Light-stable rhodopsin. II. An opsin mutant (TRP-265→PHE) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore

Ridge, Kevin D. ; Bhattacharya, S. ; Nakayama, Tomoko A. ; Khorana, H. Gobind (1992) Light-stable rhodopsin. II. An opsin mutant (TRP-265→PHE) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore Journal of Biological Chemistry, 267 (10). pp. 6770-6775. ISSN 0021-9258

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Official URL: http://www.jbc.org/content/267/10/6770.abstract?si...

Abstract

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117→Phe, Glu-122→Gln(Ala, Asp), Trp-126→Phe(Leu, Ala), Trp-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→Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126→Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122→Asp, Trp-265→Ala, Tyr-268→Phe, and Ala-292→Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265→Tyr, while the mutant Trp-265→Phe was light-insensitive. Consistent with this result, the mutant Trp-265→Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase.

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