A method for selective 19F‐labeling absent of probe sequestration (SLAPS). (26th October 2022)

• Record Type: Journal Article
• Authors: Dixon, Austin D.; Robson, Scott A.; Trinidad, Jonathan C.; Ziarek, Joshua J.
• Is Part Of: Protein science. Volume 31:Number 11(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-10-26
• Subjects: 2‐bromo‐N‐(4‐[trifluoromethyl]phenyl)acetamide -- detergent -- G protein‐coupled receptor -- in‐membrane chemical modification -- isotope labeling -- lipid mimetic -- lipophilic -- mass spectrometry -- membrane protein -- micelle -- nuclear magnetic resonance; Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.4454
• Languages: English
• ISSNs: 0961-8368

Abstract

Abstract: 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.