Topographical and active site studies on rhodopsin

Mullen, Eric (1981) Topographical and active site studies on rhodopsin. University of Southampton, Doctoral Thesis.

Abstract

The retinal-binding site of bacteriorhodopsin was characterized. A radiochemical approach was used to show that the retinyl moiety of the chromophore was attached to the c-amino group of lysine. The retinyl-opsin bond of bacteriorhodopsin was stabilised by the conversion of a retinyl moiety into a carboxymethyl group, which was achieved by treatment of the labelled protein with ozone and performic acid. This treatment was coupled with degradative strategies to isolate active site peptides of bacteriorhodopsin. The active site lysine was identified as lysine-216 of the protein. Rhodopsin was treated with papain to yield three fragments of approximate molecular weights 23,000, 15,000 and 6,000(H, M and L respectively). The H-fragment contained the SH-group modified by N-ethyl maleimide, the M-fragment theretinal-binding site and the L-fragment an SH-group modified by N-ethyl maleimide and 5-iodoacetamidosalicylate. Thethree, labelled, fragments of papain-cleaved rhodopsin were isolated by affinity chromatography and ion exchange chromatography. An active site peptide of rhodopsin was isolated from L- plus M-fragments and M-fragment. The activesite lysine was identified as the 53rd from the carboxyl-terminal. Isolation of pure L-fragment allowed the sites of papain cleavage between the M- and L-fragments to be identified as the amino terminals of Glutamine and Phenylalanine, 37 and 36 residues from the carboxyl terminal.

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