Application of surface plasmon resonance spectroscopy to study G-protein coupled receptor signalling

Methods Mol Biol. 2010:627:249-60. doi: 10.1007/978-1-60761-670-2_17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Immobilized Proteins / analysis
  • Immobilized Proteins / chemistry
  • Immobilized Proteins / metabolism
  • Light
  • Optical Fibers
  • Rhodopsin / analysis*
  • Rhodopsin / chemistry
  • Rhodopsin / metabolism
  • Surface Plasmon Resonance / instrumentation
  • Surface Plasmon Resonance / methods*
  • Surface Properties
  • Transducin / metabolism

Substances

  • Immobilized Proteins
  • Rhodopsin
  • Transducin