Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia.
Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, Diepenbeek, Belgium.
Commun Biol. 2023 Apr 3;6(1):362. doi: 10.1038/s42003-023-04727-z.
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 adenosine receptor (AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound AAR, explaining the receptor's constitutive activity. For the agonist-bound AAR, 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.
G 蛋白偶联受体（GPCR）的复杂药理学由其多态构象动力学定义。单分子Förster 共振能量转移（smFRET）非常适合量化单个蛋白质分子的动力学；然而，它在 GPCR 中的应用具有挑战性。因此，smFRET 仅限于在细胞膜中研究受体间相互作用以及在去污剂环境中的受体。在这里，我们在自由扩散的脂质纳米盘中进行了功能活跃的人 A 腺苷受体（AAR）分子的 smFRET 实验，以研究它们的分子内构象动力学。我们提出了一种 AAR 激活的动态模型，该模型涉及在apo 和拮抗剂结合的 AAR 中，活性样和非活性样构象之间的缓慢（>2 ms）交换，这解释了受体的组成型活性。对于激动剂结合的 AAR，我们检测到更快（390 ± 80 µs）的配体效力依赖性动力学。我们的工作为 GPCR 研究建立了一个通用的 smFRET 平台，该平台可用于药物筛选和/或作用机制研究。
