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In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs |
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| Authors: | Lukas Sušac Dr. Casey O'Connor Prof. Dr. Raymond C. Stevens Prof. Dr. Kurt Wüthrich |
| Affiliation: | 1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA);2. Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, 3430 South Vermont Avenue, Los Angeles, CA 90089 (USA);3. Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA);4. Institute of Molecular Biology and Biophysics, ETH Zurich, Otto‐Stern‐Weg 5, 8093 Zurich (Switzerland) |
| 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 (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 spectroscopy experiments. |
| Keywords: | fluorescence spectroscopy G‐protein‐coupled receptors membrane proteins NMR spectroscopy site‐specific labeling |