Raman spectra of copoly(D,L-alanines)

Raman spectra in the region 1000–150 cm^?1 were measured for copoly(D ,L -alanines) with the D -residue contents, 3, 7, 10, and 20%, and compared with the spectrum of the α-helical poly-L -alanine. The 532- and 378-cm^?1 peaks were assigned to the L -residues with a right-handed α-helix-like local conformation or to the D -residues with a left-handed α-helix-like local conformation. From the intensity of the latter peak the contents of these local conformations were estimated as a function of the D -residue contents for the copolymers. The 264-cm^?1 peak, which has been assigned to the breathing vibration of the α-helical poly-L -alanine, shows a marked decrease in its intensity upon the introduction of the D residues. This result suggests that the overall deformation vibration of the α-helix arises from rather long sequences of the L - and D -alanine residues with the α-helical conformation and that the intensity of this vibration depends on the content of these sequences in the copolymers.     

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 %.     

Conformational studies on a modified poly-L-tryptophan: Circular dichroism and optical rotatory dispersion of poly-2-(2-nitrophenylsulfenyl)-L-tryptophan and of random copolymers of L-tryptophan and 2-(2-nitrophenylsulfenyl)-L-tryptophan

Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements were carried out on a block copolymer, (γ-ethyl DL -glutamate)₁₆₀ (L -Trp)₃₂, in which the tryptophan sequence has been modified to various extents by using 2-nitrophenylsulfenyl chloride. The CD spectrum of the completely modified copolymer exhibits bands in some of the regions of maximum absorption of the sidechain chromophores. In the peptide absorption region the spectrum is similar to that reported in the literature for polypeptides in the α-helical conformation. When the extent of modification of the tryptophan sequence is progressively reduced, there is a gradual change in the ORD spectra of the copolymers. On the basis of these data the assumption was made that no conformational change occurs on proceeding from the pure unmodified tryptophan sequence to the completely modified sequence. The results are discussed in connection with the study of possible conformational effects arising from selective chemical modification of tryptophan residues in proteins.     

Far-infrared spectra of polyalanines with alpha-helical and beta-form structures

Far-infrared spectra of poly-L -alanines having the α-helical conformation and the β-form structure were measured. The spectra of glycine–L -alanine copolymer, silk fibroin, and copoly-D ,L -alanines with different D :L compositions were also measured. In addition to the bands so far reported, four bands at 190, 107, 120, and 90 cm^?1were found for the α-helix conformation and the two bands at 442 and 247 cm^?1 were found for the β form. The 442 cm^?1 band consists of the parallel 432 cm^?1 and perpendicular 445 cm^?1 bands. The 247 cm^?1 band is well defined and has strong dichroism parallel to the direction of stretching. These two bands appear also for silk fibroin and glycine–L -alanine copolymer. All the far-infrared bands of copoly-D ,L -alanines can be interpreted as α-helix bands, the three peaks at 580, 478, and 420 cm^?1 being ascribed to the D -residue incorporated into the right-handed α-helix or to the L -residue in the left-handed α-helix.     

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.     

Raman spectra of D and L amino acid copolymers. Poly-DL-alanine, poly-DL-leucine, and poly-DL-lysine.

The Raman spectrum of poly-DL -alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L -alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds. The Raman spectra of poly-DL -leucine (PDLL) and poly-L -leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm^?1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L -residues in right-handed segments and D -residues in left-handed segments (in PDLL). On the other hand, the 890-cm^?1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L -residues in left-handed segments and D -residues in right-handed ones. The Raman spectra of poly-DL -lysine and poly-L -lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L -lysine because the distribution of the residues in poly-DL -lysine probably tends towards blocks, and the individual blocks may take up the 3₁ helix.     

Vibrational frequencies and modes of alpha-helix

Dichroic properties of the far-infrared absorption bands of the right-handed α-helix of poly-L -alanine were measured. The normal vibration frequencies of this structure were calculated. The assignments of bands were made and the vibrational modes dis-cussed. The frequencies of the α-helix vibrations with various phase differences were calculated. The frequencies of accordionlike vibrations and Young's modulus of the α-helix were estimated. The vibrational frequency for the right-handed α-helices of poly-D -alanine and poly(L -α-amino-n-butyric acid) were calculated, and the results were used for the interpretation of the spectra of copoly-D ,L -alanines and poly(L -α-amino-n-butyric acid). For the latter compound the existence of the rotational isomers in the side chain was strongly suggested. The vibrational modes of the bands characteristic of the α-helix were discussed with regard to the results of the normal coordinate treatment.     

Extent and stability of the alpha-helix in gamma-benzyl L-glutamate-glycine copolymers

Block and random copolymers of γ-benzyl L -glutamate and glycine were studied by optical rotatory dispersion methods in order to ascertain the extent and stability of the α-helix formed by these polymers in appropriate solvent mixtures. Results indicate that when in solution such polymers do contain sections of α-helix. However, it appears that glycine does not readily fit into the α-helix which is largely due to the γ-benzyl L -glutamate segments. Further, there are indications that the helical sections in the random copolymers are due to the nonrandomness of the polymerization.     

The conformation of poly-L-alanine in hexafluoroisopropanol

High-molecular-weight poly-L -alanine dissolved in hexafluoroisopropanol exhibits infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra which are unique and unlike any other previously reported polypeptide spectra. Strong evidence that a helical conformation is present is shown by the high degree of hypochromism in the 187mμ absorption peak and by the positions of the amide infrared bands. The CD and ORD spectra are also similar to those of α-helical polypeptides, though important qualitative and qualitative differences are observed. To explain the novel spectra, which are not mixtures of the spectra of previously reported polypeptide conformations, a new α-helix-like conformation is proposed. The postulated conformation (a doubly hydrogen-bonded helix) is a distorted α-helix in which the peptide carbonyl groups point slightly out from the helix axis and are hydrogen bonded simul taneously both to the NH of the fourth peptide residue to the carboxyl terminal side (as in the classical α-helix), as well as to a solvent molecule's hydroxyl hydrogen.     

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.     

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.     

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.     

Quasielastic light scattering investigation of the isothermal "helix to extended-coil" transition of poly-L-lysine HBr

The quasielastic light scattering of poly-L -lysine HBr in aqueous solutions has been examined over a variety of pH values where the polymer is in different conformations. Analysis of the spectra demonstrates that as the pH is lowered, the polymer changes from an extended anisotropic from through a collapsed randomized state to a larger isotropic form. These data support the current view that the polymer undergoes a conformational change from an α-helical form to a “kinked” extended-coil conformation through an intermediate partially α-helical state in which internal packing of the helical portions occurs. Finally, a value for the rate of propagation of the unwinding of the α-helix has been obtained.     

Effect of temperature on the circular dichroism spectra of polypeptides in the extended state

The circular dichroism (CD) spectra of poly-L -proline and of poly-L -glutamic acid and poly-L -lysine in their charged states have been studied as a function of temperature. The variation of CD spectra with temperature is inconsistent with the assignment of the spectrum of such charged polypetides to an unordered chain conformation, but does support our earlier assignment to a locally ordered structure—what we have called the extended helix conformation. These results also strengthen our previous assignment of the CD spectrum of an unordered chain, and indicate that three conformational states (α-helix, extended helix, and unordered) should be incorporated in our thinking about conformational transitions in polypeptides.     

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.     

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.     

Spectroscopic studies of random chain and -helical polypeptides in hexafluoroisopropanol

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non-ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly-γ-morpholinylethyl-L -glutamamide has random chain type spectra in this solvent, whereas the spectra of poly-γ-methyl-L -glutamate, poly-L -methionine, poly-ε, N -Carbo-benzoxy-L -lysine, and poly-L -homoserine indicate that these four polypeptides are α-helical. Small but significant variability between the different α-helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α-helices.     

Far ultraviolet optical rotatory properties of poly-L-tryptophan. I

A block copolymer [γ-Et-DL -Glu]_m [L -Trp]_n was prepared using N-carboxy anhydrides (NCA) of L -tryptohan and γ-ethyl DL -glutamate. The block copolymer, dissolved in trifluoroethanol (TFE)–dichloroacetic acid (DCA) mixtures, exhibited a sharp change in the specific rotation at 546 mμ when the solvent composition reached 70–75% DCA content. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurement were carried out in TFE solution in the spectral range 180–350 mμ. Indole side-chain chromophores were found to be optically active in the polymer. On the other hand, these groups exhibit very small optical activity in the model compound C₆H₃? CH₂? O? CO? (L -Trp)₂? O? CH₃. Indole groups therefore appear to be in a dissymmetric environment only in the polymer. From these data it was concluded that poly-L -Trp is in some type of helical conformation in TFE. Strong overlapping of CD bands from side-chain chromophores and peptides chromophores in the wavelength range 185–240 mμ does not allow definite conclusions to be drawn about the type of helical conformation which exists in poly-L -Trp in TFE solution.     

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.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998