A method for selective ¹⁹ F-labeling absent of probe sequestration (SLAPS).

Fluorine (¹⁹ F) offers several distinct advantages for biomolecular nuclear magnetic resonance spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine-reactive ¹⁹ F-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. 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 noncovalent probe sequestration by detergent proteomicelles as a second source of offsite labeling when using the popular ¹⁹ F-probe BTFMA (2-bromo-N-(4-[trifluoromethyl]phenyl)acetamide). The chemical shift and relaxation rates of these unreacted ¹⁹ F-BTFMA molecules are insufficient to distinguish them from protein-conjugates, but they can be easily identified using mass spectrometry. 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 ¹⁹ F-BTFMA, remove excess unreacted ¹⁹ F-BTFMA molecules via ultracentrifugation, and finally solubilize in the detergent of choice. Our approach builds upon the in-membrane chemical modification method with the addition of one crucial step: removal of unreacted ¹⁹ F-probes by ultracentrifugation prior to detergent solubilization. SLAPS is broadly applicable to other lipophilic cysteine-reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics.