Monitoring GPCR conformation with GFP-inspired dyes.

Anatoliy Belousov, Ivan Maslov, Philipp Orekhov, Polina Khorn, Pavel Kuzmichev, Nadezhda Baleeva, Vladislav Motov, Andrey Bogorodskiy, Svetlana Krasnova, Konstantin Mineev, Dmitry Zinchenko, Evgeni Zernii, Valentin Ivanovich, Sergei Permyakov, Johan Hofkens, Jelle Hendrix, Vadim Cherezov, Thomas Gensch, Alexander Mishin, Mikhail Baranov, Alexey Mishin, Valentin Borshchevskiy

iScience 2024 Aug 16

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Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation. © 2024 The Authors. Published by Elsevier Inc.

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PMID: 39156645

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