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
Affiliations
Anatoliy Belousov
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Ivan Maslov
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia; Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, 3590 Diepenbeek, Belgium; Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium
Philipp Orekhov
Faculty of Biology, Shenzhen MSU-BIT University, Shenzhen 518172, China; Sechenov University, Moscow 119146, Russia
Polina Khorn
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Pavel Kuzmichev
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Nadezhda Baleeva
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; Pirogov Russian National Research Medical University, Moscow 117997, Russia
Vladislav Motov
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia; Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia
Andrey Bogorodskiy
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Svetlana Krasnova
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; National Research University Higher School of Economics, Moscow 101000, Russia
Konstantin Mineev
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia; Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia
Dmitry Zinchenko
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino 142290, Russia
Evgeni Zernii
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia
Valentin Ivanovich
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Sergei Permyakov
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino 142292, Russia
Johan Hofkens
Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium; Max Plank Institute for Polymer Research, Mainz, Germany
Jelle Hendrix
Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, 3590 Diepenbeek, Belgium; Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium
Vadim Cherezov
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA
Thomas Gensch
Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium
Alexander Mishin
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia
Mikhail Baranov
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; Pirogov Russian National Research Medical University, Moscow 117997, Russia
Alexey Mishin
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
Valentin Borshchevskiy
Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia; Joint Institute for Nuclear Research, Dubna 141980, Russian Federation; Corresponding author
Summary: 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.
