PNNL

Abstract

Oxidation of methionine residues in calmodulin (CaM) under oxidative stress diminishes the productive association of CaM with targets and lowers the affinity for calcium. To define the structural consequences of CaM oxidation, we have used infrared difference spectroscopy to identify oxidation-dependent changes in both CaM conformation and calcium coordination. An oxidation-induced increase in the hydration of ?-helices is reflected in the downshift of the amide I’ band of both apo- and Ca2+ CaM, however the overall native fold is retained upon calcium binding. Shifts in the antisymmetric carboxylate band upon CaM oxidation indicate differential modification of calcium liganding by two classes of aspartates consistant with a model where: an Asp, at position 1 of the EF-loop, experiences diminished hydrogen bonding with the polypeptide backbone; while the second, at position 3 or 5 in the EF-loop, 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, which suggests that varying sensitivities of the binding sites to oxidation may underlie the loss of CaM function upon oxidation.