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 laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes 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 preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G 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.
Heyse
S
S
Laboratory of Physical Chemistry of Polymers and Membranes, Chemistry Department, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
Ernst
O P
OP
Dienes
Z
Z
Hofmann
K P
KP
Vogel
H
H
D015374:Biosensing Techniques; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008560:Membrane Fluidity; D010743:Phospholipids; D011485:Protein Binding / Q000528:radiation effects; D011956:Receptors, Cell Surface / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D015398:Signal Transduction; D013438:Sulfhydryl Compounds; D015293:Transducin / Q000378:metabolism*
D008051:Lipid Bilayers; D010743:Phospholipids; D011956:Receptors, Cell Surface; D013438:Sulfhydryl Compounds; D012243:Rhodopsin; D015293:Transducin