G-protein-coupled receptors (GPCRs) exhibit a variety of multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) can be applied to quantify the dynamics of individual receptor molecules in the presence or absence of different ligands. However, observation times of GPCRs, which are freely diffusing in solution, are limited to a few milliseconds. Here, we performed smFRET experiments on an active human A2A adenosine receptor reconstituted into a lipid nanodisc. We captured a single receptor with a confocal anti-Brownian electrokinetic trap (ABEL trap) and recorded smFRET time traces of up to seconds for individual receptors. Pulsed laser excitation enabled FRET donor lifetime recordings in parallel to confirm that conformational dynamics of the A2A adenosine receptor were causing smFRET fluctations. Influences of agonists as well as antagonists on transient conformational changes could be discriminated.
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