Environments of Tyrosine and Tryptophan in Rhodopsin as Compared to Bacteriorhodopsin

The UV fluorescence spectrum of rhodopsin in bovine disk membranes is characterized by emission from both tyrosine and tryptophan residues. Analyses of the spectra at different excitation wavelengths in the presence and absence of guanidine hydrochloride reveal three distinct classes of tryptophan residues in rhodopsin. One of these is buried in non-polar regions of the protein, another is on the surface but in limited contact with water, and the third category is completely exposed to water. Most of the tryptophan residues belong to the first two classes in rhodopsin. When compared to bacteriorhodopsin (BR), considerable differences in the fluorescence characteristics of rhodopsin can be observed. In the presence of guanidine hydrochloride, a greater proportion of tryptophan is exposed in BR than in rhodopsin. At pH ~ 11, ionization of tyrosine to tyrosinate is suggested by a shoulder at 340 nm in the fluorescence spectrum. However, no shift of the emission maximum is observed at pH 11, indicating that, unlike BR, a majority of the tyrosine residues in rhodopsin is buried. In contrast to the BR case, the temperature dependence of the emission spectrum of tryptophan reflects no lipid phase transition; rather the change is consistent with the thermal denaturation of the rhodopsin molecule. The results suggest that, despite the apparent structural similarity, significant differences between BR and mammalian rhodopsin may exist in amino acid sequence, in disposition of the protein with respect to the membrane and in the lipid—protein interaction.