Orientations of Tyr 21 and Tyr 24 in the Capsid of Filamentous Virus Ff Determined by Polarized Raman Spectroscopy^,

Abstract

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

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

“…The abc system is fixed to the virion, with c as the virion axis, whereas a and b are equivalent and perpendicular to c. θ is the angle between the z and c axes, while is the angle between y and the line of intersection of the xy and ab planes (28). The Raman tensor coordinate systems for the 854 and 1206 cm -1 modes are the same, viz.…”

“…In the case of Ff, which has been studied most extensively, the Tyr 21, Tyr 24, and Trp 26 side-chain conformations were independently resolved (24,(26)(27)(28). The average angle of inclination of the Ff subunit R-helix from the virion axis was also determined (θ h ) 16°( 4°) (25).…”

“…Other applications of polarized Raman methods to the filamentous viruses Ff (strains fd and f1) and Pf1 have been described (19,(24)(25)(26)(27)(28) and reviewed elsewhere (29,30). The use of polarized infrared spectroscopy to study oriented gels of Ff and Pf1 virions has also been reported (9,31).…”

Raman spectroscopy of proteins: from peptides to large assemblies

“…107 This permitted the determination of main-chain and side-chain orientations and led to extensive revisions and refinements of the diffraction-based models. 105,106,108 Recent structural results, which were obtained for the filamentous viruses by Raman spectroscopy, are described below. Pf1 is a long (2 µm) and thin (6.5 nm) bacteriophage infecting Pseudomonas aeruginosa and belongs to the structural class II of filamentous viruses (lattice symmetry C 1 S 5.4 ).…”

Micro-Raman spectroscopy study of ALVAC virus infected chicken embryo cells

“…Raman spectroscopy has been successfully used for studying various types of viruses and has led to the identification of a novel conformation-dependent Raman bands in a native alpha-helix assembly. [1][2][3] Furthermore, this has led to identification of tyrosine Raman markers (Fermi doublet) near 830 and 850 cm -1 that are sensitive in intensity or frequency to the hydrogen bonding state of the phenolic group in tyrosine. 4 Technical improvement of spectrophotometers has made it possible to measure Raman spectra of a single cell [5][6][7] , a single E. coli bacterium (trapped in optical tweezers) 8,9 , and a single optically trapped bacterial spore.…”