INCORPORATION OF RHODOPSIN IN LATERALLY STRUCTURED SUPPORTED MEMBRANES - OBSERVATION OF TRANSDUCIN ACTIVATION WITH SPATIALLY AND TIME-RESOLVED SURFACE-PLASMON RESONANCE
S. Heyse et al., INCORPORATION OF RHODOPSIN IN LATERALLY STRUCTURED SUPPORTED MEMBRANES - OBSERVATION OF TRANSDUCIN ACTIVATION WITH SPATIALLY AND TIME-RESOLVED SURFACE-PLASMON RESONANCE, Biochemistry, 37(2), 1998, pp. 507-522
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.