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

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

Protein Sci

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.

Published: November 2022

Fluorine ( 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 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 F-probe BTFMA (2-bromo-N-(4-[trifluoromethyl]phenyl)acetamide). The chemical shift and relaxation rates of these unreacted 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 F-BTFMA, remove excess unreacted 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 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9601782PMC
http://dx.doi.org/10.1002/pro.4454DOI Listing

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