Structure and Interactions of the Single-Stranded DNA Genome of Filamentous Virusfd:  Investigation by Ultraviolet Resonance Raman Spectroscopy

Abstract

The filamentous bacteriophage fd is a member of the Ff class of Inovirus, which includes phages f1 and M13. Ultraviolet resonance Raman (UVRR) spectra of fd have been obtained using excitation wavelengths of 257, 244, 238, and 229 nm. Excitation at 257 nm selectively enhances Raman markers of the packaged single-stranded (ss) DNA genome, while excitation at the shorter wavelengths favors the detection of Raman signals from coat protein aromatics, particularly tryptophan (W26) and tyrosine residues (Y21 and Y24) of the viral coat subunit (pVIII). The principal findings are the following: (1) Distinctive markers of dA, dC, dG, and dT residues of the packaged genome are identified in UVRR spectra of fd excited at 257 and 244 nm, despite the low DNA mass composition (12%) of the virion. (2) Raman bands of the bases of packaged ssDNA show extraordinary resonance Raman hypochromism. Raman intensity losses as large as 80% of the parent DNA nucleotide intensities are observed. This is interpreted as evidence of extensive short-range interactions involving bases of the packaged genome. (3) Conversely, Raman bands of tryptophan and tyrosine residues of the coat protein generally exhibit strong hyperchromism. Typically, Raman markers of the aromatic amino acids are about 3-fold more intense in the UVRR spectrum of fd than in spectra of the free amino acids. The very high Raman cross sections for residues Y21, Y24, and W26 are indicative of unusual hydrophobic environments in the viral assembly. (4) UVRR band shifts that accompany the transfer of fd from H2O to D2O solution indicate that bases of 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-resonance Raman studies that fd DNA nucleosides favor the C3'-endo/anti conformation, rather than the C2'-endo/anti conformation that is characteristic of the lowest energy structure of DNA. We conclude that nucleoside conformations of the packaged fd genome are influenced by the specific organization of ssDNA and coat protein subunits in the native virion assembly.

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

“…3, B and C). The U-shaped N-terminal loop that lies at the bottom of the ␤-barrel is followed by its first ␤-strand, ␤1 (residues [13][14][15][16][17]. From ␤1 the polypeptide chain crosses the top of the ␤-barrel to form ␤2 (26 -29) on its opposite side.…”

“…In M13, the major coat protein pVIII forms the long, cylindrical phage coat that packages the phage genome. pIII, pVI, and pI are minor coat proteins located at the phage tips (12)(13)(14). Although pVI and pI are very small, pIII is a large protein, present in four-five copies at one end of the phage particle.…”

Raman, polarized Raman and ultraviolet resonance Raman spectroscopy of nucleic acids and their complexes

“…Excitation at 257 nm results in a UVRR spectrum dominated by viral DNA, while excitation at 229 nm yields a UVRR profile primarily of tryptophan and tyrosine residues of the capsid subunit, as labeled. 74,159,198,199 52 Earlier studies report scattering cross-sections for several additional UV excitation wavelengths. 68,69 Spectral data collection and processing Raman spectra of nucleic acids exhibiting acceptable signalto-noise ratios (¾10 : 1 or greater for the most intense bands in the spectrum) are typically generated by averaging independently collected sets of data on the same or identical samples.…”

“…Because filamentous viruses are not amenable to study by high-resolution structural methods, Raman spectroscopy has long been recognized as a convenient structural alternative for both the protein and DNA constituents. 157,158 In the well-studied filamentous phages fd, Pf1, Pf3 and PH75, Raman markers of the deoxynucleosides indicate, respectively, C3 0 -endo/anti (665 cm 1 ), 158,159 C2 0 -endo/anti (682 cm 1 ), 158,160 C3 0 -endo/anti (663 cm 1 ) 158,161 and C2 0 -endo/anti (679 cm 1 ) 162 conformations. In a related application, interactions of packaged ssDNA genomes of fd and Pf1 with metal ions have also been probed by Raman methods.…”

Discrimination between UV radiation‐induced and thermally induced spectral changes in AT‐paired DNA oligomers using UV resonance Raman spectroscopy

“…For example, where overlapping neighboring peaks respond differently to UV and heat exposure, a difference spectrum feature suggestive of a peak shift may be observed where, in fact, none is present. This is particularly relevant near 1330-1340 cm 1 and again near 1480-1510 cm 1 . Further, where the same band responds with different intensity changes to UV exposure and heat, temperature profiles could appear to be more complex than they in fact are, or appear to be unaffected when the opposite may be true.…”