Methionine oxidation in calmodulin-binding domains

Calmodulin is a calcium-binding protein that binds to and activates over 40 target proteins. The affinity of targets for calmodulin is modulatable by posttranslational modifications such as phosphorylation or Tyr nitration in or near the target protein calmodulin-binding domain. In addition, calmodulin itself can be phosphorylated on Tyr or oxidized at Met residues and those modifications inhibit calmodulin binding to targets. The hypothesis for this project is that Met oxidation in calmodulin-binding domains will result in loss of calmodulin affinity for those targets.;Purified calcineurin, a prominent calmodulin target in the brain, was subjected to oxidation with hydrogen peroxide. The oxidation rate of Met 406, in the calmodulin-binding domain, was measured by LC/MS, and determined to be 4.4x10-3 M-1s-1, similar to the oxidation rate for Met in a free peptide analog (4.9x10-3 M-1s-1). Oxidation of Met406 in calcineurin caused a 3.3 fold decrease in affinity for calmodulin and disrupted the calmodulin activation profile.;In preparation for global assessment of the susceptibility of calmodulin-binding domains to oxidation, calmodulin-binding proteins were isolated from rat liver and brain by affinity purification on calmodulin-sepharose. Three separate isolations were performed for each tissue. Affinity isolations were analyzed by ion trap mass spectrometry, using both gel-based and MuDPIT separations to reduce the complexity of the sample introduced into the mass spectrometer. A total of 608 proteins were identified from the brain and 542 from the liver. Using a computational approach to identify primary calmodulin-binding proteins, 137 proteins from the brain (23%) and 25 proteins from the liver (5%) were identified. Attempts to develop a physical method to determine primary calmodulin-binding proteins were unsuccessful.;Labeling of native Met residues with stable isotope labeled phenacyl bromide was used to test the sensitivity of Met residues in the pools of protein isolated by affinity chromatography to oxidation. This approach allowed us to confirm the sensitivity of Met residues in the regulatory region of calcineurin to oxidation, and also identify other sensitive Met residues. The susceptibility of calmodulin-binding domains to oxidation at Met residues and effects of Met oxidation on calmodulin binding is predicted to modulate Ca2+ -dependent signaling.