Abstract
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.