Surface plasmon resonance study of g protein/receptor coupling in a lipid bilayer-free system.
Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (Gt) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of Gt following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent KD of the Gt/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.
Komolov
Konstantin E
KE
AG Biochemistry, Faculty V, IBU, University of Oldenburg, D-26111 Oldenburg, Germany.
Senin
Ivan I
II
Philippov
Pavel P
PP
Koch
Karl-Wilhelm
KW
D000818:Animals; D002417:Cattle; D008051:Lipid Bilayers; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D008051:Lipid Bilayers; D043562:Receptors, G-Protein-Coupled