An Improved Cysteine‐Based Labeling Strategy for GPCR Structural Dynamics Studies

Spectroscopic investigations of the dynamic signaling process of G protein-coupled receptors (GPCRs) often require cysteine-based labeling. However, the presence of a highly conserved but labile disulfide bond in the receptor extracellular domain could result in strong background signals, hindering the application of spectroscopic methods in studying GPCR dynamics. Herein we report an improved strategy for site-specific labeling of muscarinic acetylcholine receptors by using phenylarsine oxide (PAO) to reversibly protect the conserved disulfide bond. This method can efficiently reduce background signals while maintaining receptor functionality, enabling both fluorophore and ¹⁹F labeling for structural dynamics studies of the M2 muscarinic receptor (M2R) by single-molecule fluorescence resonance energy transfer (smFRET) and ¹⁹F nuclear magnetic resonance (NMR). This improved strategy is anticipated to broaden the applicability of cysteine-based spectroscopic methods for structural dynamics studies of a wider range of GPCRs facing similar challenges.