SynopsisOriented fibers drawn from aqueous gels of calf-thymus DNA were maintained at constant relative humidities of 75 and 92% to yield canonical A-DNA and B-DNA structures, respectively. Raman spectra of the two forms of DNA were recorded over the spectral range KKL4OOO cm-l. The authenticated DNA fibers were deuterated in hygrostatic cells containing DzO at appropriate relative humidities, and the corresponding spectra of deuterated DNAs were also obtained. The spectra reveal all of the Raman scattering frequencies and intensities characteristic of A-and B-DNA structures in both nondeuterated and deuterated forms, as well as the frequencies and intensities of adsorbed solvent molecules from which the hydration content of DNA fibers can be calculated. Numerous conformation-sensitive vibrational modes of DNA bases and phosphate groups have been identified throughout the 300-1700-~m-~ interval. Evidence has also been obtained for conformation sensitivity of deoxyribosyl CH stretching modes in the 2800-3000-~m-~ region. Raman lines of both the backbone and the bases are proposed as convenient indicators of A-and B-DNA structures. The results are extended to Z-DNA models investigated previously. Some implications of these findings for the determination of DNA or RNA structure from Raman spectra of nucleoproteins and viruses are considered.
Laser Raman spectroscopy indicates that the inner histones which are bound to DNA in chromatin or in isolated nu bodies are similar in conformation to the inner histones which are dissociated from DNA in high-salt solutions. This structure contains, on the average, 51+/-5% alpha-helix and no substantial beta-sheet conformation. It is proposed that the protein core of the nu body has a high alpha-helix content.
The cyclopentadecapeptide, c(VPGVG)3, a model structure for protein type‐II β‐turns [W. J. Cook et al. (1980) J. Am. Chem. Soc. 102, 5502–5505], has been investigated by laser Raman spectroscopy. Data obtained from both normal and deuterated crystals identify amide I, III, I′, and III′ bands characteristic of the β(II)turn. The structurally related polypentapeptide poly(VPGVG) in normal and deuterated forms has also been investigated, and exhibits the same Raman amide bands as c(VPGVG)3. The coacervate of poly(VPGVG), obtained by heating the solution to 40°C, likewise exhibits a Raman spectrum very similar to that of the c(VPGVG)3 crystal. Raman spectra thus indicate closely similar secondary structures for crystalline c(VPGVG)3 and aqueous poly(VPGVG), and provide an empirical basis for interpreting the conformation sensitive amide bands of globular proteins in terms of β(II)turn structures that may be present. An important conclusion from the present findings is that the amide I Raman profile of a protein may not be sufficient in general to distinguish turns from helix and sheet secondary structures, since the major amide I peaks of the β(II)turn at 1676 and 1652 cm−1 overlap, respectively, with amide I profiles generated by β‐sheet and α‐helix conformations.
The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations. Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids. Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure [Thomas, G.J., Jr., & Wang, A.H.-J. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol. 2, Springer-Verlag, Berlin]. The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3. The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti. However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers. Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)
SynopsisLaser Raman spectra of the trinucleoside diphosphate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are. facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA a t low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra-structure correlations for future conformational studies of RNA by laser Raman spectroscopy.
Laser Raman spectroscopy of the cowpea chlorotic mottle virus (CCMV) in native (pH 5.0) and partially swollen (pH 7.5) states reveals the presence of small percentages of protonated adenine (less than 15%) and cytosine (less than 7%) bases in the encapsidated RNA molecule of the native virion. The protonated bases are titrated with pH-induced swelling of the virus. Titration of putative COOH groups of aspartic and glutamic side chains of the virion subunit cannot be detected over the same pH range, which suggests that carboxyl anions (CO-2) and protonated bases are both available at pH 5 to stabilize the ribonucleoprotein particles by electrostatic interactions. The highly (95%) ordered secondary structure of encapsidated RNA may undergo a small additional increase (less than 3%) in ordered structure with release from the virion, suggesting at most a marginal structure-distorting influence from protein contacts in the native particle. The Raman spectra of the virion are also compared by difference spectroscopy with spectra of capsids (empty shells devoid of RNA), subunit dimers, and protein-free RNA. The results indicate that the subunit structure is altered by the release of RNA from the virion, as well as by the swelling of the virion. Amino acid residues and protein secondary structures that are affected in these in vitro assembly and disassembly processes are identified from their characteristic Raman lines. Two classes of cysteinyl SH groups, solvent exposed and solvent protected, are revealed for the capsid and virion subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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