1 research outputs found
Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry
The Ca<sup>2+</sup>/calmodulin activated
phosphatase, calcineurin,
is inactivated by H<sub>2</sub>O<sub>2</sub> or superoxide-induced
oxidation, both <i>in vivo</i> and <i>in vitro</i>. However, the potential for global and/or local conformation changes
occurring within calcineurin as a function of oxidative modification,
that may play a role in the inactivation process, has not been examined.
Here, the susceptibility of calcineurin methionine residues toward
H<sub>2</sub>O<sub>2</sub>-induced oxidation were determined using
a multienzyme digestion strategy coupled with capillary HPLC–electrospray
ionization mass spectrometry and tandem mass spectrometry analysis.
Then, regions within the protein complex that underwent significant
conformational perturbation upon oxidative modification were identified
by monitoring changes in the modification rates of accessible lysine
residues between native and oxidized forms of calcineurin, using an
amine-specific covalent labeling reagent, <i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS),
and tandem mass spectrometry. Importantly, methionine residues found
to be highly susceptible toward oxidation, and the lysine residues
exhibiting large increases in accessibility upon oxidation, were all
located in calcineurin functional domains involved in Ca<sup>2+</sup>/CaM binding regulated calcineurin stimulation. These findings therefore
provide initial support for the novel mechanistic hypothesis that
oxidation-induced global and/or local conformational changes within
calcineurin contribute to inactivation via (i) impairing the interaction
between calcineurin A and calcineurin B, (ii) altering the low-affinity
Ca<sup>2+</sup> binding site in calcineurin B, (iii) inhibiting calmodulin
binding to calcineurin A, and/or (iv) by altering the affinity between
the calcineurin A autoinhibitory domain and the catalytic center