Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Random copolymers of alpha-benzyl-L-aspartate with ortho- and para-nitrobenzyl-L-aspartates. Intrinsic and extrinsic dichroic bands and the left-to-right-handed alpha-helical transition.

Copolymers of benzylaspartate with the corresponding para-nitro and ortho-nitro derivatives are known to undergo, at increasing nitro content, a transition from a left-handed α-helical conformation to a right-handed α-helical conformation. The circular dichroism associated with the n, π* transition of the amide group, as well as the extrinsic bands associated with the nitrobenzyl chromophore, have been studied for these copolymers as a function of the nitro content. The circular dichroism associated with the 330-nm extrinsic band is shown to present an abrupt change, which parallels the change in the 222-nm band characteristic of the left- to right-handed helix transition. The intensity of the 222-nm CD band seems unaffected by the interaction with the side-band chromophore. The circular dichroism associated with the 330-nm band is much stronger in the right-handed conformation and is unaffected by increasing interactions between side chains. Interpretation in the light of the possible mechanism for optical activity of helical molecules seems to indicate that the 330-nm extrinsic effect is essentially due to the Condon, Altar, and Eyring (CAE) mechanism and μ-m perturbation (in Schellmann terminology) on the side-chain chromophore n, π* transition.     

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.     

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.     

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).     

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 %.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Conformational studies of polymers and copolymers of L-aspartate esters. I. Preparation and solution studies

An alcoholysis method is described for the modification of high molecular weight poly(β-benzyl L -asparatate); by this method the benzyl groups in the polypeptide have been replaced by methyl, ethyl, isopropyl, n-propyl, and phenethyl groups to give a series of copolymers of each of the corresponding aspartate esters with benzyl L -aspartate. By repeating the reactions, replacement of better than 99% has been achieved in some cases to give in effect the homopolymer. Optical rotatory dispersion studies show that of all the systems studied only poly(β-methyl L -aspartate) has the left-handed helix sense, the others are right-handed. It is shown further that the helix sense is not an intrinsic property of the nature of the aspartate side chain. Raising the temperature of chloroform solutions of the right-handed form of the copolymers of benzyl L -aspartate and ethyl L -aspartate results in a transition to the left-handed helix, the temperature of the transition being dependent on the composition of the copolymer. Also poly(β-n-propyl L -aspartate) undergoes a transition from the right- to the left-handed helix form at 59°C. These results suggest a general pattern of behavior of poly(aspartate esters) and that with suitable conditions of solvent and temperature they may be in either the right- or left-handed helical form.     

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.     

Rotatory dispersion and circular dichroism of low-molecular-weight poly- -benzyl-L-glutamate

Rotatory dispersion and circular dichroism of low-molecular-weight poly-γ-benzyl-L -glutamate, which was prepared from the N-carboxyanhydride by n-hexylamine initiation at [A]/[I] 3, 4, and 8, have been measured in ethylene dichloride and dioxane at different concentrations. The rotatory properties of the polypeptides are all characterized by a trough at 233 mμ of a negative Cotton effect or by a negative circular dichroic band at 223 mμ. With increasing A/I value or concentration, dextrorotation increases and the negative dichroic band becomes deeper. Both the trough magnitude and the negative ellipticity are linearly dependent on the content of β-structure, and the rotatory parameters for the pure β-structure are estimated by extrapolation of the linear relations. Circular dichroism and infrared spectra of the polypeptides have also been measured in trifluoroethanol, and the effect of solvents on the polypeptide conformation is discussed.     

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.     

Conformations of poly-L-valine in solution

In order to test theoretical predictions that poly-L -valine can exist in an α-helical conformation, water-soluble block copolymers of L -valine and D , L -lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D , L -lysine HCl)₁₈-(L -valine)₁₅-(D , L -lysine-HCl)1₆-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.     

Optical activity of simple cyclic amides in solution

The cyclic dipeptide, L -alanylglycyl anhydride, has been studied by optical rotatory dispersion; both L -alanylglycyl anhydride and the lactam, L -3-aminopyrrolidin-2-one, have been studied by circular dichroism. In hydroxylic solvents the circular dichroism spectra of 3-aminopyrrolidin-2-one can be attributed to an n–π* transition near 220 mμ and a π–π* transition near 190 mμ. In these solvents the optical activity of L -alanylglycyl anhydride can be explained as being due to contributions of n–π* transitions and a split π–π* transition. In acetonitrile, however, the circular dichroism spectrum of 3-aminopyrrolidin-2-one shows an additional apparent minimum near 200 mμ. The CD spectrum of the dipeptide is also quite distinctive in this solvent. The possible nature of the band at 200 mμ and the implications of these findings are discussed.     

Nuclear magnetic resonance and optical spectroscopic studies of block copolymers of polypeptides. I. Block copoly((benzyl-L-glutamate) n : (benzyl-L-aspartate) m )

In a study of A–B type block copolymers of γ benzyl-L -gultamate and β-benzyl-L -aspartate use has been made of the observations: (1) that for poly aspartate esters the chemical shifts of the α-CH and NH resonances are sensitive to the helix sense, (2) that in both helical and random coil conformations the same resonances of poly-γ-benzyl-L -glutamate are well separated from those of poly aspartates. Since the sense of poly-β-benzyl-L -aspartate is very sensitive to the inclusion of γ-benzyl-L -glutamate residues, the degree of overlap between the blocks can be studied by monitoring the helix sense of the aspartate. The ability of the NMR method to make separate observation of the two blocks removes the necessity of relying on an overall ORD parameter such as b₀. The copolymers studied include those having lefthanded, righthanded, and mixed-sense aspartate, corresponding to differing degrees of overlap.     

Circular dichroism of type 13 β-turn in linear tripeptides containing L-proline and D-alanine

The linear tripeptides tBoc-L-Prolyl-D-alanyl-L-leucine and tBoc-L-prolyl-D-alanyl-L-valine have been shown, from circular dichroism (CD) and infrared spectral data, to take up the 4 → 1 hydrogen bonded β-turn conformation in organic solvents. The CD spectra of these tripeptides in trifluoroethanol exhibit a positive n → π^★ band around 220 nm contrary to the usual negative band observed for the type II β-turn. The observed CD spectra of the tripeptides provide the first examples of those predicted theoretically by Woody for peptides containing L,D sequences and adopting the Venkatachalam type 13 β-turn. This conformation is seen to revert to the type II β-turn when the N-terminal protecting group is acetyl or when the C-terminal residue is glycine. These data are shown to have a direct bearing on the interpretation of the CD spectra of globular proteins.     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

Evidence from circular dichroism for the binding of hydrogen ions and calcium ions by poly (L-proline).

The positive circular dichroism band observed near 228 nm with poly(L -proline) responds in a similar fashion to HCl and CaCl₂. The spectra in the HCl solutions are compatible with a simple binding equation and a pK near ?2 for the dissociation of a proton from a protonated peptide bond in poly(L -proline). The data obtained in CaCl₂ is susceptible to the same analysis, suggesting a pK near ?1.5 for the dissociation of a calcium ion from its complex with poly(L -proline).     

Synthèse et structure de la poly-O-carbobenzoxy L- tyrosine et de copolymères de O-carbobenzoxy L-tyrosine et de glutamate de benzyle ou d'aspartate de benzyle

The optical rotatory dispersion of copolymers of O-carbobenzoxy-L -tyrosine and benzyl L (or D )-glutamate as well as benzyl L -aspartate, dissolved in nonpolar solvent, has been studied. Moffitt's equation permits the determination of b₀ coefficients whose variation, with varying composition in amino acid residues, suggests that the molecules of poly-O-carbobenzoxy-L -tyrosine have a helical structure similar to that of poly-(benzyl L -glutamate). Results obtained from infrared spectroscopy and x-ray diffraction show that the copolymers possess a helical conformation in the solid state, even when they are very rich in carbobenzoxy-L -tyrosine residues. The value of the b₀, coefficient for poly-O-carbobenzoxy-L -tyrosine may be explained by a regular stacking of the chromophore groups around the helical backbone. The ordering of the molecules of this polymer in a purely helical structure seems favored by the insertion of a small number of foreign residues in the polypeptide chain.     

Conformational properties of L-leucine,L-isoleucine,and L-norleucine side chains in L-lysine copolymers

The structure inducing properties of L -leucine, L -isoleucine, and L -norleucine residues incorporated into poly(L -lysine) were investigated by the observation of the circular dichroism of the respective random copolypeptides. The comparison involves the coil-helix transition in water/methanol mixtures, the formation of ordered structures at higher pH, and the kinetics of the α-helix to β-conformation transition of the leucine and norleucine copolymers induced by temperature changes at pH 10.5. The results confirm the known properties of the leucine residue, strongly supporting the α-helix conformation. They also support the idea that the isoleucine residue is one of the most powerful candidates for β-structure formation, and they show that the unbranched norleucine residue has intermediate properties. The results are discussed on the basis of steric and hydrophobic properties of the three side chains.