The goal of this research is to determine the molecular mechanism of active calcium transport regulation in cardiac sarcoplasmic reticulum (SR), with the specific goal of a PhD thesis in Biochemistry, Molecular Biology, and Biophysics. The focus is on the Ca-ATPase (SERCA), the large integral membrane enzyme that pumps calcium into the SR and thus relaxes the muscle, and phospholamban (PLB), the small integral membrane protein that regulates SERCA. Specific mechanistic hypotheses will be tested for the functional roles of structural dynamics in this system. The approach is to attach paramagnetic probes (nitroxide spin labels) to selected sites on synthetic variants of PLB, reconstitute spin-labeled PLB into lipid bilayers in the presence or absence of SERCA, and perform electron paramagnetic resonance (EPR) experiments to probe the structure and dynamics of PLB. I will focus on several aspects of PLB cytoplasmic domain structure that have recently been proposed to play important roles in cardiac function: (a) Using a spin label synthetically incorporated into the PLB backbone, I will detect dynamic order-disorder transitions in the helical segments of the cytoplasmic domain, (b) By detecting the dipolar interaction between pairs of spin labels, I will measure spin-spin distances and thus detect oligomeric structures and peptide chain extension, (c) By measuring the effect of membrane-bound paramagnetic relaxation agents on spin label saturation, I will probe the dynamic interaction between the cytoplasmic domain and the membrane surface. These three biophysical assays will be combined with functional measurements to pursue the following three aims: (1) Compare the structural dynamics of PLB monomer and pentamer, to resolve a current controversy about the structure of the pentamer. (2) Test models for functional dynamics of PLB while interacting with SERCA. (3) Test models for the effect of PLB phosphorylation on its structural dynamics in the presence and absence of SERCA. This research focuses on two cardiac proteins that play central roles in a wide range of heart diseases. In particular, current medical research has identified phospholamban as a major target of therapeutic approaches to heart failure. The findings of this research will provide the kind of detailed molecular information that is needed for the rational design of drugs, gene therapies, and other means of treating the failing heart.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-DIG-H (29))
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Meadows, Tawanna
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University of Minnesota Twin Cities
Schools of Medicine
United States
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Ablorh, Naa-Adjeley; Miller, Tyler; Nitu, Florentin et al. (2012) Accurate quantitation of phospholamban phosphorylation by immunoblot. Anal Biochem 425:68-75
Ablorh, Naa-Adjeley; Nitu, Florentin; Engebretsen, Kristin et al. (2009) Insulin-dependent rescue from cardiogenic shock is not mediated by phospholamban phosphorylation. Clin Toxicol (Phila) 47:296-302