ROOM-TEMPERATURE TRAPPING OF RHODOPSIN PHOTOINTERMEDIATES

By suspending bovine rhodopsin in trehalose-water glass films, it is p ossible to trap photostates in the light-activation process. Because o f the unusually high vitrification temperature of trehalose-water mixt ures, this trapping can be accomplished at room temperature. This allo ws for a facile investigation of the spectroscopic properties of rhodo psin's photointermediates. Depending on experimental conditions, it is possible to trap photolysis products that have visible absorbance spe ctra closely resembling the two different photointermediates, metarhod opsin I and metarhodopsin II. When rhodopsin is maintained in the nati ve rod outer segment membrane, the photolysis product has the spectral properties of metarhodopsin I. Upon detergent solubilization, the pho tolysis product closely resembles metarhodopsin II. Ultraviolet circul ar dichroism spectra show that the metarhodopsin I product had no chan ge in secondary structure compared with unbleached rhodopsin. The meta rhodopsin II product did show a significant decrease in alpha-helical content. Resonance energy transfer was measured from extrinsic probes located on each of the cytoplasmic cysteine residues to the retinal in the trapped photoproducts. It is seen that these distances are the sa me for rhodopsin and metarhodopsin I while metarhodopsin II shows cons iderably shorter distances. Metarhodopsin II is intimately associated with the signal transduction process, and the present results suggest that large structural changes have occurred in the transition to this state. These results demonstrate the utility of room temperature trapp ing of photostates in trehalose-water glasses.