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利用受绿色荧光蛋白启发的染料监测G蛋白偶联受体构象

Monitoring GPCR conformation with GFP-inspired dyes.

作者信息

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

机构信息

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.

出版信息

iScience. 2024 Jul 4;27(8):110466. doi: 10.1016/j.isci.2024.110466. eCollection 2024 Aug 16.

DOI:10.1016/j.isci.2024.110466
PMID:39156645
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11326922/
Abstract

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 A adenosine receptor (AAR). Two dyes showed Ca-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 AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of AAR 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.

摘要

溶剂致变色化合物已成为生物学研究中重要的环境敏感探针。在此，我们使用绿色荧光蛋白（GFP）发色团的硫醇反应性溶剂致变色类似物来追踪两种蛋白质（恢复蛋白和A1腺苷受体（A1AR））的构象变化。当连接到恢复蛋白上时，两种染料均显示出Ca2+诱导的荧光变化。我们表现最佳的染料DyeC，当连接到A1AR上时，其荧光光谱的强度和形状均表现出激动剂诱导的变化；而其他常见的环境敏感染料均未观察到这些效应。分子动力学模拟表明，A1AR的激活导致DyeC所处的环境更加受限且亲水性增强。此外，A1AR的变构调节剂在DyeC光谱中诱导出明显的荧光变化，表明存在独特的受体构象。我们的研究表明，受GFP启发的染料可有效检测G蛋白偶联受体（GPCR）的结构变化，具有基于强度和比率追踪、荧光光谱红移以及对变构调节敏感等优点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4c85/11326922/e59c5c328218/fx13.jpg
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