Light-induced conformational changes of rhodopsin probed by fluorescent Alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-indu ced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of flu orescence changes at a red excitation light wavelength of 605 nm, thus avoi ding significant rhodopsin bleaching. Upon absorption of a photon by rhodop sin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of similar to6.8 at 4 degreesC, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fl uorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluo rescence emission intensity was reduced by addition of GTP, in agreement wi th the fact that transducin enhances the formation of metarhodopsin II. Und er conditions that favored the formation of a metarhodopsin II-Alexa594 com plex, transducin slightly decreased the fluorescence. In the presence of ar restin, under conditions that favored the formation of metarhodopsin I or I I, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly de creased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin an d transducin. These results demonstrate the application of Alexa594-modifie d rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interact ions with other proteins.