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Department of Molecular and Cellular Biochemistry

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  • New paper from Joshua Ziarek’s lab published in Protein Science

New paper from Joshua Ziarek’s lab published in Protein Science

Sunday, September 18, 2022

Joshua Ziarek
Joshua Ziarek

The lab of Joshua Ziarek has published a new article in Protein Science, entitled “A method for selective 19F-labeling absent of protein sequestration (SLAPS).”

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 non-covalent probe sequestration by detergent proteomicelles as a second source of offsite labeling when using the popular 19F-probe BTFMA (2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide). The chemical shift and relaxation rates of these unreacted 19F-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 19F-BTFMA, remove excess unreacted 19F-BTFMA molecules via ultracentrifugation, and finally solubilize in the detergent of choice. Our approach builds upon the In-Membrane Chemical Modification (IMCM) method with the addition of one crucial step: removal of unreacted 19F-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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The College of Arts & Sciences

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    • Simon Hall Facilities
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    • BMB Research Series
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