Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System

Abstract

Membrane-associated proteins are important because they mediate interactions between a cell’s external and internal environment and they are often targets of therapeutics. Characterizing their structures and binding interactions, however, is challenging because they typically must be solubilized using artificial membrane systems that can make measurements difficult. Mass spectrometry (MS) is emerging as a valuable tool for studying membrane-associated proteins, and covalent labeling MS has unique potential to provide higher order structure and binding information for these proteins in complicated membrane systems. Here, we demonstrate that diethylpyrocarbonate (DEPC) can be effectively used as a labeling reagent to characterize the binding interactions between a membrane-associated protein and its binding partners in an artificial membrane system. Using chemotaxis histidine kinase (CheA) as a model system, we demonstrate that DEPC-based covalent labeling MS can provide structural and binding information about the ternary complex of CheA with two other proteins that is consistent with structural models of this membrane-associated chemoreceptor system. Despite the moderate hydrophobicity of DEPC, we find that its reactivity with proteins is not substantially influenced by the presence of the artificial membranes. However, correct structural information for this multiprotein chemoreceptor system requires measurements of DEPC labeling at multiple reagent concentrations to enable an accurate comparison between CheA and its ternary complex in the chemoreceptor system. In addition to providing structural information that is consistent with the model of this complex system, the labeling data supplements structural information that is not sufficiently refined in the chemoreceptor model.

Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting

“…Oxidation of buried methionine and quenching of the oxidation of proximal residues by methionine and tryptophan has been observed with all approaches to protein oxidative footprinting. 18 All eight cysteine (C) residues are disulfide-bonded and solvent-inaccessible in the structure. Although C residues are intrinsically reactive, six of the eight are not oxidized, which is consistent with their inaccessibility.…”

“…W108 and W111 are spatially close to M105, which is reactive even though its ASA is zero, as is M12 (Figure F). Oxidation of buried methionine and quenching of the oxidation of proximal residues by methionine and tryptophan has been observed with all approaches to protein oxidative footprinting …”

“…Reversible methods include hydrogen–deuterium exchange (HDX) and “protein painting” . Among protein footprinting methods that introduce covalent modification (e.g., refs − ) oxidation by the hydroxyl radical ( • OH) is most common. Oxidation by • OH is promiscuous, with all side chains but glycine detectable by mass spectrometry (MS) .…”

Investigating Daptomycin–Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs

“…Oxidative modifications to these species result in predictable mass shifts that are detected by MS. FPOP has been used to investigate a variety of proteins with model membrane systems such as nanodiscs, , micelles, bicelles, and even living organisms . Other rapid footprinting chemistries have also been successfully used for membrane proteins, including the trifluoromethyl radical (·CF 3 ), carbenes derived from diazirines, and carbocations. − Slower covalent labeling reagents, including glycine ethyl ester diethylpyrocarbonate have also proven effective. The field is rapidly advancing with the development of new reagents that differ in reactivities, kinetics, and selectivity to enhance structural investigations of diverse targets.…”

Investigating Daptomycin-Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs

“…17 Other rapid footprinting chemistries have also been successfully used for membrane proteins, including the trifluoromethyl radical (•CF3), carbenes derived from diazirines, and carbocations. [18][19][20][21][22][23][24][25] Slower covalent labeling reagents, including glycine ethyl ester 26 diethylpyrocarbonate 27 have also proven effective. The field is rapidly advancing with the development of new reagents that differ in reactivities, kinetics, and selectivity to enhance structural investigations of diverse targets.…”