Muscle contraction and relaxation are regulated by changes in intracellular calcium levels. To facilitate muscle contraction, calcium is released from the intracellular calcium reservoir into the cytosol by the homotetrameric calcium channel known as the ryanodine receptor (RyR). The sarcoplasmic reticulum membrane-embedded RyR is a target for many small molecule and protein modulators, including the ubiquitously expressed calcium binding protein calmodulin (CaM). CaM can bind four calcium ions via its four EF-hand motifs and has calcium-dependent effects on RyR. It is well established that CaM potentiates channel opening below µM calcium and inhibition above µM calcium. Despite this, the structural mechanism of the calcium-dependent CaM-mediated RyR regulation remain poorly understood. The primary goal of the work presented here is to elucidate the structural mechanisms of the CaM-RyR interaction, using bifunctional spin labels and electron paramagnetic resonance (EPR). In the first study, we investigated the structural dynamics of a spin labeled ryanodine receptor peptide (RyRp) bound to CaM using EPR (Chapter 4). By detecting the rotational dynamics of specific sites along the backbone, we show that the interaction of RyRp with CaM is nonuniform along the peptide, and the primary effect of calcium is to increase the interaction of the N-lobe of CaM with RyRp. In the second study (Chapter 5), we placed spin probes on both CaM and RyRp and investigated the calciumdependent structural changes of the complex using a distance measurement EPR technique known as double electron-electron resonance (DEER). Our DEER distance results provide support for the conformational selection mechanism of CaM binding to RyRp (i.e. the binding of RyRp shifts CaM to preexisting structural states). We discovered differential Ca effects on the two lobes of CaM with respect to RyRp binding. More specifically, we discovered that Ca was required for complete interaction of the N-lobe with RyRp, while the C-lobe bound RyRp independent of Ca. These findings are consistent with results from Chapter 4 and provide support for the hypothesis that CaM functions as a subunit of RyR through binding of the C-lobe, and complete interaction of the N-lobe of CaM (in response to increased cytosolic Ca levels) is responsible for maximum inhibition of RyR. Thus, our results provide novel insight into the structural mechanism of CaM-mediated RyR regulation while showcasing an innovative approach with wide applicability to other biological systems.
University of Minnesota Ph.D. dissertation.August 2018. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David Thomas. 1 computer file (PDF); xiii, 92 pages.
Structural Dynamics of the Calmodulin-Ryanodine Receptor Interaction Using Bifunctional Spin Labels and EPR.
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