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.     

Normal mode spectrum of the parallel-chain β-sheet

Normal mode calculations have been carried out for parallel-chain β-sheet structures. These include the parallel-chain pleated sheet of poly(L -alanine) and the parallel-chain rippled sheet of polyglycine. Dipole derivative coupling has been included for amide I and II modes, and the effects of parallel-sheet and antiparallel-sheet arrangements of varying separation have been examined for the poly(L -alanine) case. Some amide and nonamide modes are distinctly different from their antiparallel-chain counterparts, thus providing a basis for distinguishing between such structures from their ir and Raman spectra. As in our previous studies, these results emphasize the need for both kinds of spectral data in order to draw definitive conclusions about conformation.     

Vibrational analysis of peptides,polypeptides, and proteins. I. Polyglycine I

A force field has been refined for the antiparallel chain-rippled sheet structure of polyglycine I. Transition dipole coupling and hydrogen bonding are explicitly taken into account. Amide I and amide II mode splittings are well accounted for, the latter also providing a quantitative explanation of the amide A and amide B mode frequencies and intensities. In addition to predicting other features of the vibrational spectrum of polyglycine I, this force field is completely transferable to other β polypeptides, even though these have the antiparallel chainpleated sheet structure.     

Vibrational analysis of peptides,polypeptides, and proteins. XVIII. Conformational sensitivity of the α-helix spectrum: αI- and αII-Poly(L-alanine)

The α_II-helix (? = ?70.47°, ψ = ?35.75°) is a structure having the same n and h as the (standard) α_I-helix (? = ?57.37°, ψ = ?47.49°). Its conformational angles are commonly found in proteins. Using an improved α-helix force field, we have compared the vibrational frequencies of these two structures. Despite the small conformational differences, there are significant predicted differences in frequencies, particularly in the amide A, amide I, and amide II bands, and in the conformation-sensitive region below 900 cm^?1. This analysis indicates that α_II-helices are likely to be present in bacteriorhodopsin [Krimm, S. & Dwivedi, A. M. (1982) Science 216 , 407–408].     

Ab initio-based vibrational analysis of α-poly(L-alanine)

Polarized ir and Raman spectra have been obtained on oriented films of α-helical poly(L -alanine) (α-PLA) and its N-deuterated derivative. These improved spectra permit a more complete assignment of observed bands to A-, E₁-, and E₂-species modes. A new empirical force field has been refined, based on ab initio force fields of N-methylacetamide and L -alanyl-L -alanine, which reproduces observed frequencies above 200 cm⁻¹ to less than 5 cm⁻¹. A new transition dipole coupling treatment avoids the weak coupling and perturbation approximations, and can now account for the newly observed and reassigned amide I (E₂) mode. As a result of this improved force field, several other observed bands have also been reassigned. © 1998 John Wiley & Sons, Inc. Biopoly 46: 283–317, 1998     

Vibrational spectroscopy of L-valyl-glycyl-glycine,a parallel-chain β-structure

Bands in the ir and Raman spectra of L -valyl-glycyl-glycine (VGG) and VGG-ND have been assigned on the basis of a normal mode analysis of the known parallel-chain β-structure of this tripeptide. Amide I, II, III, and V mode shifts are obtained by the interactions of dipole derivatives in symmetry coordinates, referred to as dipole derivative coupling. These derivatives, obtained from ab initio studies, are also used to calculate ir intensities of amide I, II, and V modes. The agreement between predicted and observed frequencies and intensities is very good, providing confidence in the application of our force fields to the calculation of the vibrational modes of the general parallel-chain β-sheet structure (following paper).     

Vibrational analysis of peptides,polypeptides, and proteins. XXI. β-Calcium-poly(L-glutamate)

The observed Raman and ir spectra of Ca-poly(L -glutamate) in the β conformation have been analyzed by means of a normal mode calculation. The force field for the main chain was transferred without refinement from β-poly(L -alanine), yet it provides a good prediction of the observed bands and, in particular, explains subtle differences in the spectra of these two β-sheet structures. Main- and side-chain modes are well characterized, and the dependence of the amide III frequency on side-chain composition is again demonstrated.     

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 %.     

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

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

Vibrational analysis of peptides,polypeptides, and proteins. XV. Crystalline polyglycine II

A force field has been refined for the 3₁-helix structure of polyglycine II, using the polyglycine I force field plus previous C^αH^α…?O force constants as a starting point. Besides force constants associated with the hydrogen bonds, which must change since the hydrogen-bond characteristics are different in the two structures, we have had to modify only 10 force constants from the polyglycine I force field to make it suitable for reproducing the polyglycine II frequencies. Most involve the NC^α bond, which is the torsion angle that changes from the I to the II structure. Calculations were done for parallel chain and antiparallel chain crystal structures of polyglycine II, the observed spectra being found to agree best with the latter structure. Since this provides strong evidence for the loss of strict threefold symmetry in the chain, our analysis strengthens the support for the existence of C^αH^α…?O hydrogen bonds in the structure of polyglycine II.     

Vibrational analysis of peptides,polypeptides, and proteins. V. Normal vibrations of β-turns

The normal modes have been calculated for β-turns of types I, II, III, I′, II′, and III′. The complete set of frequencies is given for the first three structures; only the amide I, II, and III modes are given for the latter three structures. Calculations have been done for structures with standard dihedral angles, as well as for structures whose dihedral angles differ from these by amounts found in protein structures. The force field was that refined in our previous work on polypeptides. Transition dipole coupling was included, and is crucial to predicting frequency splittings in the amide I and amide II modes. The results show that in the amide I region, β-turn frequencies can overlap with those of the α-helix and β-sheet structures, and therefore caution must be exercised in the interpretation of protein bands in this region. The amide III modes of β-turns are predicted at significantly higher frequencies than those of α-helix and β-sheet structures, and this region therefore provides the best possibility of identifying β-turn structures. Amide V frequencies of β-turns may also be distinctive for such structures.     

Conformational analysis of helical poly(β-L- aspartate)s by IR dichroism

Poly(β–l–aspartate)s are known to take up helical conformations reminiscent of the α-helix of polypeptides. The isobuttyl, n-butyl, and 2-methoxyethyl esters have been examined by polarized ir spectroscopy in order to discriminate between the left ( 1L ) and right ( 2R ) -handed conformations, which are known to be compatible with the 13/4-helix adopted by these polyamides when crystallized in the hexagonal form. Dichroic ratios obtained from samples stretched in poly(ethylene oxide) together with orientation measurements made by x-ray diffraction were used to estimate the transition moment directions of amide A, I, and II bands with respect to the fiber axis. These were compared to those calculated by modeling simulations to conclude that the right-handed conformation consisting of 14-membered hydrogen-bonded rings is the correct model for the 13/4-helix. These results give definite support to earlier molecular mechanics calculations, which had shown that the 2R model is energetically favored over the 1L by about 2. 5 kcal/(mol residue). © 1995 John Wiley & Sons, Inc.     

Fluctuations and mechanical strength of α-helices of polyglycine and poly(L-alanine)

By using the static correlations of fluctuations in the dihedral angles of the α-helices of polyglycine and poly(L -alanine) calculated previously, geometrical fluctuations of a section (consisting of up to 18 peptide units) of the α-helices of infinite length are calculated. These fluctuations are found to differ in some respects (e.g., the dependence of amplitudes on the length of section) from those of a circular rod made of homogeneous continuous material. However, the moduli of the mechanical strengths (tensile Young's modulus, bending Young's modulus, and the shear modulus) of a circular rod are calculated, whose geometrical fluctuations are approximately equal to the fluctuations of a section consisting of 18 peptide units. They are of the order of 10¹¹ dyn/cm². The tensile rigidity, flexural rigidity, and torsional rigidity are calculated to be 1.20 × 10^?3 dyn, 2.46 × 10^?19 dyn·cm² and 1.79 × 10^?19 dyn·cm² for polyglycine, and 1.96 × 10^?3 dyn, 4.05 × 10^?19 dyn·cm² and 3.28 × 10^?19 dyn·cm² for poly(L -alanine), respectively.     

Crystal structure of dehydromonopetide, (Z)-N-acetyl-α,β-dehydrophenylalanine methylamide

The crystal structure of (Z)-acetyl-α,β-dehydrophenylalanine methylamide (monoclinic Cc, a = 10.241(1), b = 15.252(1), c = 8.643(1) Å, β = 120.98(1)°, Z = 4) has been solved by x-ray diffraction to an R-factor = 0.148, and compared to that of the homologous L -phenylalanine dervative. Molecules are intermolecularly hydrogen-bonded to four neighboring molecules in a three-dimensional network with alternating layers of interacting amide bonds and orthogonally arranged phenyl rings. The existence of the C^α = C^β double bond results in a phenyl orientation that is forbidden for phenylananine (χ¹ = ?7,8°), and in shorter C^α ? C^β and C^β ? C^γ distances. The geometrical paramenters of the peptide backbone are not drastically modified by α,β-unsaturation. However, the N-C^α-C′ angle is increased by nearly 5°, and the dimensions, and therefore probably the electronic conjugation, of the N-terminal amide group to be affected by the occurrence of the vicinal C^α = C^β double bond.     

Normal vibrations of crystalline polyglycine I

A valence force field has been refined for crystalline polyglycine I using its known antiparallel chain pleated-sheet structure and without replacing the CH₂ group by a point mass. Polyglycine I and four of its isotopic derivatives were used in the refinement. The calculated frequencies are in good agreement with the observed, except for the amide I modes. It is shown that this is a consequence of the fact that no reasonable force field predicts a large D₁₀ term of the Miyazawa perturbation treatment. The amide I splittings can, however, be satisfactorily accounted for by introducing a direct interaction force constant between adjacent C?O groups in neighboring chains. This can reasonably arise from transition dipole coupling and corresponds to the heretofore neglected D₁₁ term.     

A quantitative anharmonic analysis of the amide A band in α‐helical poly(L‐alanine)

Polarized ir spectra of oriented films of α‐helical poly(l ‐alanine) (α‐PLA) have been obtained as a function of residual solvent dichloroacetic acid (DCA). The amide A, B, II, and V regions exhibit multiple bands whose structure depends on the residual DCA content, and those associated with the α_I‐PLA structure have been identified. A calculation of the relevant cubic anharmonic force constants indicates that, contrary to previous assignments, the overtone of amide II(A) is in Fermi resonance with the NH stretch fundamental, whose unperturbed frequency we now find to be at 3314 cm⁻¹, significantly higher than the previously suggested 3279 cm⁻¹. The presence of a structure in addition to the standard α_I‐PLA is indicated by our analysis. © 1999 John Wiley & Sons, Inc. Biopoly 49: 195–207, 1999     

Helix–coil transition in copolypeptides. II. Poly(γ-benzyl L-glutamate-co-ε-carbobenzoxy-L-lysine)

The thermal helix–coil transition of poly(γ-benzyl L -glutamate-co-ε-carbobenzoxy-L -lysine) copolypeptides was studied in solvent mixtures of different compositions. The cooperativity parameter v changes linearly with polymer (and solvent) composition, whereas the heat of the transition shows a very pronounced minimum as a function of polymer composition. This minimum cannot be due only or mainly to the solvent changes and must be attributed to the effect on the transition of the side chains of the polypeptides.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Insights into the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) by two‐dimensional Raman correlation spectroscopy

In this study, the transition process of the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) was investigated by Raman scattering and 2‐dimensional correlation spectroscopy. Temperature‐dependent Raman spectra were obtained during the helix‐sense inversion. The results of 2‐dimensional correlation analysis in the spectral regions of 1600‐1800 and 3200‐3400 cm^?1 showed that the intensity changes of the side‐chain ester C═O stretching bands occurred prior to those of amide A and amide I bands in the unwinding process of αR‐helix on heating. The sequential order of the intensity changes for amide A, amide I, and the side‐chain ester C═O stretching bands during the inversion process was determined. It was found that the conformation change of the side chain occurred prior to that of the main chain for the αR‐helix on heating. Thus, we concluded that the transformation of the backbone chain from right‐handed to left‐handed is triggered by the conformational change of the side chains.     

Fourier transform IR attenuated total reflectance study on the secondary structure of poly(γ -methyl L-glutamate) surfaces treated with formic acid

Fourier transform ir attenuated total reflectance (FTIR ATR) spectra have been obtained to investigate the secondary structure of poly(γ -methyl L -glutamate) (PMLG) surfaces untreated and treated with formic acid in a quantitative manner. Curve analysis including Fourier self-deconvolution and the band fitting was applied to the ir spectra in the amide II region, revealing that the amide II band of those surfaces consists of five components. The essentially α-helical form in the PMLG surface layer transformed readily into the β-structure by the formic acid treatment, and the β-structure content increased with increasing time of the treatment. The content of random coil structure of treated PMLG was generally very little and/or negligible. The depth profile obtained by considering the depth of ir beam penetration indicated that the β-structure content also increased with approaching the outermost surface.