Incorporation of Rhodopsin in Laterally Structured Supported Membranes: Observation of Transducin Activation with Spatially and Time-Resolved Surface Plasmon Resonance
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- 作者：Stephan Heyse ; Oliver P. Ernst ; Zoltan Dienes ; Klaus Peter Hofmann ; and Horst Vogel
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：January 13, 1998
- 年：1998
- 卷：37
- 期：2
- 页码：507 - 522
- 全文大小：245K
- 年卷期：v.37,no.2(January 13, 1998)
- ISSN：1520-4995
文摘
Rhodopsin-transducin coupling was used as an assay toinvestigate a laterally patternedmembrane reconstituted with a receptor and its G protein. Itserved as a model system to show thefeasibility to immobilize G protein-coupled receptors on solid supportsand investigate receptor activationand interaction with G proteins by one-dimensional imaging surfaceplasmon resonance. Supportedmembranes were formed by the self-assembly of lipids and rhodopsin fromdetergent solution ontofunctionalized gold surfaces. They formed micrometer-sizedalternating regions of pure fluid phospholipidbilayers separated by bilayers composed of an outer phospholipidleaflet on a gold-attached inner thiolipid.Rhodopsin was found to incorporate preferentially into thephospholipid bilayer regions, whereas transducinwas uniformly distributed over the entire outer surface of thesupported patterned membrane. The influenceof rhodopsin on the dark binding of transducin to lipid membranes wasdescribed quantitatively andcompared with previously published data. Coupling reactions withtransducin resembled closely the nativesystem, indicating that the native functionality of rhodopsin waspreserved in the supported membranes.The spatially varying properties of the membranes resulted in apattern of rhodopsin activity on the surface.This combination of techniques is very promising for theinvestigation of the lateral diffusion of transducin,can be extended to include signalling proteins downstream of the Gprotein, and may be applied to functionalscreening of other G protein-coupled receptors. In the future, itmay also serve as a basis for constructingbiosensors based on receptor proteins.