Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

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 %.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

The two beta forms of poly(L-glutamic acid).

β-Helical poly(L -glutamic acid) in a gel state was found to be easily converted to the antiparallel β form by heating. Two β forms were obtained, depending on the temperature of heating. Temperatures between 40° and 85°C produced a β form with a spacing between pleated sheets (d₀₀₁) of 9.03 Å, termed β₁. If the heating was carried out at temperatures higher than 85°C, the β₁ form underwent another conformational transition reducing the d₀₀₁ value from 9.03 to 7.83 Å (termed β₂) without any prominent change in the fiber repeat distance (i.e., the polypeptide backbone conformation). The time course of these two transitions was followed by measuring the infrared spectra of the samples, and it was concluded that the α → β₁ transition in its initial stage obeys a pseudo-first order rate process with activation enthalpy and entropy of 54 kcal/mol and 92 eu, respectively. On the other hand, the typical sigmoidal conversion curves observed for the transition between the two types of β forms (β₁ → β₂) indicate that this transition proceeds via a socalled “nucleation and growth” process. The kinetic theory of phase transitions developed by Avrami can be applied with success to explain this transition. The infrared spectra, in the region from 1800 to 200 cm^?1, were measured for these two β forms and the results showed that the conformation of the side chains and the mode of the hydrogen bonding between the side-chain carboxyl groups undergo appreciable change during the transition. The heat-induced conformational transition of poly(L -Glu⁷⁸ L -Val²²) was also studied. The copolymer was transformed from the α-helical conformation directly to the β₂ form. The reason for this was thought to be due to the fact that the L -valine residues and the L -glutamyl residues near the L -valine residues have a strong tendency to take the more compact β₂ form.     

A vitrational study of poly (L-proline) I and II in the far infrared.

The far infrared spectra of poly(L -proline) I (190–35 cm^?1) and II (400–35 cm^?1) were obtained in the solid state at both 300° and 110°K. A significant difference in the region below 100 cm^?1 was observed. A very intense band located at 60 cm^?1 in the infrared spectrum of form II has no counterpart in form I. This indicates the sensitivity of low-frequency vibrations to the difference in conformation assumed by both forms in the solid state. Additional bands observed in this study are correlated with ir and Raman data previously reported and tentative assignments are made using the results of normal mode calculations (in the single-chain approximation) which have been reported.     

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.     

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.     

Conformational study of poly(α,β-L-aspartic acid)

The conformation of several samples of poly(α,β-L -Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L -Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm^?1 was shifted to 620 cm^?1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.     

Laser-excited raman spectroscopy of biomolecules. XII. Thermally induced conformational changes in poly(L-glutamic acid)

The α-helical from of poly(L -glutamic acid) [α-poly(Glu)] gives rise to the same amide I and III lines as α-poly(γ-benzyl-L -glutamate) at 1652 and 1296 cm^?1, respectively. The latter is a superposition of the amide III line near 1290 cm^?1 and a line deu to vibrational made of CH₂ groups of the side chain near 1300 cm^?1. A line at 924 cm^?1 is tentatively identified as characteristics of α-poly(Glu). Both the β₁- and β₂- forms of poly(Glu) give rise to characteristic of β-amide. III frequencies that are similar because of their similar backbone structures. Differences in the conformations of their side chains and in the environments of the backbone are reflected in the region 800–1200 cm^?1 and in the amide I. A line at 1042 cm^?1 and a pair at 1021 and 1059 cm^?1 are tentatively assigned as characteristic of β₁-poly(Glu) and β₂-poly(Glu), respectively. The α-β₂ transition in poly(L -Glu⁷⁸L -Val²²) is shown by the appearance of all the β₂-characteristic lines in the thermally transformed sample. The same features observed in poly(L -Glu⁹⁵L -Val⁵) also indicate that the α-β₂ transition of poly(Glu) is facilitated by the presence of L -valine and that the content of L -valine is not critical for this purpose. Investigation of the Raman spectra of the calcium, strontium, barium and sodium slats of poly(Glu) shows that these salts, under the conditions of preparation used, all the have random-coil conformations.     

A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)₈ and (dA)₂, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co-workers [M. Berjot, L. Marx, and A. J. P. Alix (1985) J. Ramanspectrosc., submitted; A. J. P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149–154], 1 gp32 contains ≈ 45% helix, ≈ 40% β-sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4–6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO^? groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide–protein complexes the nucleotide shields the 4–6 tyrosine residues from coordination by the COO^? groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single-stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more β-sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate–sugar–base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm^?1 in poly(rA), at 1230 cm^?1 in poly(rU), and at 1240 and 1378 cm^?1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp32² in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)₈.     

Intensity Changes of Base and Backbone Raman Modes in the B to Z Transition of poly(dG-dm5C)

Abstract

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm-⁵C). The transition midpoint was about 37°C for a solvent containing 20 mM Mg²⁺. A 10-fold change in Mg²⁺ concentration altered the transition midpoint by at least 60°C. Raman spectra of the B and Z forms of poly(dG-dm⁵C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm^?1 guanine mode and 835 cm^?1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm^?1, 805 and 819 cm¹. Several bands unique to poly(dG-dm⁵C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Low-frequency vibrational spectra of some homopolypeptides in the solid state

Low-frequency Raman and far-infrared spectra of polyglycine, poly-L -alanine, and poly-L -valine have been measured. The Raman spectra exhibit an intense band near 100 cm^?1 for these homopolypeptides. Lattice calculations of the polyglycines are used to assign the intense Raman band to a rotary lattice mode. For homopolypeptides in the β conformation, an infrared band is observed whose frequency varies inversely with the square root of the mass of the peptide repeating unit. This infrared band is assigned to the hydrogen bond stretching lattice vibration.     

Laser-Raman spectroscopy of biomolecules. XI. Conformational study of poly(L-valine) and copolymers of L-valine and L-alanine

Raman spectroscopic studies have been carried out on polymers of L -valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40–1750 cm^?1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L -alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L -valine.     

Far-infrared absorption of nucleotides and poly(I)·poly(C) RNA

We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ～25 to ～500 cm^?1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm^?1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm^?1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm^?1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Far-infrared spectra of copoly(- -amino acids)

Far-infrared spectra in the region from 700 to 200 cm^?1 were measured for the copolymers of L -alanine and glycine, those of L -alanine and L -valine, those of L -alanine and L -leucine, and those of L -alanine and L -phenylalanine. The observed spectra were interpreted on the basis of the analysis of the far-infrared spectra of the corresponding homopolymers, and the correlation between the conformations of the copolymers and the kinds of the component amino acids was discussed.     

Spectral study of interaction between poly [acrylamide‐(2‐methyl acryloxyethyl) dimethyl buthylammonium bromide] and nucleic acids

A polycation, poly[acrylamide‐(2‐methylacryloxyethyl) dimethyl buthylammonium bromide] [P (AM‐DMB)], was synthesized. The interaction between P (AM‐DMB) and DNA was studied by spectral methods. Resonance light scattering spectra, FTIR‐spectra and UV‐spectra were used to study the mechanisms of the interaction. The results indicate that the conformation of the DNA alters during the interaction because the microenvironment of DNA changes. The binding constant K between P (AM‐DMB) and DNA is 8.0 × 10³ L mol^?1. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Secondary structure of peptides. 4: 13C-Nmr CP/MAS investigation of solid oligo- and poly(L-alanines)

Primary and tertiary amine-initiated polymerizations of L -alanine-N-carboxyanhydride (L -Ala-NCA) were conducted at 20 or 100°C in a variety of solvents. The 75.5-MHz ¹³C-nmr CP/MAS spectra of the resulting poly(L -alanines) revealed that all samples contain both α-helix and pleated-sheet structures. Depending on the reaction conditions the α-helix content varied between ca. 1 and 99%. Reprecipitation from aprotic nonsolvents does not change the α-helix/β-sheet ratio, indicating that this ratio is thermodynamically controlled. Since relatively large amounts of oligopeptides of degree of polymerization (DP ) 4–6 can be extracted by means of acetic acid, it is concluded that (a) most poly(L -alanines) possess a bimodal molecular weight distribution, (b) the oligopeptide fraction with DP ? 11 is responsible for the β-sheet fraction of all samples, and (c) the two-stage crystal growth proposed by Komoto and Kawai is not correct. Solubilizing initiators such as poly(ethylene oxide) NH₂ prevent the precipitation of oligoalanine and, thus, the formation of a β-sheet structure. ¹³C-nmr CP/MAS measurements also show that tri- and tetra-L -alanines form insoluble β-sheet structures.