Application of surface plasmon resonance spectroscopy to study G-protein coupled receptor signalling.
The G-protein coupled receptor rhodopsin is a classical example of a seven transmembrane helix receptor; it is photoexcited and transmits this light signal to a G-protein mediated cascade. Many components of this receptor-triggered cascade can be purified in their native forms from natural sources making this system most suitable for biophysical studies. A central aspect of cellular signal transduction routes is to understand protein-protein interactions in a quantitative way. Surface plasmon resonance (SPR) spectroscopy is a biosensor-based technique that allows investigating molecular interactions by determining kinetic parameters. We here show how dark-adapted rhodopsin can be immobilized on the sensor chip surface. A laser device implemented in the SPR system allowed us to trigger light-induced conformational changes in rhodopsin and to monitor light-dependent binding of the photoreceptor cell G-protein transducin to rhodopsin. The sensor chip surface can be regenerated and used for several rounds of interaction analysis. Furthermore, illuminated rhodopsin can be regenerated by applying 9-cis-retinal on the sensor chip surface.
Komolov
Konstantin E
KE
Biochemistry Group, IBU, University of Oldenburg, Oldenburg, Germany.
Koch
Karl-Wilhelm
KW
D000818:Animals; D002417:Cattle; D055767:Immobilized Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008027:Light; D055100:Optical Fibers; D012243:Rhodopsin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties; D015293:Transducin / Q000378:metabolism
D055767:Immobilized Proteins; D012243:Rhodopsin; D015293:Transducin