Pulsed EPR Distance Measurements Resolve the Impact of Site-Specific Calmodulin Methionine Oxidation

Abstract

We have examined the structural impact of oxidizing specific protein methionines (Met) in the C-ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease.We have linked oxidation-induced changes in RyR regulation to changes in CaM-RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation-induced changes in protein structural dynamics. Pulsed EPR distance measurements across CaM's lobes (multiple pairs of labeling sites, one label on each lobe) were sensitive to large-scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized the elongated conformation, while RyR peptide binding to calcium-loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M-to-Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM's functional interaction with RyR through changes in the orientation and flexibility of CaM's lobes.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160 and P30AR057220), RJ Moen (F31AG037303), the Minnesota Biophysical Spectroscopy Center and the Minnesota Supercomputing Institute.