Conformational Dynamics of a G Protein-Coupled Receptor at the Single-Molecule Level

Abstract

G protein-coupled receptors (GPCRs) are the integral membrane proteins that detect extracellular ligands and mediate signal transduction. Binding of ligands promotes transitions from inactive to active receptor conformations, which are recognized by intracellular effectors. Chemically distinct ligands may trigger different signaling responses by altering dynamics of the receptor. We developed a single-molecule fluorescence system to observe conformational switching of the β2-adrenergic receptor in real-time within a native-like membrane environment. The receptor was covalently labeled with a photostable and environmentally-responsive Cy3 fluorophore at the cytoplasmic end of either transmembrane (TM) helix VI or helix VII. Individual receptor molecules were incorporated in phospholipid nanodiscs, tethered to a microscope cover slip and visualized over time by total internal reflection fluorescence microscopy. We observed spontaneous transitions of TM helices VI and VII between inactive and active conformations, even in the absence of any ligands. Studies of ligands that span a comprehensive range of pharmacological efficacies showed that full agonists shorten the time spent in the inactive conformations and prolong the time in the active conformations, leading to an increased population of active species, while an inverse agonist prolonged the time spent in inactive conformations. These observations provide new insights into the mechanism of GPCR activation and the molecular basis for the variable pharmacological efficacies of different drug molecules.