title: Impact of methionine oxidation on calmodulin structural dynamics.
abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron-electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM's structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4nm (closed) and another at ∼6nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1 to 3nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM's functional interaction with its target proteins by perturbing this Ca-dependent structural shift.
authors.affiliation: Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Megan R
authors.initials: MR
authors.lastname: McCarthy
authors.affiliation: Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Thompson
authors.affiliation: Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Florentin
authors.initials: F
authors.lastname: Nitu
authors.affiliation: Chemistry and Geology Department, Minnesota State University, Mankato, MN 56001, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Moen
authors.affiliation: Biology Department, University of Wisconsin, La Crosse, WI 54601, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Olenek
authors.affiliation: Biology Department, University of Wisconsin, La Crosse, WI 54601, USA. Electronic address: jklein@uwlax.edu.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Klein
authors.affiliation: Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: ddt@umn.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D008715:Methionine / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013113:Spin Labels
chemical_list: D002147:Calmodulin; D013113:Spin Labels; D005973:Glutamine; D008715:Methionine
keywords: Aging; DEER; Muscle; Oxidative stress; Pulsed EPR; Ryanodine receptor