Modification of ovine opsin with the photosensitive hydrophobic probe 1-azido-4-[125I]iodobenzene. Labelling of the chromophore-attachment domain.
The hydrophobic photosensitive probe 1-azido-4-[125I]iodobenzene (AIB) partitioned preferentially into photoreceptor disc membranes and, upon u.v. irradiation, became covalently bound to opsin and phospholipid. The labelling of both protein and phospholipid was linearly related to AIB concentration. The amount of probe incorporated into protein was not significantly different when membranes were irradiated at -100 degrees, 4 degrees or 25 degrees C, but irreversible aggregation of monomeric opsin was dramatically reduced by performing the photolysis at -100 degrees C. Labelling of opsin after irradiation at -100 degrees or 4 degrees was not significantly reduced by the presence of lysine in the aqueous buffer, indicating that significant amounts of reactive species did not enter the aqueous phase. The incorporation into phospholipid, unlike that into opsin, decreased as the temperature of irradiation increased. Some labelling of opsin occurred on incubation with pre-photoactivated AIB, indicating that reaction may also occur with reactive species of longer lifetimes than the nitrene. Proteolysis of labelled opsin with Staphylococcus aureus V8 proteinase yielded two radiolabelled membrane-bound fragments. The location of the modified sites (cysteine, tryptophan, tyrosine, lysine and histidine residues: all nucleophiles) in the smaller fragment was entirely consistent with putative models for the protein derived from other studies.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D005136:Eye Proteins; D008956:Models, Chemical; D010446:Peptide Fragments / Q000032:analysis; D010786:Photoreceptor Cells; D012168:Retinal Pigments; D017299:Rod Opsins; D012756:Sheep
D000345:Affinity Labels; D001386:Azides; D005136:Eye Proteins; D010446:Peptide Fragments; D012168:Retinal Pigments; D017299:Rod Opsins; C020866:1-azido-4-iodobenzene