Direct Monitoring of GPCR Reconstitution and Ligand-Binding Activity by Plasmon Waveguide Resonance.

Abstract

The study of G-protein-coupled receptor (GPCR ) mechanisms of activation and signaling often the isolation, purification, and reconstitution of GPCRs in model lipid membranes. GPCR reconstitution from a detergent-micelle system into model membranes is usually a laborious process whose success is tested after the whole process is over by rather indirect methods such as SDS-PAGE and western blotting or by biophysical approaches. Following that, protein activity is measured in yet a different experimental setup. Overall, the whole procedure is tedious, long, and often requires high quantities of material and the use of labeled proteins. Herein, a protocol is described to follow, in a single experimental setup, both GPCR reconstitution from detergent micelles into planar lipid bilayers and its ligand-binding activity using plasmon waveguide resonance. Alternatively, receptor/ligand interactions can also be investigated from cell membrane fragments overexpressing the receptor of interest, bypassing isolation and reconstitution procedures. The method is direct, sensitive, and does not rely on the use of any labeled material.