A Method for Selective 19F-Labeling Absent of Probe Sequestration (SLAPS)

Fluorine (19F) offers several distinct advantages for biomolecular nuclear magnetic resonance (NMR) spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine reactive 19F-probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein coupled receptors (GPCRs). As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks. Especially when analyzing systems with intermediate to slow timescale conformational exchange. Here, we uncovered a second source of offsite labeling: non-covalent probe sequestration by detergent micelles. We present a simple four-step protocol for Selective Labeling Absent of Probe Sequestration (SLAPS): physically-disrupt cell membranes in the absence of detergent, incubate membranes with cysteine-reactive 19F-probes, remove excess unreacted 19F-probe molecules via ultracentrifugation, and finally solubilize in the detergent of choice. SLAPS should be broadly applicable to other lipophilic cysteine-reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics. ### Competing Interest Statement The authors have declared no competing interest.