Light-stable rhodopsin. II. 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 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.
Ridge
K D
KD
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Bhattacharya
S
S
Nakayama
T A
TA
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics*; D051606:G-Protein-Coupled Receptor Kinase 1; D007536:Isomerism; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D017299:Rod Opsins; D013057:Spectrum Analysis
D005136:Eye Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1