Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal.
Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly121 in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambdamax, increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C9 methyl group is a crucial structural anchor for the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly121 of opsin and the C9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambdamax of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C9 methyl group on the chromophore could be partially restored by replacing Gly121 with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C9 methyl group of the chromophore and the opsin in the vicinity of Gly121 on transmembrane helix 3.
Han
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Laboratory of Molecular Biology and Biochemistry, Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Groesbeek
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Smith
S O
SO
Sakmar
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TP
D007536:Isomerism; D008954:Models, Biological; D009154:Mutation; D010782:Photolysis; D011994:Recombinant Proteins / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism
C110176:9-demethylretinal; D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde