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

## TL;DR

The study uses a new technique to observe structural changes in a receptor over longer timescales, revealing new details about its dynamic behavior.

## Contribution

ABEL-FRET extends single-molecule FRET observation timescales for A2A adenosine receptors from milliseconds to seconds.

## Key findings

- ABEL-FRET enables observation of A2AAR conformational heterogeneity in apo and ligand-bound states.
- Dwell times for long-lived receptor states are updated to hundreds of milliseconds.

## Abstract

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 A2A adenosine receptors (A2AAR) reconstituted in lipid nanodiscs from milliseconds to seconds. We characterized conformational heterogeneity in apo and ligand-bound A2AAR 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.

Single-molecule Förster resonance energy transfer (smFRET) can be used to probe the structural dynamics underlying the functional complexity of G protein-coupled receptors (GPCRs), however, the approach is limited to specific timescales. Here, the authors use smFRET with anti-Brownian electrokinetic (ABEL) trapping to extend the observation time of untethered A2A adenosine receptors (A2AAR) embedded in lipid nanodiscs from milliseconds to seconds, characterizing conformational heterogeneity in both apo and ligand-bound A2AAR and updating previous estimates of dwell times for long-lived receptor states.