Figure 6

Ca²⁺ protects against structural degradation from oxidation. (a) In the absence of Ca²⁺, [¹H, ¹⁵N] HSQC overlay of apo ¹³C/¹⁵N Met labeled WT-CaM before and after exposure to 50 mM H₂O₂. (b) Change in normalized [¹H, ¹⁵N] HSQC peak intensity for each Met residue in apo CaM-WT illustrating the increased susceptibility of C-terminal Met residues to oxidation. The complete oxidation of CaM corresponds to the plateau of the intensity of the resonances and was confirmed by MALDI. (c) In the presence of saturating Ca²⁺, [¹H, ¹⁵N] HSQC overlay of Ca²⁺-bound ¹³C/¹⁵N Met labeled WT-CaM before and after exposure to 50 mM H₂O₂. All NMR spectra were acquired on a Bruker 900 MHz spectrometer at 25 °C. (d) CD spectra tracking the loss of α-helical secondary structure for WT-CaM in the absence and presence of Ca²⁺ , following exposure to 50 mM H₂O₂ for 5 h. CD spectra were acquired at 25 °C and tracked the α-helical signal at 222 nm. Spectral images were generated using NMRFAM-Sparky⁴⁶, and graphs in panels (b) and (d) were generated using Origin 2015 (https://www.originlab.com/) and Excel 2016 (https://office.microsoft.com/excel), respectively.