WATER AND PEPTIDE BACKBONE STRUCTURE IN THE ACTIVE-CENTER OF BOVINE RHODOPSIN

Difference FTIR spectra in the conversion of rhodopsin or isorhodopsin to bathorhodopsin were recorded for recombinant wild-type and E113Q b ovine rhodopsins. Differences in various vibrational modes between E11 3Q and the wild-type proteins whose Schiff bases interact with chlorid e and Glu113, respectively, were analyzed. Water molecules in rhodopsi n that change upon formation of bathorhodopsin are detected by a chang e in frequency of the O-H stretching vibration from 3538 to 3525 cm(-1 ). This change in the wild-type protein is absent in E113Q. One or a f ew water molecules are therefore suggested to be located in the proxim ity of Glu113, the counterion of the Schiff base. Another water vibrat ion at 3564 cm(-1), which is shifted to 3542 cm(-1) in bathorhodopsin in the wild type, persists in E113Q but with similar to 5-cm(-1) shift toward higher frequency. This is due to water molecules that may be l ocated at a site somewhat more remote from Glu113. Structural changes of some peptide carbonyls and amides are also absent in E113Q. On the other hand, the E113Q protein shows shifts of the N-H+ stretching vibr ational band, that is probably due to the protonated Schiff base, upon conversion of rhodopsin to bathorhodopsin. No corresponding changes w ere observed in the wild type. We propose a model in which a water mol ecule interacts with Glu113, the protonated Schiff base, and peptide c arbonyls, and amides. These residues undergo structural changes upon f ormation of bathorhodopsin.