Rapid identification of oxidation-induced conformational changes by kinetic analysis.
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.
Venkatesh
Sanjay
S
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Tomer
Kenneth B
KB
Sharp
Joshua S
JS
D000465:Algorithms; D007700:Kinetics; D008832:Microchemistry / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011506:Proteins; D017382:Reactive Oxygen Species