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

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 k-opioid receptor (KOR) and the human A(2A) adenosine receptor (A(2A)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.