In-Membrane Chemical Modification (IMCM) for Site-Specific Chromophore Labeling of GPCRs

We present in-membrane chemical modification(IMCM) for obtaining selective chromophorelabeling 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 chromophoresfor NMR and fluorescence spectroscopyin the human κ-opioid receptor(KOR) and the human A2A adenosine receptor(A2AAR). 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 spectroscopyexperiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.