Characterization of the Mutant Visual Pigment Responsible for Congenital Night Blindness: A Biochemical and Fourier-Transform Infrared Spectroscopy Study†
A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly 90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D
... tion of the G90D pigment chromophore [Sieving, P. ]. Based on existing models of rhodopsin and in Vitro biochemical studies of site-directed mutants, it appears likely that Gly 90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu 113 and increased hydrogen-bond strength of Asp 83 . Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin. 1 Abbreviations: bp, base pairs; con A, concanavalin A; FTIR, Fourier-transform infrared; HOOP, hydrogen-out-of-plane; MII, metarhodopsin II; ROS, rod outer segment. 7536 Biochemistry 1996, 35, 7536-7545 S0006-2960(96)00391-1 CCC: $12.00 FIGURE 1: UV-visible absorption spectra of mutant pigments. Spectra of pigments G90D (A) and G90D/E113A (B) after regeneration with 11-cis-retinal and purification in dodecyl maltoside detergent buffer in darkness are shown. The λ max values were 484 and 474 nm, respectively.