Dataset Information

Sub-millisecond conformational dynamics of the A_2A adenosine receptor revealed by single-molecule FRET.

ABSTRACT: The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A_2A adenosine receptor (A_2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A_2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A_2AAR, explaining the receptor's constitutive activity. For the agonist-bound A_2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

SUBMITTER: Maslov I

PROVIDER: S-EPMC10070357 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Sub-millisecond conformational dynamics of the A<sub>2A</sub> adenosine receptor revealed by single-molecule FRET.

Maslov Ivan I Volkov Oleksandr O Khorn Polina P Orekhov Philipp P Gusach Anastasiia A Kuzmichev Pavel P Gerasimov Andrey A Luginina Aleksandra A Coucke Quinten Q Bogorodskiy Andrey A Gordeliy Valentin V Wanninger Simon S Barth Anders A Mishin Alexey A Hofkens Johan J Cherezov Vadim V Gensch Thomas T Hendrix Jelle J Borshchevskiy Valentin V

Communications biology 20230403 1

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active ...[more]

PMID: 37012383

Similar Datasets

Project description:G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Dataset Information

Sub-millisecond conformational dynamics of the A_2A adenosine receptor revealed by single-molecule FRET.

Publications

Sub-millisecond conformational dynamics of the A<sub>2A</sub> adenosine receptor revealed by single-molecule FRET.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET.

Publications

Sub-millisecond conformational dynamics of the A<sub>2A</sub> adenosine receptor revealed by single-molecule FRET.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Sub-millisecond conformational dynamics of the A_2A adenosine receptor revealed by single-molecule FRET.