Oxidation of methionine residues in calmodulin (CaM) lowers the affinity for calcium and results in an inability to activate target proteins fully. To evaluate the structural consequences of CaM oxidation, we used infrared difference spectroscopy to identify oxidation-dependent effects on protein conformation and calcium liganding. Oxidation-induced changes include an increase in hydration of α-helices, as indicated in the downshift of the amide I′ band of both apo-CaM and Ca2+-CaM, and a modification of calcium liganding by carboxylate side chains, reflected in antisymmetric carboxylate band shifts. Changes in carboxylate ligands are consistent with the model we propose: an Asp at position 1 of the EF-loop experiences diminished hydrogen bonding with the polypeptide backbone, an Asp at position 3 forms a bidentate coordination of calcium, and an Asp at position 5 forms a pseudobridging coordination with a calcium-bound water molecule. The bidentate coordination of calcium by conserved glutamates is unaffected by oxidation. The observed changes in calcium ligation are discussed in terms of the placement of methionine side chains relative to the calcium-binding sites, suggesting that varying sensitivities of binding sites to oxidation may underlie the loss of CaM function upon oxidation. © 2008 by the Biophysical Society.
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