Single-molecule analysis of ligand efficacy in βAR-G-protein activation.

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β adrenergic receptor (βAR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound βAR-G complexes are formed and the efficiency of nucleotide exchange leading to G activation. These data also reveal transient nucleotide-bound βAR-G species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.