Analysis of the oxidative damage-induced conformational changes of apo- and holocalmodulin by dose-dependent protein oxidative surface mapping.
Calmodulin (CaM) is known to undergo conformational and functional changes on oxidation, allowing CaM to function as an oxidative stress sensor. We report the use of a novel mass spectrometry-based methodology to monitor the structure of apo- and holo-CaM as it undergoes conformational changes as a result of increasing amounts of oxidative damage. The kinetics of oxidation for eight peptides are followed by mass spectrometry, and 12 sites of oxidation are determined by MS/MS. Changes in the pseudo-first-order rate constant of oxidation for a peptide after increasing radiation exposure reveal changes in the accessibility of the peptide to the diffusing hydroxyl radical, indicating conformational changes as a function of increased oxidative damage. For holo-CaM, most sites rapidly become less exposed to hydroxyl radicals as the protein accumulates oxidative damage, indicating a closing of the hydrophobic pockets in the N- and C-terminal lobes. For apo-CaM, many of the sites rapidly become more exposed until they resemble the solvent accessibility of holo-CaM in the native structure and then rapidly become more buried, mimicking the conformational changes of holo-CaM. At the most heavily damaged points measured, the rates of oxidation for both apo- and holo-CaM are essentially identical, suggesting the two assume similar structures.
Sharp
Joshua S
JS
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Tomer
Kenneth B
KB
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D017665:Hydroxyl Radical / Q000737:chemistry; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D053719:Tandem Mass Spectrometry
D002147:Calmodulin; D010455:Peptides; D017665:Hydroxyl Radical