title: Contribution of glutamic acid in the conserved E/DRY triad to the functional properties of rhodopsin.
abstract: Rhodopsin is a G protein-coupled receptor specialized for photoreception and contains a light-absorbing chromophore retinal that binds to the lysine residue of opsin through a protonated Schiff base linkage. Light converts rhodopsin to an equilibrium mixture of the active state metarhodopsin II (MII) and its precursor, metarhodopsin I (MI), which have deprotonated and protonated Schiff base chromophores, respectively. This equilibrium was thought to depend on the pKa of not the Schiff base chromophore but glutamic acid E134 in the highly conserved E/DRY triad in helix III. We performed mutational analyses of E134 and nearby residues to examine whether the equilibrium is really dependent on the pKa of E134 and to obtain clues about the contribution of E134 to the G protein activation characteristics of rhodopsin. All the single mutants at position 134 except for E134D lost the characteristic pH-dependent equilibrium, indicating that the carboxyl group of E134 is responsible for the equilibrium. Interestingly, mutation at position 134 caused little change in the MI or MII spectra or G protein activation efficiency of MII, while it caused a shift of the MI-MII equilibrium. The mutants containing hydrophobic or amide-containing residues at position 134 formed an equilibrium in favor of MII, resulting in an increase in light-induced G protein activation efficiency. On the other hand, the wild type exhibited an opsin activity lower than those of the mutants, which exhibited reasonable light-dependent activities. These results strongly suggest that the evolutionary significance of E134 is not an increase in G protein activity but rather suppression of the opsin activity.
authors: Department of Biophysics, Graduate School of Science, Kyoto University , Kyoto 606-8502, Japan.
authors: Keita
authors: K
authors: Sato
authors: Takahiro
authors: T
authors: Yamashita
authors: Yoshinori
authors: Y
authors: Shichida
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D018698:Glutamic Acid; D012243:Rhodopsin; D015293:Transducin