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bioRxiv · 10.1101/2022.06.17.496653

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

Dixon, A.·Trinidad, J.·Ziarek, J. J.
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Abstract

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.

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Dixon, A., Trinidad, J., Ziarek, J. J.. 2022-06-19. A Method for Selective 19F-Labeling Absent of Probe Sequestration (SLAPS). https://doi.org/10.1101/2022.06.17.496653

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