Sulfate Radical Anion as a New Reagent for Fast Photochemical Oxidation of Proteins

Abstract

The focus is to expand the original design of fast photochemical oxidation of proteins (FPOP) and introduce SO4−•, generated by 248 nm homolysis of low mM levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical footprinting approach to footprinting proteins and protein complexes by “snapshot” reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. We show that FPOP can accommodate other reagents, increasing its versatility. The new persulfate FPOP system is a potent, non-specific, and tunable footprinting method; 3–5 times less persulfate is needed to give the same global levels of modification as seen with OH radicals. Although solvent-exposed His and Tyr residues are more reactive with SO4−• than with •OH, oxidation of apomyoglobin and calmodulin shows that •OH probes smaller accessible areas than SO4−•, with the possible exception of histidine. His64, an axial ligand in the heme-binding pocket of apomyoglobin, is substantially up-labeled by SO4−• relative to •OH. Nevertheless, the kinds of modification and residue selectivity for both reagent radicals are strikingly similar. Thus, the choice of these reagents relies on the physical properties, particularly the membrane permeability, of the radical precursors.

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

“…In this application, we make two-state (bound and unbound) comparisons of labeling extents at identical sites. The comparisons reflect changes in solvent accessibility accompanying intermolecular interactions or allosteric changes [47]. The irreversible nature of FPOP labeling allows for complicated sample handling before MS detection, including deglycosylation, digestion, and long chromatography.…”

“…The table indicates that 350 μM His gave the closest scavenging ability comparing to 20 mM Gln. According to previous studies [47], one important criterion indicating the sampling of a single (native) conformation of the protein is the Poisson distribution of +0, +16, +32 species after FPOP labeling. Therefore, a more rigorous analysis was conducted, presented in the supporting information section, to model the product distribution for all four experiments.…”

Artificial Photosynthesis: Molecular Systems for Catalytic Water Oxidation

“…Because the sacrificial acceptor undergoes irreversible bond cleavage after electron transfer, the problem with recombination, or back-donation occurring, can be ruled out. The sulfate radical is a strong oxidant by itself and has sufficient potential (E°> 2.40 V vs NHE 95 …”

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…More commonly, ⋅OH abstracts a hydrogen, and the resulting radical undergoes a series of reactions involving dissolved O 2 , culminating in +16 Da (or other) products [25,47]. Importantly, hydrogen abstraction and subsequent oxidation can also be triggered by radicals other than ⋅OH [46][47][48]. Hence, one has to ask if Bsecondary radicals^may be formed under FPOP conditions, and whether such species can cause protein labeling after ⋅OH has disappeared.…”