Elucidating the structural dynamics of SERCA-PLB regulation by electron paramagnetic resonance

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Elucidating the structural dynamics of SERCA-PLB regulation by electron paramagnetic resonance

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Muscle contraction and relaxation is initiated by changes in intracellular calcium, making adequate calcium transport essential for proper muscle function. A primary calcium transporter is the sarcoendoplasmic reticulum Ca<super>2+</super>-ATPase (SERCA), located within the muscle cell and embedded in an organelle called the sarcoplasmic reticulum (SR). To facilitate muscle relaxation, calcium is sequestered from the cellular cytosol into the SR by SERCA. In cardiac muscle, enhanced regulation of calcium transport is needed to accommodate &beta;-adrenergic stimulation (adrenaline demand), which is provided by the regulatory protein phospholamban (PLB). PLB binds to SERCA and inhibits calcium transport through reduction in calcium affinity. However, a phosphate group can be attached to PLB during adrenaline response (phosphorylation) which relieves PLB's inhibitory effect. The structural mechanisms for SERCA regulation by PLB, particularly with respect to phosphorylation, are not well-resolved. Under the Dissociation Model, PLB phosphorylation relieves SERCA inhibition by dissociating the SERCA-PLB complex. In contrast, the Subunit Model proposes that SERCA inhibition is relieved by a subtle structural change, where the SERCA-PLB complex is preserved. The primary goal of my thesis work is to elucidate the structural mechanisms of the SERCA-PLB complex using electron paramagnetic resonance (EPR) spectroscopy. The first study (Chapter 3) aims to discriminate between the Dissociation and Subunit models by measuring changes in the rotational diffusion of EPR spin labels rigidly coupled to PLB and SERCA. The second study (Chapter 4) further develops an anisotropic membrane system called bicelles for EPR orientation measurements on PLB and SERCA. The third study (Chapter 5) uses a combination of oriented bicelles and a novel rigid spin label (bifunctional spin label, or BSL) to measure PLB topology in the lipid membrane, with comparison to previous structural measurements by NMR and x-ray crystallography. Ongoing studies (Chapter 6) reconstitute both spin-labeled PLB and SERCA in bicelles to make PLB orientation measurements by EPR, as affected by PLB phosphorylation and binding of SERCA.


University of Minnesota Ph.D. dissertation. August 2014. Major: Physics. Advisor: David D. Thomas. 1 computer file (PDF); xiv, 138 pages.

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McCaffrey, Jesse Earl. (2014). Elucidating the structural dynamics of SERCA-PLB regulation by electron paramagnetic resonance. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/167283.

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