Monitoring ligand-dependent conformational dynamics of a single G-protein-coupled receptor by time-resolved FRET in an ABEL trap

Michael Börsch; Ivan Maslov; Valentin Borshchevskiy; Thomas Gensch

doi:10.1117/12.3005264

13 March 2024 Monitoring ligand-dependent conformational dynamics of a single G-protein-coupled receptor by time-resolved FRET in an ABEL trap

Michael Börsch, Ivan Maslov, Valentin Borshchevskiy, Thomas Gensch

Author Affiliations +

Proceedings Volume PC12847, Multiphoton Microscopy in the Biomedical Sciences XXIV; PC128470W (2024) https://doi.org/10.1117/12.3005264
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

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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Citation Download Citation

Michael Börsch, Ivan Maslov, Valentin Borshchevskiy, and Thomas Gensch "Monitoring ligand-dependent conformational dynamics of a single G-protein-coupled receptor by time-resolved FRET in an ABEL trap", Proc. SPIE PC12847, Multiphoton Microscopy in the Biomedical Sciences XXIV, PC128470W (13 March 2024); https://doi.org/10.1117/12.3005264

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KEYWORDS

Fluorescence resonance energy transfer

Confocal microscopy

Molecular energy transfer

Molecules

Pulsed laser operation

Resonance energy transfer

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