Single-molecule FRET experiments with human A2A adenosine receptor in its apo and agonist-bound states in lipid nanodiscs provide insights into its conformational dynamics and activation. The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Forster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A(2A) adenosine receptor (A(2A)AR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A(2A)AR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A(2A)AR, explaining the receptor's constitutive activity. For the agonist-bound A(2A)AR, we detected faster (390 +/- 80 mu s) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies. A.L., A.B., and V.B. are thankful for the Ministry of Science and Higher Education of the Russian Federation (agreement #075-03-2023-106, project FSMG-2020-0003). IM acknowledges the UHasselt Special Research Fund. Measurements of surface expression and Gs-signaling were supported by the Russian Science Foundation (project no. 22-74- 10036; https://rscf.ru/project/22-74-10036Test/). Computational simulations were supported by the National Natural Science Foundation of China, grant #32250410316 (to PO). We acknowledge the Advanced Optical Microscopy Centre at Hasselt University for support with microscopy experiments. Microscopy was made possible by the Research Foundation Flanders (FWO, projects G0B4915, G0B9922N, and G0H3716N).
