Orientations of Tyr 21 and Tyr 24 in the capsid of filamentous virus Ff determined by polarized Raman spectroscopy

The capsid of filamentous virus Ff is assembled from similar to 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 2 1 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, ut ilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman si gnature of Tyr 21 was obtained by incorporating C-delta1,C-delta2,C-epsilon 1,C-epsilon2-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signatur e of Tyr 24 was obtained by incorporating C-delta1,C-delta2,C-epsilon1,C-ep silon2-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 220 0-2400 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 ba nds of Ff near 642 and 855 cm(-1), which originate from vibrational modes o f the tyrosine phenolic ring. The results indicate the following: (i) For b oth 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) t he mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman marke rs 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 e ither Tyr 21 or Tyr 24, indicating that nonrandom base orientations of pack aged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A m olecular model consistent with these findings is proposed.