用于G蛋白偶联受体（GPCRs）位点特异性发色团标记的膜内化学修饰（IMCM）
In-Membrane Chemical Modification (IMCM) for Site-Specific Chromophore Labeling of GPCRs.
作者信息
Sušac Lukas, O'Connor Casey, Stevens Raymond C, Wüthrich Kurt
机构信息
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA).
Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, 3430 South Vermont Avenue, Los Angeles, CA 90089 (USA).
出版信息
Angew Chem Int Ed Engl. 2015 Dec 7;54(50):15246-9. doi: 10.1002/anie.201508506. Epub 2015 Nov 6.
Abstract
We present in-membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G-protein-coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site-specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ-opioid receptor (KOR) and the human A2A adenosine receptor (A2A AR). IMCM is applicable to a wide range of in vitro studies of GPCRs, including single-molecule spectroscopy, and is a promising platform for in-cell spectroscopy experiments.
摘要
我们提出了一种膜内化学修饰（IMCM）方法，用于在最少诱变的情况下对G蛋白偶联受体（GPCR）的细胞内表面半胱氨酸进行选择性发色团标记。该方法利用了膜环境对大多数半胱氨酸的天然保护作用。通过在人κ-阿片受体（KOR）和人A2A腺苷受体（A2A AR）中引入用于核磁共振（NMR）和荧光光谱的发色团，展示了IMCM的实际应用。IMCM适用于GPCR的广泛体外研究，包括单分子光谱，并且是细胞内光谱实验的一个有前途的平台。
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