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 structuraldynamics, spanning timescales from nanoseconds to minutes. Single-molecule Förster ResonanceEnergy Transfer (smFRET) enables direct observation of these dynamics in individual receptors, eitherfreely diffusing in solution, using confocal microscopy, or immobilized on surfaces, using Total InternalReflection Fluorescence (TIRF) camera-based microscopy. However, these modalities are limited todistinct timescales – faster than milliseconds or slower than hundreds of milliseconds, respectively. Toovercome these limitations, we employed smFRET with Anti-Brownian Electrokinetic (ABEL) trappingto extend the observation time of untethered human A2A adenosine receptors (A2AAR) reconstituted inlipid nanodiscs from milliseconds to seconds. We characterized conformational heterogeneity in apoand ligand-bound A2AAR and updated previous estimates of dwell times for long-lived receptor statesfrom milliseconds to hundreds of milliseconds. Our results highlight the power of ABEL-FRET to probeGPCRs dynamics and offer valuable insights into GPCR conformational landscapes.