Premium
Pulsed EPR distance measurements resolve the impact of site‐specific calmodulin methionine oxidation (1009.4)
Author(s) -
Klein Jennifer,
McCarthy Megan,
Reuter Mitch,
Olenek Michael,
Moen Rebecca,
Thomas David
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1009.4
Subject(s) - ryanodine receptor , calmodulin , chemistry , biophysics , ryr1 , calcium , biochemistry , protein structure , oxidative stress , oxidative phosphorylation , receptor , biology , organic chemistry
We have examined the structural impact of oxidizing specific protein methionine (Met) residues 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). 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. 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. Targeted interventions that block or repair specific oxidative damage, while supporting normal levels of oxidation, could decrease morbidity associated with muscle dysfunction_this level of discrimination demands molecular‐level insights into the mechanisms that underlie the decline in muscle force associated with oxidative stress. Pulsed EPR distance measurements across CaM’s lobes 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 an 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. Grant Funding Source : This work is supported by a University of Wisconsin‐La Crosse Faculty Research Grant to JC Klein, NI