Application of Surface Plasmon Resonance Spectroscopy to Study G-Protein Coupled Receptor Signalling

Abstract

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.