We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca2+-sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM’s protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM’s regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca2+ binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM’s Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.
Bibliographical noteFunding Information:
Many thanks to Drs. Megan McCarthy, Jennifer Klein, Jonggul Kim, and Rebecca Moen, for many helpful discussions. We thank Dr. Youlin Xia for help on NMR experiment setup. This work was funded by NIH R37-AG026160 and NIH R01-HL139065 (formerly GM027906) (DDT and RLC), NIH R01-HL092097 (RLC), AHA Predoctoral Fellowship 16PRE27770056 (SEDN), and AHA Postdoctoral Fellowship 16POST31010019 (RTR). NMR data were acquired at the Minnesota NMR Center. CD data were acquired at the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics.
© 2020, The Author(s).
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't