LIGHT-STABLE RHODOPSIN .2. AN OPSIN MUTANT (TRP-265 -] PHE) AND A RETINAL ANALOG WITH A NONISOMERIZABLE 11-CIS CONFIGURATION FORM A PHOTOSTABLE CHROMOPHORE

In order to prepare a completely light-stable rhodopsin, we have synth esized an analog, II, of 11-cis retinal in which isomerization at the C-11-C-12 cis-double bond is blocked by formation of a cyclohexene rin g from the C-10 to C-13-methyl. We used this analog to generate a rhod opsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K. D., Knox, B. E., and K horana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lamb da(max), 512 nm) formed from opsin and analog II (rhodopsin-II) showed ground state properties very similar to those of rhodopsin, but was n ot entirely stable to light. In the present work, 12 opsin mutants (Al a-117 --> Phe, Glu-122 --> Gln(Ala, Asp), Trp-126 --> Phe(Leu, Ala), T rp-265 --> Ala(Tyr, Phe), Tyr-268 --> Phe, and Ala-292 --> Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117 --> Phe, Glu-122 --> Gln(Ala), Trp-126 --> Leu(Ala), and Trp-265 --> Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117 --> Phe and Trp-126 --> Al a. Upon illumination, the reconstituted pigments showed varying degree s of light sensitivity; the mutants Trp-126 --> Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutan ts Glu-122 --> Asp, Trp-265 --> Ala, Tyr-268 --> Phe, and Ala-292 --> Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduce d light sensitivity was observed with the mutant Trp-265 --> Tyr, whil e the mutant Trp-265 --> Phe was light-insensitive. Consistent with th is result, the mutant Trp-265 --> Phe showed no detectable light-depen dent activation of transducin or phosphorylation by rhodopsin kinase.