Regulation of the calcium release channel (ryanodine receptor, RyR), by calmodulin (CaM) is disrupted by oxidation and disease-causing mutations, but the structural mechanisms are poorly understood. I hypothesize that these functional effects are caused by structural changes in the CaM-RyR complex, and that elucidation of these structure-function connections will be crucial in the analysis and treatment of heart disease. I further hypothesize that some of the most functionally important structural features of the CaM-RyR interaction are the relative position, dynamics, and disorder of the two lobes of CaM. I will use site-directed spectroscopic probes to test and refine these hypotheses. CaM mutagenesis will be used for two purposes ? to introduce reactive Cys residues for attaching probes, and to introduce mutations that mimic oxidation or are known to cause disease. Electron paramagnetic resonance (EPR, using spin labels) and time-resolved fluorescence anisotropy (TFA, using fluorescent labels) with single probes will be used to resolve lobe-specific rotational dynamics within CaM. Distances between pairs of site-directed probes on the two lobes of CaM will be measured using two time-resolved spectroscopic techniques for which our lab has played leading roles in both developing and applying to muscle protein biophysics: an EPR experiment called double electron-electron resonance (DEER, using spin labels) and fluorescence resonance energy transfer (FRET, using fluorescent labels). These techniques are complementary; DEER offers greater structural resolution and uses smaller probes, while FRET offers greater sensitivity, as required for studies involving the intact RyR.
In Aim 1 I will use all of these techniques to determine how RyRp, a peptide corresponding to the CaM binding site on RyR, affects the Ca- dependent structural dynamics of CaM.
In Aim 2, I will use the more sensitive fluorescence techniques to determine the structural changes associated with CaM when bound to either skeletal or cardiac RyR.
In Aim 3, I will use both techniques, where applicable, to detect mutation-induced structural changes in CaM bound to RyRp or RyR, focusing on two disease-associated mutations (N98S and N54I) and two oxidation mimics (M124Q and M109Q). These mutations are linked to stress-induced arrhythmia known as CPVT, which can lead to sudden cardiac death. My goal is to determine the structural changes in CaM (free and bound to RyR or RyRp) that are caused by these mutations, using a combination of DEER and FRET, as described in Aims 1 and 2. My previously published work involved only DEER experiments on isolated CaM. The present proposal greatly expands my training by expanding my repertoire to multiple EPR and fluorescence techniques, and to structural and functional analysis of intact RyR. This work is designed to (a) provide a robust training program for me, introducing me to experimental approaches and concepts that will prepare me for a career in molecular biophysics, and (b) answer key questions that are needed to guide future work on the diagnosis and treatment of heart disease.
(relevance to public health) My work focuses on calmodulin, a protein that plays an important role in controlling the release of calcium in each muscle cell. I want to find out how chemical changes in calmodulin, caused by age-related stress or heredity, cause decline in the function of skeletal and cardiac muscle. This information is needed to devise improved therapies that can prolong life and improve its quality for individuals that suffer from muscle disorders.
