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Normal cardiac function involves the highly regulated switching of contraction on and off by Ca2+-dependent conformational changes in the troponin-tropomyosin complex. A myriad of factors, both biochemical and mechanical, adaptively modulate the regular systole-diastole transition , though all ultimately act through the proteins of the sarcomere. Among the most influential is the ternary troponin complex, lesions of which account for the etiology of a number of cardiomyopathies, including dilated cardiomyopathy, familial hypertrophic cardiomyopathy, and myocardial stunning. The latter can often be traced in particular to dysfunction of cardiac troponin I, the "inhibitory" subunit and directly key to the on/off movements of tropomyosin on the thin filament. This research examines the Ca2+-dependent regulatory role of cardiac troponin I's C-terminus in vitro, as well as its development as a clinical marker for use in SnO2 nanobelt FET biosensors. Investigations into how cardiac troponin I's C-terminal mobile domain and adjacent regions contribute to the Ca2+-dependent "switch" were conducted using thein vitromotility assay. Specifically, we test the hypothesis that cTnI's mobile domain is tightly-coupled to the so-called switch peptide domain, to which it is immediately adjacent. The ability of wild-type cardiac troponin complexes to regulate filament sliding was compared to that of complexes containing one of two mutant cardiac troponin Is: a C-terminally truncated cardiac troponin I lacking the mobile domain, and a cardiac troponin I that incorporated an 8-residue flexible linker between the switch peptide and mobile domain. We find that neither mutation affected maximum filament sliding speeds, indicating that neither the mobile domain nor its adjacency to the switch peptide are required for full activation in the presence of Ca2+. Each mutant complex was found to increase the Ca2+-sensitivity of activation, presumably in different ways. Cooperativity coefficients decreased with each compared to wild type, which agrees with a more permissive range of motion for the switch peptide (i.e. a more ready association with the N-lobe of troponin C) in which fewer activated regulatory units are required to effect the same activating level of crossbridge association. The truncated troponin I complex also failed to fully inhibit filament sliding at low [Ca2+], showing that while the mobile domain is necessary for full Ca2+-dependent inhibition, some inhibitory interaction with actin-tropomyosin remains. These efforts have been timely because, with increasing structural data, it has recently become possible to test detailed hypotheses about the functional role of the of cardiac troponin I C-terminus in Ca2+regulation of thin filaments. It has also been of clinical importance to optimize point-of-care troponin I biosensor devices for the rapid and reliable detection of this myocardial infarction marker.
A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science.
Includes bibliographical references.
P. Bryant Chase, Professor Directing Thesis; Kenneth Taylor, Committee Chair; Piotr Fajer, Committee Member.
Florida State University
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