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 mutants, Trp-182→Phe or Trp-189→Phe, from that of the wild-type. Analysis of the spectra shows that the χ2,1 torsion angle about the Cβ−C3 bond is ±93° for Trp-182 and ±100° for Trp-189. Both Trp residues are moderately hydrogen bonded to proton acceptors at their indolyl nitrogens in hydrophobic environments. The environmental hydrophobicity is particularly strong for Trp-182, as judged from the splitting of the W7 Raman band to a triplet. The Raman information on the structure and environment of Trp-189 is consistent with the molecular model from electron diffraction [Grigorieff et al. (1996) J. Mol. Biol. 259, 393−421]. On the other hand, the χ2,1 angle and the hydrogen-bonding state of Trp-182 found here differ from those in the model structure. Revision of the model to correspond to the Raman findings would require a 60° rotation of the Trp-182 indole ring about th...