Cardiomyopathies (CM) are the leading cause of death in America, and can develop from mutations in sarcomeric proteins, leading to altered protein structure and function. Current therapies target upstream signaling pathways to treat the symptoms of heart failure, but are associated with increased mortality by affecting downstream signaling pathways and other muscle types. Rational drug design can develop therapies to treat CM at the protein level. However, a detailed knowledge of how sarcomeric proteins regulate muscle contraction is required. Muscle contraction occurs through a cyclic interaction between actin thin and myosin thick filaments, regulated by intracellular Ca 2+ concentration. Troponin (Tn), the Ca2+-binding protein in muscle, allosterically regulates actin and myosin interactions (crossbridge formation) by facilitating the release of two troponin I (TnI) actin binding sites at high Ca2+, the inhibitory region (IR) and the second actin binding site (SABS). The mechanism to remove TnI crossbridge inhibition is not well understood. A multi-site Förster resonance energy transfer (FRET) assay in cardiac Tn in reconstituted thin filaments was used to investigate the Ca2+-dependent structure and dynamics of the SABS, and show current theories behind Tn activation are biased using structures developed in isolated Tn. The SABS underwent large Ca2+-dependent conformational changes, suggesting this region plays an important structural role in muscle regulation. The mechanisms behind thin filament Ca2+ sensitivity were also assessed to facilitate rational drug design. Titrations monitoring FRET efficiency (Tn activation) by Ca2+ and myosin showed the drug bepridil works in a similar mechanism to rigor myosin binding, which in native muscle increases Ca2+ sensitivity. The Ca2+-desensitizing drug EGCG, however, does not work in a similar mechanism to protein kinase A (PKA)-mediated phosphorylation of cardiac TnI, which in native cardiac muscle disrupts residues in TnC responsible for binding Ca2+ at Site II. A single point drug screen was developed for Tn in reconstituted thin filaments using a novel correlation between the Ca2+-depleted FRET efficiency and Ca2+ sensitivity. This study shows the utility of performing Tn structural studies in an environment that mimics native muscle.
|Advisor:||Cartrette, David P.|
|Commitee:||Hoppe, Adam D., Klein, Nicole L., Rice, James A.|
|School:||South Dakota State University|
|Department:||Chemistry & Biochemistry|
|School Location:||United States -- South Dakota|
|Source:||DAI-B 77/10(E), Dissertation Abstracts International|
|Subjects:||Pharmacology, Biochemistry, Biophysics|
|Keywords:||Cardiac muscle regulation, Cardiac thin filament, Cardiac troponin, Drug screening, Fluorescence, Protein conformation|
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