Effect of rhodopsin Cysteine modifications on cooperative binding of transducin.

Abstract

Abstract: : Light activated rhodopsin interacts with heterotrimeric G–protein transducin to initiate phototransduction. Rhodopsin–transducin binding is subject to complex allosteric regulation and shows strong positive cooperativity. The nature of this effect is unclear but is consistent with multi–site interactions. We hypothesize that interaction of rhodopsin with two distinct sites on transducin alpha and gamma subunits contributes to the cooperativity of binding. We have previously shown that selective chemical modification of Cysteine 140 by NEM abolishes interactions between Metarhodopsin II and a synthetic peptide derived from the C–terminus of the alpha– but not gamma subunit. We analyze GTP–dependent binding and release of transducin to light activated rhodopsin in native membranes, and to rhodopsin with Cys 140 modified by NEM. We show that while maximal extent of binding to modified rhodopsin is preserved, the positive cooperativity of binding is lost. The implications of this result for the mechanism of rhodopsin–catalyzed activation of transducin is discussed. This work is supported in part by NIH and Foundation for Research to Prevent Blindness.

Keywords: signal transduction • protein structure/function • photoreceptors