along the side chain flanking the α-helical backbone

The side chain conformations of α-helical poly(L -glutamic acid) esters $ \rlap{--}[NHCH(CH_2 CH_2 COOR)CO\rlap{--}]_x $, carrying a homologous series of ester residues such as R = ? (CH₂)_n? with n = 1–3, have been studied in the lyotropic liquid crystalline state (chloroform 20 v/v%) by the deuterium nmr method. In order to study the surface chirality of the molecule, the phenyl groups situated at the terminal of the side chain have been deuterated. From the observed deuterium quadrupolar splittings, the average inclination θ_p of the para-axis of the phenyl group with respect to the α-helical backbone was elucidated. A distinct odd–even oscillation in the quantity such as 〈 cos² θ_p〉 was observed with the number of methylene units n. A rotational isomeric state analysis has indicated that the observed orientational correlation arises from the interdependence of the neighboring bond rotation along the side chain. Preference of the “extended” conformations is also enhanced by the mutual conformational exclusion of neighboring side chains.     

Synthesis and characterization of four sequential glycyl/glutamate polypeptides

Poly(Glu(OBzl)-Gly)_n, poly(Glu-Gly)_n, poly(Gly)-(Glu(OBzl)-Gly), and poly(Gly-Glu-Gly) were synthesized from the pentachlorophenyl esters of the sequential monomer. Both of the polymers containing free glumatic-acid residues are soluble in water, as is the lower molecular weight fraction of the polytripeptides with the benzyl ester in place. Circular dichroism studies and infrared dichroism studies suggest that the 2₁ helix is favored for the polydipeptide with removal of the benzyl ester reducing the conformational integrity. The polytripeptide showed evidence of 3₁ helix in addition to the 2₁ form, depending on solvent. A rationale for the conformations observed is developed based on the bulkiness of the side-chain residues and conformational stabilization, in certain cases, by hydrophobic interactions between the benzyl ester groups.     

Conformation study of poly(γ-methyl-L-glutamate) in helicogenic solvents by small-angle X-ray scattering

Small-angle x-ray scattering of poly(γ-methyl-L -glutamate), [Glu(OMe)]_n, in m-cresol and in pyridine was measured to determine the mass per unit length, M_q, and the radius of gyration of the cross section, 〈S〉^1/2. It was confirmed from the values of M_q that [Glu(OMe)]_n exists in an α-helical conformation in these solvents. It was elucidated from the calculations on 〈S〉^1/2 that the side chains come in moderately close contact with the main chain in these solvents. It was indicated from the analysis of the outer portion of the scattering curves that the side-chain conformation varied depending on the solvent.     

Conformations of cyclic peptide/calcium complexes in solution

Synthetic cyclic octapeptides of general structure cyclo[Glu(γOBzl)-Sar-Gly-(N-R)Gly]₂ (R = n-hexyl and cyclohexyl) transport calcium ions selectively across organic phases and phospholipid membranes. We have now used proton nmr spectroscopy (360 MHz) to study the solution conformation(s) of their calcium complexes. When Ca(ClO₄)₂ was added to solutions of these peptides in CDCl₃, nmr spectra of the resulting calcium complexes were characteristic of a single C₂-symmetric conformer. From a Karplus-Bystrov analysis of vicinal coupling constants in both the peptide backbone and Glu side chain (treated as an ABCC′MX spin system), in conjuction with model-building studies, a structure was proposed in which the calcium ion is bound in an octahedral-type complex by the four (coplanar) carbonyl groups of the (all-trans) Glu-Sar and Gly-(N-R)Gly peptide bonds. Occurrence of preferred rotamers about Glu side chain C^α–C^β bonds indicated that restricted rotation in peptide side chains arises upon calcium binding.     

Cation-binding cyclic peptides with lipophilic tails

We have synthesized and characterized a series of cation-binding cyclic octapeptides which may function as potential ionophoric substances. The materials contain varying degrees of hydrophobic character, which was controlled systematically through the incorporation of N-alkylglycine residues where N-alkyl = methyl, n-hexyl, cyclohexyl, or n-decyl. The peptides reported include cyclo(Phe-Sar-Gly-Sar)₂, cyclo(Glu(OBzl)-Sar-Gly-Sar-Glu(OBzl)-Sar-Gly-(N-decyl)Gly), cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-hexyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-cyclohexyl)Gly)₂, and the corresponding free diacid forms of the Glu-containing compounds. Using ¹³C- and ¹H-nmr spectra, we demonstrated that the mixture of cis/trans peptide bond-isomer conformers, characteristic of the free-peptide benzyl esters in solution, was converted to unique C₂-symmetric, presumably all-trans conformers on complexation with calcium ions. Cation-transport experiments, using the thick-liquid model of transport in a Pressman cell, established that these compounds transport a variety of cations and that one peptide examined in detail, cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂ (selectivity Ca²⁺ > Na⁺ > K⁺ > Mn²⁺ > Cu²⁺ > Mg²⁺ > Co²⁺ > Zn²⁺), transports calcium about an order of magnitude more efficiently than magnesium.     

Relationship between conformation and physicochemical properties of polypeptides. I. Synthesis of homo- and co-oligopeptides by the liquid-phase method

The synthesis of the following oligo- and co-oligopeptides by the liquid-phase method is described: (L -Met)₁₅ (I), [L -Glu(OBzl)]₂₀ (II), (L -Val)₈-Gly (IV), (L -Ile)₈-Gly (V), (L -Ile)₄-Gly-(L -Ile)₄ (VI), (L -Ile)₄-Pro-(L -Ile)₄ (VII), (L -Met)₅-L -Pro-(L -Met)₅ (VIII), [L -Glu(OBzl)]₇-L -Pro-[L -Glu(OBzl)]₇ (IX). The oligomers are covalently bound to bifunctional polyethylene glycol (PEG) and monofunctional PEG-M of M_r 5 × 10³?2 × 10⁴. Analytical controls were carried out after each step of synthesis in order to ensure quantitative coupling yields. All products could be obtained in high purity as indicated by amino acid analysis, thin-layer chromatography and chiroptical methods. The solubility of the oligomers was strongly enhanced by the presence of the C-terminal PEG group, enabling conformational investigations in a variety of solvents. A significant relationship between conformation and physicochemical properties of the oligopeptides was observed. Oligomers with tendencies to adopt α-helical (I, II) or unordered structures (VI–IX) showed no pronounced change in solubility or coupling kinetics during chain elongation, whereas the onset of a β-structure (IV, V) was paralleled by a drastic decrease in solubility and reactivity of the terminal amino groups. Most notably, the insertion of a proline or glycine in the middle of a β-forming peptide chain (VI, VII) resulted in a considerable increase in solubility compared to the corresponding homo-oligomers. The impact of the conformational properties of a peptide chain on strategic considerations of peptide synthesis in solution is delineated.     

Intrachain end-to-end charge transfer interaction in oligosarcosines

Oligosarcosines with two to seven sarcosine units terminated by an electron donor (p-dimethylaminoanilide group) and an electron acceptor (3,5-dinitrobenzoyl group) were synthesized by a liquid- or solid-phase method. The oligomers showed a distinct charge-transfer (CT) absorption in chloroform and ethanol solutions at low enough concentrations to eliminate the contribution of intermolecular CT interaction. Fractions of the oligomers which form the intrachain CT complex were evaluated at infinite dilution and plotted as a function of the number of sarcosine units n. The plot showed an alternating higher and lower tendency with the increase of n from n = 3 to 7; the total maximum was found at n = 4. The maximum reached 0.24 in chloroform, which is higher than that expected for an unperturbed oligosarcosine chain free from the end-to-end interaction, by an approximate factor of 50. The alternating chain-length dependence was found to be governed by an alternating tendency in the conformational enthalpy required for cyclization.     

Synthesis and nmr conformational studies of polyoxyethylene-bound, alpha-deuterated glutamate oligopeptides

The syntheses of three series of glutamate oligopeptides attached to a macromolecular solubilizing polyoxyethylene (POE) group Boc-[Glu(OMe)]_n-OPOE, Ac-[Glu(OMe)]_n-OPOE, pGlu-[Glu(OMe)]_n?1-OPOE (n ? 1–7) and their various analogs specifically deuterated at individual α-CH positions using the liquid-phase method of peptide synthesis are described. It was shown that stepwise synthesis using the symmetrical anhydride gave homo-oligopeptides that are analytically pure. Fragment condensation methods using DCC-HOBt yield POE-peptides with POE-HOBt impurities but the peptide synthesis may be carried stoichiometrically with smaller quantities of amino acid derivatives. 360 MHz ¹H-nmr conformational studies of these homo-oligopeptides in DMSO-d₆ are presented. The α-deuterated peptides are shown to allow unequivocal homoligopeptide backbone NH assignments.     

The infuence of interchain compositional heterogeneity on the conformation in random copolymers of γ-benzyl-L-glutamate and L-valine

Variation in the solvent used for the copolymerization of γ-benzyl-L -glutamate and L -valine N-carboxyanhydrides provides copolymers which have variable interchain compositions, and this variation in interchain compositional heterogeneity is reflected in the solid-state conformations of the respective copolymers. Poly[Glu(OBzl)²⁹Val⁷¹] prepared in dioxane exhibits a β-structure, whereas a copolymer of the same average composition prepared in benzene/methylene chloride shows predominantly an β-helix conformation with a small amount of β-structure. The use of the monomer reactivity ratio permits the calculation of the average and incremental copolymer compositions at any conversion; thus, correlations between conformation and interchain compositional heterogeneity can be made. In general, copolymers prepared in dioxane show a greater distribution of chain composition and therefore permit a wider variety of conformation than copolymers prepared in benzene/methylene chloride under identical conditions.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). III. The temperature dependence of the transient and steady-state behavior in cyclohexanone

The reversing-pulse electric birefringence (RPEB) technique was applied to the study of the temperature effect on the electrooptical and hydrodynamic properties of a fractionated [Glu(OBzl)]_n sample, which is molecularly dissolved in cyclohexanone. The aim was to develop a standard analytical method for thermal denaturation and temperature-induced conformational transitions. The field-on reverse and steady-state signal, and the field-off decay signal, were measured at 535 nm and at a constant low field strength (ca. 3 kV/cm) over a wide temperature range (5–90°C). The steady-state birefringence and the relaxation time in the decay process were also measured at two constant temperatures (5 and 70°C) over a wide field strength range (E ≤ 20 kV/cm). By the combination of these two different sets of RPEB measurements, the unwanted effect of the high pulse field on polymer conformation at elevated temperatures could be minimized. Together with the density and viscosity of cyclohexanone measured between 5 and 95°C, the following quantities could be evaluated: the weight-average permanent dipole moment and polarizability anisotropy, the reduced optical anisotropy factor (Δg/n), the weight-average length, and the degree of polydispersity. All these quantities, except for Δg/n, were found to be almost independent of temperature (5–90°C) and concentration (1.54–4.27 mM).     

Block copolymerization of α-amino acid N-carboxyanhydride initiated by vinyl polymers having a terminal amino group and the characterization of the block copolymers

Poly(methyl methacrylate) and polystyrene having terminal amino groups were synthesized by the radical polymerization of those monomers in the presence of 2-mercaptoethylammonium chloride as a chain-transfer agent. By the terminal group analysis and the molecular weight determination of the polymers, 0.5–1.3 amino groups were found in a chain of poly(methyl methacrylate) and 0.5–2.5 amino groups in a chain of polystyrene. Using these polymers having a terminal amino group as an initiator, the block polymerization of α-amino acid N-carboxyanhydride (NCA) was carried out. In the polymerizations of Glu(OBzl) NCA and Lys(Z) NCA by the poly(methyl methacrylate) initiator, the terminal amino group underwent a nucleophilic addition reaction to NCA and initiated the polymerization, yielding A-B-type block copolymers in a high yield. The same was observed in the polymerizations of Gly(OBzl) NCA and Lys(Z) NCA by the polystyrene initiator. By eliminating the protecting groups of the side chains of the polypeptide segment, the block copolymers poly(methyl methacrylate)-poly(Glu), poly(methyl methacrylate)-poly(Lys), polystyrene-poly(Glu) and polystyrene-poly(Lys) were synthesized with little side reactions. The side chain amino groups of poly(Lys) segment in the poly(methyl methacrylate)-poly(Lys) block copolymers were sulphonated or stearoylated successfully.