PROPERTIES OF EARLY PHOTOLYSIS INTERMEDIATES OF RHODOPSIN ARE AFFECTED BY GLYCINE-121 AND PHENYLALANINE-261

Glycine 121 in transmembrane (TM) helix 3 and phenylalanine 261 in TM helix 6 of bovine rhodopsin have been shown to be critical residues fo r creating an appropriate chromophore binding pocket for 11-cis-retina l [Han, M., Lin, S. W., Smith, S. O., and Sakmar, T. P. (1996) J. Biol . Chem. 271, 32330-32336; Han, M., Lin, S. W., Minkova, M., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32337-32342]. To furt her explore structure-function relationships in the vicinity of recept or helices 3 and 6, time-resolved absorption difference spectra of rho dopsin mutants G121A, G121V, and G121L/F261A were obtained at 20 degre es C. Data were collected from 30 ns to 690 ms after laser photolysis with 7 ns pulses (lambda(max) = 477 nm) and analyzed using a global ex ponential fitting procedure after singular value decomposition (SVD), For each mutant, the decay of its bathorhodopsin photoproduct (bathe) into an equilibrium with its blue-shifted intermediate (bsi) was too f ast to resolve (<20 ns). The reaction scheme found for the mutants G12 1A and G121L/F261A was batho/bsi --> lumirhodopsin (lumi) --> metarhod opsin I (MI) --> metarhodopsin II (MII). For G121V, an additional earl y 380 nm absorber, with a back-reaction to lumi, had to be included in the above scheme, For the three Gly(121) mutants, the main pathway to reach the active MII state is via lumi and MI. This is in contrast to rhodopsin where the main pathway in detergent samples is via lumi and an early 380 nm absorber, MI380. From the accelerated batho decay pre sent in all three mutants, we conclude that Gly(121) is likely to part icipate in the earliest chromophore-protein interactions, In addition, bsi decay is further accelerated in mutant G121L/F261A, suggesting th at Phe(261) is an essential determinant of the protein processes invol ved in bsi decay.