Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin.
By using a photoactivatable analog of 11-cis-retinal in rhodopsin, we have previously identified the amino acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, and Trp-265 as major sites of cross-linking to the chromophore. To further investigate the amino acids that interact with retinal, we have now used site-directed mutagenesis to replace a variety of amino acids in the membrane-embedded helices in bovine rhodopsin, including those that were indicated by cross-linking studies. The mutant rhodopsin genes were expressed in monkey kidney cells (COS-1) and purified. The mutant proteins were studied for their spectroscopic properties and their ability to activate transducin. Substitution of the two amino acids, Trp-265 and Glu-122 by Tyr, Phe, and Ala and by Gln, Asp and Ala, respectively, resulted in blue-shifted (20-30 nm) chromophore, and substitution of Trp-265 by Ala resulted in marked reduction in the extent of chromophore regeneration. Light-dependent bleaching behavior was significantly altered in Ala-117----Phe, Trp-265----Phe, Ala, and Ala-292----Asp mutants. Transducin activation was reduced in these mutants, in particular Trp-265 mutants, as well as in Glu-122----Gln, Trp-126----Leu (Ala), Pro-267----Ala (Asn, Ser), and Tyr-268----Phe mutants. These findings indicate that Trp-265 is located close to retinal and Glu-122, Trp-126, and probably Tyr-268 are also likely to be near retinal.
Nakayama
T A
TA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004252:DNA Mutational Analysis; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012172:Retinaldehyde / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D012172:Retinaldehyde