Ligand modulation of the conformational dynamics of the A(2A) adenosine receptor revealed by single-molecule fluorescence

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A(2A) receptor (A(2A)R) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A(2)R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A(2A)R to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229(6.31) on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A(2A)R, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-G(s)). Most notably, the agonist binding and the coupling to mini-G(s) accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288(7.53), and Y290(7.55)) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229(6.31) on TM6. This study provides a quantitative description of conformational dynamics in A(2A)R and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.