ULTRAVIOLET RESONANCE RAMAN-SPECTRA OF TRP-182 AND TRP-189 IN BACTERIORHODOPSIN - NOVEL INFORMATION ON THE STRUCTURE OF TRP-182 AND ITS STERIC INTERACTION WITH RETINAL

Ultraviolet (244 nm) resonance Raman spectra of Trp-182 and Trp-189 in bacteriorhodopsin were obtained by subtracting the spectrum of the mu tants, Trp-182-->Phe or Trp-189-->Phe, from that of the wild-type. Ana lysis of the spectra shows that the chi(2,1) torsion angle about the C -beta-C-3 bond is +/-93 degrees for Trp-182 and +/-100 degrees for Trp -189. Both Trp residues are moderately hydrogen bonded to proton accep ters at their indolyl nitrogens in hydrophobic environments, The envir onmental hydrophobicity is particularly strong for Trp-182, as judged from the splitting oi the W7 Raman band to a triplet. The Raman inform ation on the structure and environment of Trp-189 is consistent with t he molecular model from electron diffraction [Grigorieff et al. (1996) J. Mel. Biol, 259, 393-421]. On the other hand, the chi(2,1) angle an d the hydrogen-bonding state of Trp-182 found here differ from those i n the model structure, Revision of the model to correspond to the Rama n findings would require a 60 degrees rotation of the Trp-182 indole r ing about the C-beta-C-3 bond toward the chromophore retinal and the p resence of a water molecule that is hydrogen bonded to the indolyl nit rogen. The tripler feature of the W7 band of Trp-182 is attributable t o unusually strong steric repulsion between the indole ring and the 9- and 13-methyl groups of the retinal. Resonance Raman spectra in the v isible suggest that this steric conflict destabilizes the 13-cis isome ric state of the retinal.