The cyclical interaction between the force-generating protein myosin and actin is the mechanism responsible for muscle contraction among all muscle types. Cardiac muscle contraction is tightly controlled to ensure that blood pumps effectively and efficiently from the heart to peripheral organs. Mutations in various cardiac proteins can lead to cardiac dysfunction and a number of cardiomyopathies.
The first part of this dissertation studies two disease-linked mutations in the regulatory light chain of the cardiac myosin molecule, D166V and K104E, and assesses the kinetic and mechanochemical effects of the mutations via the in vitro motility assay. The data show that D166V mutant myosin force generation is reduced compared to wild type, and exogenous phosphorylation of the mutant light chain rescues force generation. In contrast, the K104E mutation showed no deficit in force production but exhibited increased calcium sensitivity of activation. These results are consistent with contractile defects associated with cardiomyopathies caused by various mutation-induced changes to protein function and mechanism of interaction.
The second part uncovers the actin-binding mechanism of one of the chief muscle regulatory proteins tropomyosin. In cardiac and skeletal muscle, tropomyosin and troponin modulate muscle contraction. Tropomyosin binds along the length of actin filaments and blocks myosin-binding sites. Following an excitatory stimulus, calcium binds troponin and causes tropomyosin to shift its position on actin, allowing myosin to bind. The precise mechanism of how tropomyosin monomers with low actin affinity bind to form a stably bound, high affinity chain is unknown. By directly observing fluorescently labeled tropomyosin binding to actin filaments, it was shown that tropomyosin molecules bind randomly along the actin filament. Subsequent monomer binding, and formation of tropomyosin end-to-end bonds, increases the probability of sustained chain growth by decreasing the probability of detachment prior to additional monomer binding. Tropomyosin molecules added to the growing chain at approximately 100 monomers/(μM*s).
Different tropomyosin isoforms segregate to distinct functional and structural regions of cells. The last chapter presents data that show spatial segregation of two different tropomyosin isoforms on actin filaments. This suggests that tropomyosin sorting in cells is, at least partly, an intrinsic property of the binding mechanism.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/15291 |
| Date | 12 March 2016 |
| Creators | Schmidt, William Murphy |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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