PHOTOAFFINITY-LABELING STUDIES OF BACTERIORHODOPSIN WITH [15-3H]-3-DIAZO-4-KETO-ALL-TRANS-RETINAL

Photoaffinity mapping can be used to help clarify the tertiary structures of the retinal proteins bacteriorhodopsin (bR) and rhodopsin (Rh). An efficient photoaffinity labeled retinal analogue, [15-3H]-all-trans-3-diazo-4-ketoretinal (specific activity >4 Ci/mmol), has been developed as a probe for clarifying the binding site structure around the chromophoric ionone ring in bR. This analogue is functional (20% of the natural retinal) in bR and can be readily photolyzed, covalently binding the chromophore to amino acids in the receptor site. Enzymatic and chemical cleavages of labeled bR followed by HPLC purification and Edman degradation of the radiolabeled peptide fragments identified radiolabeled amino acids Ala126/Leu127 and Trp137/Trp138. The pattern of the labeled amino acid positions permits predictions of the structure of the retinal binding region in bR. © 1990, American Chemical Society. All rights reserved.