Rapid identification of oxidation‐induced conformational changes by kinetic analysis

Abstract

Protein oxidation by reactive oxygen species is known to result in changes in the structure and function of the oxidized protein. Many proteins can tolerate multiple oxidation events before altering their conformation, while others suffer gross changes in conformation after a single oxidation event. Additionally, reactive oxygen species have been used in conjunction with mass spectrometry to map the accessible surface of proteins, often after verification that the oxidations do not alter the conformation. However, detection of oxidation-induced conformational changes by detailed kinetic oxidation analysis of individual proteolytic peptides or non-mass spectrometric analysis is labor-intensive and often requires significant amounts of sample. In this work, we describe a methodology to detect oxidation-induced conformational changes in proteins via direct analysis of the intact protein. The kinetics of addition of oxygen to unmodified protein are compared with the kinetics of addition of oxygen to the mono-oxidized protein. These changes in the rate of oxidation of the oxidized versus the non-oxidized protein are strongly correlated with increases in the random coil content as measured by the molar ellipticity at 198 nm. This methodology requires only small amounts of protein, and can be done rapidly without additional sample handling or derivatization.

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

“…β-lactoglobulin is known to be conformationally sensitive to oxidation 25. It follows that 5 mM Na 2 S 2 O 8 FPOP labeling, like 15 mM H 2 O 2 FPOP, labels faster than most secondary and tertiary protein motions26, 27 and corroborates the theoretical prediction of labeling at the μs level.…”

Fast Photochemical Oxidation of Protein Footprints Faster than Protein Unfolding

“…Small in size and tractable for analysis, β-lactoglobulin (β-lg), apo-calmodulin (apo-CaM), and lysozyme (LysC) are not structurally similar, yet are representative of proteins exhibiting this sensitivity. 21, 22 Therefore, we chose these proteins to test the hypothesis.…”

Effects of Fe(II)/H2O2 Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

“…35 On the other hand, γ-induced oxidation of ubiquitin does not lead to any protein unfolding of the native state as also measured by CD. 36 A recent IMS-MS investigation of radical-induced electrical oxidation of ubiquitin has measured similar collision cross sections for monooxidized and unmodified ubiquitin ions of charge states +5 and +6, which are formed upon electrospraying a 2.5–5 μM solution in a 10 mM ammonium acetate buffer. 38 The observed structures for monooxidized and unmodified ions were compact in conformation for +5 ions.…”