INCORPORATION OF RHODOPSIN IN LATERALLY STRUCTURED SUPPORTED MEMBRANES - OBSERVATION OF TRANSDUCIN ACTIVATION WITH SPATIALLY AND TIME-RESOLVED SURFACE-PLASMON RESONANCE

Rhodopsin-transducin coupling was used as an assay to investigate a la terally patterned membrane reconstituted with a receptor and its G pro tein. It served as a model system to show the feasibility to immobiliz e G protein-coupled receptors on solid supports and investigate recept or activation and interaction with G proteins by one-dimensional imagi ng surface plasmon resonance. Supported membranes were formed by the s elf-assembly of lipids and rhodopsin from detergent solution onto func tionalized gold surfaces. They formed micrometer-sized alternating reg ions of pure fluid phospholipid bilayers separated by bilayers compose d of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholip id bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The infl uence of rhodopsin on the dark binding of transducin to lipid membrane s was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was pres erved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surfa ce. This combination of techniques is very promising for the investiga tion of the lateral diffusion of transducin, can be extended to includ e signalling proteins downstream of the Cr protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.