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, which span timescales from nanoseconds to minutes. Single-molecule Forster Resonance Energy Transfer (smFRET) enables direct observation of these dynamics in individual diffusing 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 - due to constraints of observation time and camera frame rate, while the slow dynamics observed in immobilized receptors may be perturbed by tethering. To overcome these limitations, we employed smFRET in combination with Anti-Brownian Electrokinetic (ABEL) trapping to extend the observation window of untethered A2A adenosine receptors (A2AAR) embedded in lipid nanodiscs from milliseconds to seconds. This approach enabled us to characterize the conformational heterogeneity in both apo and ligand-bound A2AAR, effectively bridging the gap between fast dynamics measured in freely diffusing receptors and slow dynamics observed in immobilized receptors. Our results, taken together with previous studies, demonstrate that both inactive-like and active-like conformations are populated in apo and ligand-bound A2AAR and dynamically interconvert. Apo and antagonist-bound receptors are predominantly locked in inactive-like or active-like states, exhibiting only slow exchange dynamics on timescales longer than hundreds of milliseconds. In contrast, agonist-binding not only shifts the receptor population toward the active-like state but also accelerates the conformational exchange by nearly three orders of magnitude: from hundreds of milliseconds to sub-millisecond timescales. These results underscore the dual role of agonists in modulating both the equilibrium and dynamics of receptor conformations. Our study highlights the power of ABEL-FRET to probe structural dynamics of GPCRs, offering valuable insights into GPCR conformational landscapes and aiding the development of more effective GPCR- targeted therapeutics.