Biochemical Characterization of GPCR-G Protein Complex Formation.

The complex of G protein-coupled receptors (GPCR) and G proteins is the core assembly in GPCR signaling in eukaryotes. With the recent development of cryo-electron microscopy, there has been a rapid growth in structures of GPCR-G protein complexes solved to near-atomic resolution, giving important insights into this signaling complex. Here we describe the biochemical protocol to study the interaction between GPCRs and G proteins before preparation of GPCR-G protein complexes for structural studies. We use gel filtration to analyze the binding properties between GPCR and G protein with the presence of agonist or antagonist, as well as the complex dissociation in the presence of GTP analogue. Methods used in the protocol are affinity purification and gel filtration, which are also commonly used in protein sample preparation for structural work. Therefore, the protocol can be easily adapted for large-scale sample preparation.