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

Abstract

The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() 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 (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-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 E(2)()-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 (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-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.

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

“…Furthermore, the DNA molecule itself could be dynamic in the hydrated form of the Pf1 virus, even though there is order in the phosphate backbone and nucleosides of the DNA (15). For instance, solid-state studies of a hydrated, Watson-Crick basepaired DNA molecule show considerable submicrosecond dynamics in the backbone and the deoxyribose ring (39,42).…”

“…For instance, solid-state studies of a hydrated, Watson-Crick basepaired DNA molecule show considerable submicrosecond dynamics in the backbone and the deoxyribose ring (39,42). By comparison, an extended DNA in Pf1 with bases directed outward toward the capsid (11,15) may have additional dynamic modes or different amplitudes in the normal modes, such as greater torsional motion about the N-glycosidic bonds (43). Given the intimate contacts between the DNA and protein implicit in the unit stoichiometric ratio, increased motion in the Pf1 DNA molecule could further increase dynamics of the protein side chains in contact with the DNA.…”

“…For example, it has been recently proposed that there is a minor ''outward-bulge'' conformer in the backbone of residues 15-17 that is highly dynamic (16), but such a minor conformer was not observed in the 13 C and 15 N chemical shift assignments (18). Is the backbone indeed dynamic in this region?The DNA in Pf1 is known to be highly extended (4, 9), and it has been proposed to be highly twisted to follow the helical symmetry of the capsid (11) but the manner of the match between DNA and capsid helical symmetries remains a matter of debate (11,15,(24)(25)(26). There is evidence supporting a P form DNA with exposed bases on the outside and ordered phosphates at the center (11,15,26,27).…”

“…The virion consists of a circular single-stranded DNA genome of 7,349 nt (3) within a cylindrical capsid of Ϸ7,300 major coat protein subunits that have 1:1 stoichiometric interactions (4,5) with nucleotides along the filament, as well as a small number of other proteins interacting with reverse turns of DNA at the ends. A variety of spectroscopic and diffraction studies of Pf1 have been carried out (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), including studies by solid-state NMR of the protein in the intact virion (16)(17)(18)(19), as in the present study, and by solution NMR of the isolated protein in detergent micelles (8,14) as a model of its structure in the membrane before virion assembly. Virion assembly occurs at the membrane in such a way that major coat protein subunits are packed in the filament as ␣-helices with their Nterminal regions on the outside and their C-terminal regions buried in the interior to interact with the DNA.…”

“…There is evidence supporting a P form DNA with exposed bases on the outside and ordered phosphates at the center (11,15,26,27). How might such an unusual DNA conformation affect virion dynamics?…”