Covalent Labeling with Diethylpyrocarbonate: Sensitive to the Residue Microenvironment, Providing Improved Analysis of Protein Higher Order Structure by Mass Spectrometry

Abstract

Covalent labeling with mass spectrometry is increasingly being used for the structural analysis of proteins. Diethylpyrocarbonate (DEPC) is a simple to use, commercially-available covalent labeling reagent that can readily react with a range of nucleophilic residues in proteins. We find that in intact proteins weakly nucleophilic side chains (Ser, Thr, and Tyr) can be modified by DEPC in addition to other residues such as His, Lys, and Cys, providing very good structural resolution. We hypothesize that the microenvironment around these side chains, as formed by a protein's higher order structure, tunes their reactivity such that they can be labeled. To test this hypothesis, we compare DEPC labeling reactivity of Ser, Thr, and Tyr residues in intact proteins with peptide fragments from the same proteins. Results indicate that these residues almost never react with DEPC in free peptides, supporting the hypothesis that a protein's local microenvironment tunes the reactivity of these residues. From a close examination of the structural features near the reactive residues, we find that nearby hydrophobic residues are essential, suggesting that the enhanced reactivity of certain Ser, Thr, and Tyr residues occurs due to higher local concentrations of DEPC.

Accurate protein structure prediction with hydroxyl radical protein footprinting data

“…In addition to incorporating the findings presented here into the ab initio protocol, future work will pursue the Rosetta mover ensemble in conjunction with other covalent labels to continue to improve Rosetta protein structure prediction. Future work will focus on investigations of the microenvironmental effects of covalent labels and aim to incorporate these effects into structure prediction 13 . Finally, in future work, we plan to explore long-timescale dynamics governing linked secondary or other higher-order structure perturbations using long MD simulations.…”

“…Covalent labeling can be achieved with a multitude of reagents, including carbenes, diethylpyrocarbonate, and hydroxyl radicals [11][12][13] . Hydroxyl radicals, a commonly used covalent labeling reagent, are frequently derived from radiolysis or photolysis of hydrogen peroxide or water 14,15 .…”

Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

“…Because protein 3D structure is a network of residue interactions, the chemical reactivity of a footprinter is determined by not only intrinsic chemical reactivity and ASA but also the protein microenvironment [30] (e.g., hydrogen bonding, reagent local concentration). Correlations of reactivity, structure, and ASA will require more detailed studies, and these should be productive and are planned in the future.…”

Zn2+-induced changes in Cav2.3 channel function: An electrophysiological and modeling study

“…Second, we used DEPC to probe the role of histidine residues for high-affinity Zn 2+ binding, which is frequently employed for this purpose ( Choi and Lipton, 1999 ; Bancila et al, 2005 ; Harvey et al, 1999 ) but has no perfect target specificity and can also react with other amino acid residues ( Mendoza and Vachet, 2008 ; Limpikirati et al, 2019 ). Protons often compete with Zn 2+ for binding to the histidine imidazole ring, so that their effects on Zn 2+ -induced modulation can to some extent support or oppose an action at histidine residues.…”