Phage phi X174 probed by laser Raman spectroscopy: evidence for capsid-imposed constraint on DNA secondary structure.
The Raman spectrum of the isometric bacteriophage phi X174 contains a number of well-resolved bands which have been assigned unambiguously to proteins of the capsid or to the single-stranded DNA (ssDNA) genome. Additional Raman bands of protein and DNA, which are partially overlapped in the spectrum of virus, have been resolution enhanced by Fourier deconvolution to permit improved semiquantitative measurement of spectral intensities and frequencies for structural conclusions. Raman conformation markers indicate that the ssDNA molecule within the capsid contains nucleosides of C2'-endo sugar pucker and anti-glycoside bond orientation, but the nucleic acid backbone lacks the geometry characteristic of B-form DNA. The Raman profile of encapsidated phi X DNA indicates a backbone more similar to heat-denatured DNA than to DNA containing hairpinlike secondary structure. This finding suggests limited interbase interactions in the packaged genome, which is presumably the result of constraints imposed by the viral capsid. Thus, the extensive pairing and stacking of bases indicated by Raman profiles from ssRNA viruses are not evident for the phi X174 chromosome. Overall, the proteins of the virion contain extensive beta-sheet and irregular secondary structures. Fourier deconvolution of the Raman amide I band provides an estimate of the percentage of total beta-sheet structure (approximately 60%) in all proteins of the virion. The amide III region of the spectrum confirms that beta-sheet and irregular domains are the predominant protein secondary structures. Samples of phi X174 concentrated for Raman spectroscopy by either ultracentrifugation or ultrafiltration exhibit nearly identical Raman spectra, indicating that either method can be employed to prepare intact virus without significant loss of DNA or protein components.
D010584:Bacteriophage phi X 174 / Q000648:ultrastructure*; D002213:Capsid / Q000502:physiology*; D004279:DNA, Viral / Q000302:isolation & purification / Q000648:ultrastructure*; D004926:Escherichia coli / Q000648:ultrastructure; D005796:Genes; D005814:Genes, Viral; D007834:Lasers; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods; D014764:Viral Proteins / Q000032:analysis
D004279:DNA, Viral; D014764:Viral Proteins