In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly

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... ng the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state. . 4 The abbreviations used are: Rho-P, phosphorylated rhodopsin; Rho*-P, lightactivated rhodopsin; max , wavelength of maximum absorption or emission; ex , wavelength of excitation; em , wavelength of emission; I72B, bimane-labeled arrestin I72C; DTNB, 5,5Ј-dithiobis(2,2Ј-nitrobenzoic acid); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; DTT, dithiothreitol.