Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal.
A photoactivatable analog of 11-cis-retinal has been used to probe the orientation of retinal in bovine rhodopsin. The analog binds to the opsin to regenerate a chromophore with lambda max at 458 nm. The linkage site of the analog to the opsin was confirmed to be Lys-296 as in 11-cis-retinal rhodopsin. The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination. On photolysis of rhodopsin containing the radioactively labeled analog at 365 nm at -15 degrees C, 20-25% of the analog was covalently linked to the protein. Proteolysis of the labeled protein and characterization of the appropriate peptides showed that cross-linking of the analog was predominantly to helices C or F. When analog reconstituted rhodopsin in rod outer segments was photolyzed, cross-linking was predominantly to helix C. However, when analog-reconstituted rhodopsin, purified in lauryl maltoside, was photolyzed, labeling occurred mainly in helix F. Sequence analysis showed major sites of cross-linking to be Phe-115, Ala-117, Glu-122, Trp-126, and Ser-127 in helix C while Trp-265 was the major site in helix F. The results suggest that the beta-ionone ring of retinal orients toward helices C and F.
Nakayama
T A
TA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010782:Photolysis; D011487:Protein Conformation; D012168:Retinal Pigments / Q000378:metabolism*; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D017299:Rod Opsins; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism
D003432:Cross-Linking Reagents; D005136:Eye Proteins; D012168:Retinal Pigments; D012176:Retinoids; D017299:Rod Opsins; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde