STRUCTURE AND INTERACTIONS OF THE SINGLE-STRANDED-DNA GENOME OF FILAMENTOUS VIRUS FD - INVESTIGATION BY ULTRAVIOLET RESONANCE RAMAN-SPECTROSCOPY

The filamentous bacteriophage fd is a member of the Ff class of Inovir us, which includes phages f1 and M13. Ultraviolet resonance Raman (UVR R) spectra of fd have been obtained using excitation wavelengths of 25 7, 244, 238, and 229 nm. Excitation at 257 nm selectively enhances Ram an markers of the packaged single-stranded (ss) DNA genome, while exci tation at the shorter wavelengths favors the detection of Raman signal s from coat protein aromatics, particularly tryptophan (W26) and tyros ine residues (Y21 and Y24) of the viral coat subunit (pVIII). The prin cipal findings are the following: (1) Distinctive markers of dA, dC, d G, and dT residues of the packaged genome are identified in UVRR spect ra of fd excited at 257 and 244 nm, despite the low DNA mass compositi on (12%) of the virion. (2) Raman bands of the bases of packaged ssDNA show extraordinary resonance Raman hypochromism. Raman intensity loss es as large as 80% of the parent DNA nucleotide intensities are observ ed. This is interpreted as evidence of extensive short-range interacti ons involving bases of the packaged genome. (3) Conversely, Raman band s of tryptophan and tyrosine residues of the coat protein generally ex hibit strong hyperchromism. Typically, Raman markers of the aromatic a mino acids are about 3-fold more intense in the UVRR spectrum of fd th an in spectra of the free amino acids. The very high Raman cross secti ons for residues Y21, Y24, and W26 are indicative of unusual hydrophob ic environments in the viral assembly. (4) UVRR band shifts that accom pany the transfer of fd from H2O to D2O solution indicate that bases o f the packaged ssDNA are readily exchanged by the solvent. Similarly, the indole N1H group of W26 is accessible to solvent, as shown by N1H --> N1D exchange in D2O solution. (5) The UVRR markers of the packaged fd genome confirm the conclusion reached previously from off-resonanc e Raman studies that fd DNA nucleosides favor the C3'-endo/anti confor mation, rather than the C2'-endo/anti conformation that is characteris tic of the lowest energy structure of DNA. We conclude that nucleoside conformations of the packaged fd genome are influenced by the specifi c organization of ssDNA and coat protein subunits in the native virion assembly.