Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.

No abstract

No abstract

Polarized Raman scattering measurements have been made of a single crystal of L‐tyrosine by the use of a Raman microscope with the 488.0‐nm exciting beam from an argon ion laser. The L‐tyrosine crystal belongs to the space group P212121 (orthorhombic), and Raman scattering intensities corresponding to the aa, bb, cc, ab and ac components of the crystal Raman tensor have been determined for each prominent Raman band. A similar set of measurements has been made of L‐tyrosine‐d4, in which four hydrogen atoms on the benzene ring are replaced by deuterium atoms. The effects of NH3 → ND3 and OH → OD on the Raman spectrum have also been examined. In addition, depolarization ratios of some bands of L‐tyrosine in aqueous solutions of pH 13 and pH 1 were examined. For comparison with these experimental results, on the other hand, ab initio molecular orbital calculations have been made of the normal modes of vibration and their associated polarizability oscillations of the L‐tyrosine molecule. On the basis of these experimental data and by referring to the results of the calculations, discussions have been presented on the Raman tensors associated to some Raman bands, including those at 829 cm−1 (benzene ring breathing), 642 cm−1 (benzene ring deformation), and 432 cm−1 (Cα‐Cβ‐Cγ bending). © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 61–71, 1998

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.