G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

中文翻译：

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使用单分子荧光探索 GPCR 构象动力学

G 蛋白偶联受体 (GPCR) 是一种膜蛋白，通过改变其构象将特定的外部刺激传递到细胞中。这种构象变化使它们能够偶联并激活 G 蛋白以启动信号转导。研究和推断这些结构动力学的一个关键挑战来自细胞环境的复杂性，包括各种内源因素的存在。由于细胞表达系统、膜蛋白纯化技术和标记方法的最新进展，现在可以在单分子水平和活细胞中研究 GPCR 的结构动力学。在这篇综述中，我们讨论了在单分子研究背景下表达、纯化和标记 GPCR 的最先进技术和策略。我们还重点介绍了最近的四项研究，这些研究展示了单分子显微镜在揭示 GPCR 动态方面的应用。这些技术也可作为补充方法来验证从其他结构生物学工具（如冷冻电子显微镜和 X 射线晶体学）获得的结果。