Resolution enhancement in Raman spectra of biological macromolecules by Fourier deconvolution: applications to single-stranded DNA and RNA viruses

Abstract

The application of a constrained, iterative Fourier deconvolution method to Raman spectra of viruses permits separation of overlapped vibrational bands assigned to viral protein and nucleic acid constituents. The intrinsically broad and extensively overlapped Raman lines, which cannot be resolved by instrumental methods, are sufficiently well separated in the deconvoluted spectra so that band areas may be measured with sufficient accuracy to allow conclusions about the secondary structures and hydrogen bonding interactions of viral molecular components. Deconvolution of Raman scattering in the region 1500–1750 cm −1 of filamentous bacteriophages resolves the contributions of(i)amide I modes ofalpha-helix and beta-strand conformations of the viral coat proteins, (ii) aromatic ring modes of tryptophan, tyrosine and phenylalanine side chains and (iii) purine ring modes of the coated DNA molecule. The results show that among six filamentous viruses the amount of alpha-helix in coat protein subunits decreases in the following order: Pfl (100%) > IKe (93%) > fd (92%) > Ifl (90%) > Pf3 (82%) > Xf (71%). In an application to tobacco mosaic virus (TMV), the complex band in the 800–850 cm −1 region is deconvoluted to resolve contributions from the encapsidated RNA genome at 813 and 822 cm −1, which indicate at least two distinct nucleoside conformers and a contribution from the tyrosine rings of protein subunits at 831 cm −1. Integrated intensity measurements suggest that at least two-thirds of the nucleosides do not contain the usual C3′-endo ring pucker and anti orientation of the glycosidic bond normally associated with nucleoside residues of single-stranded RNA.