PT  - JOURNAL ARTICLE
AU  - Austin D. Dixon
AU  - Jonathan C. Trinidad
AU  - Joshua J. Ziarek
TI  - A Method for Selective <sup>19</sup>F-Labeling Absent of Probe Sequestration (SLAPS)
AID  - 10.1101/2022.06.17.496653
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.17.496653
4099  - http://biorxiv.org/content/early/2022/06/19/2022.06.17.496653.short
4100  - http://biorxiv.org/content/early/2022/06/19/2022.06.17.496653.full
AB  - 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 StatementThe authors have declared no competing interest.