Protein and DNA Residue Orientations in the Filamentous Virus Pf1 Determined by Polarized Raman and Polarized FTIR Spectroscopy

• Published in: Biochemistry (Easton) Vol. 42; no. 4; pp. 940 - 950
• Main Authors: Tsuboi, Masamichi, Kubo, Yoshiko, Ikeda, Teruki, Overman, Stacy A, Osman, Olivia, Thomas, George J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.02.2003
• Subjects: Alanine - chemistry; Amides - chemistry; Arginine - chemistry; Bacteriophage Pf1 - chemistry; Bacteriophage Pf1 - physiology; Capsid - chemistry; DNA, Single-Stranded - chemistry; DNA, Viral - chemistry; Genome, Viral; Microspectrophotometry; Protein Structure, Secondary; Protein Subunits - chemistry; Spectroscopy, Fourier Transform Infrared - methods; Spectrum Analysis, Raman - methods; Tyrosine - chemistry; Viral Proteins - chemistry; Viral Proteins - physiology; Virion - chemistry; Virion - physiology; Virus Assembly - physiology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi020566v

The Pseudomonas bacteriophage Pf1 is a long (∼2000 nm) and thin (∼6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue α-helical subunit. The coat subunits are arranged as a superhelix of C1 S5.4 symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2‘-endo/anti conformation, and the average DNA phosphodioxy group (PO2 -) is oriented so that the line connecting the oxygen atoms (O···O) forms an angle of 71° ± 5° with the virion axis. Raman and infrared amide band polarizations show that the subunit α-helix axis is inclined at an average angle of 16° ± 4° with respect to the virion axis. The α-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm-1 into apparent A-type and E2 -type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C5 S2 symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed α-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.