Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.
The focus is to expand the original design of fast photochemical oxidation of proteins (FPOP) and introduce SO(4)(-•), generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical 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, nonspecific, 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 SO(4)(-•) than with (•)OH, oxidation of apomyoglobin and calmodulin shows that (•)OH probes smaller accessible areas than SO(4)(-•), with the possible exception of histidine. His64, an axial ligand in the heme-binding pocket of apomyoglobin, is substantially up-labeled by SO(4)(-•) 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.
Gau
Brian C
BC
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, USA.
Chen
Hao
H
Zhang
Yun
Y
Gross
Michael L
ML
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D005609:Free Radicals / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000528:radiation effects; D055668:Photochemical Processes; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D005609:Free Radicals; D007202:Indicators and Reagents; D011506:Proteins; D012997:Solvents; D013431:Sulfates