University of Leipzig , Faculty of Life Sciences, Institute of Biochemistry , Brüderstr. 34 , 04103 Leipzig , Germany.
Max-Delbrück-Center for Molecular Medicine , Robert-Rössle-Str. 10 , 13125 Berlin , Germany.
ACS Chem Biol. 2019 Jun 21;14(6):1141-1149. doi: 10.1021/acschembio.8b01115. Epub 2019 May 23.
High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β-adrenergic and the muscarinic M class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye-tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye-tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.
高端显微镜研究 G 蛋白偶联受体（GPCR）需要将明亮且光稳定的染料安装到受体上。标记必须以定量产率进行，以允许化学计量数据分析，并且以最小侵入性的方式进行，以避免干扰 GPCR 功能。我们在这里证明，通过遗传掺入反式环辛-2-烯赖氨酸（TCO*）可以实现β肾上腺素能和毒蕈碱 M 类 A GPCR 的细胞外环以及促肾上腺皮质释放因子 B 类 GPCR 的单残基标记。通过与活细胞表面的染料四嗪缀合物反应，在几分钟内即可发生标记，并保持受体的功能。为了准确量化标记产率，我们设计了一种基于荧光波动显微镜的方法，可在单细胞水平上提取标记位点的数量。此外，我们表明，单残基标记比荧光蛋白标签更适合 GPCR 扩散研究，因为后者会影响受体的流动性。最后，通过在单个 TCO位点上进行双色竞争性标记，我们设计了一种方法来估计 GPCR 的寡聚状态，而无需生物学单体参考，这便于将荧光方法应用于寡聚体研究。由于 TCO和本研究中使用的染料四嗪可商购获得，并且描述的显微镜技术可在商业显微镜上进行，因此我们预计我们的方法将广泛适用于一般膜蛋白的荧光显微镜研究。
