ABEL-FRET bridges the timescale gap in single-molecule measurements of the structural dynamics in the A2A adenosine receptor

The functional complexity of G protein-coupled receptors (GPCRs) arises from their structural dynamics, spanning timescales from nanoseconds to minutes. Single-molecule Förster Resonance Energy Transfer (smFRET) enables direct observation of these dynamics in individual receptors, either freely diffusing in solution, using confocal microscopy, or immobilized on surfaces, using Total Internal Reflection Fluorescence (TIRF) camera-based microscopy. However, these modalities are limited to distinct timescales - faster than milliseconds or slower than hundreds of milliseconds, respectively. To overcome these limitations, we employed smFRET with Anti-Brownian Electrokinetic (ABEL) trapping to extend the observation time of untethered human A_2A adenosine receptors (A_2AAR) reconstituted in lipid nanodiscs from milliseconds to seconds. We characterized conformational heterogeneity in apo and ligand-bound A_2AAR and updated previous estimates of dwell times for long-lived receptor states from milliseconds to hundreds of milliseconds. Our results highlight the power of ABEL-FRET to probe GPCRs dynamics and offer valuable insights into GPCR conformational landscapes.