title: In-Membrane Chemical Modification (IMCM) for Site-Specific Chromophore Labeling of GPCRs.
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.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA).
authors.forename: Lukas
authors.initials: L
authors.lastname: Sušac
authors.affiliation: 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).
authors.forename: Casey
authors.initials: C
authors.lastname: O'Connor
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, 3430 South Vermont Avenue, Los Angeles, CA 90089 (USA).
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA). wuthrich@scripps.edu.|Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA). wuthrich@scripps.edu.|Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, 8093 Zurich (Switzerland). wuthrich@scripps.edu.
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D043705:Receptor, Adenosine A2A / Q000737:chemistry*; D017473:Receptors, Opioid, kappa / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D043705:Receptor, Adenosine A2A; D017473:Receptors, Opioid, kappa
keywords: G-protein-coupled receptors; NMR spectroscopy; fluorescence spectroscopy; membrane proteins; site-specific labeling