Dissertation
In vitro and in situ fluorescence spectroscopic investigations of the structural basis of the thin filament regulatory switching activity of cardiac troponin
Doctor of Philosophy (PhD), Washington State University
01/2014
Handle:
https://hdl.handle.net/2376/117076
Abstract
Fluorescence spectroscopic methods have been used in three primary studies to probe the role of the N-terminal domain of cardiac troponin C (N-cTnC) and the C-terminal domain of cardiac troponin I (C-cTnI) in the molecular mechanism underlying thin filament regulation. In the first study, we determined that the behavior of single fluorophore anisotropy probes attached to the inhibitory region of cTnI (cTnI-Ir), the switch region (cTnI-Sr), or the mobile domain (cTnI-Md) arises from the conformational dynamics of the labeled protein region. Our results from subsequent anisotropy experiments indicated that the N-cTnC-cTnI-Sr interaction counterbalances the cTnI-Md-actin and cTnI-Ir-actin interactions, such that cTnI-Sr pulls C-cTnI toward N-cTnC, whereas cTnI-Ir and cTnI-Md pull C-cTnI in the opposite direction toward actin. Thin filament activation and deactivation are thus caused by shifts in the balance between these protein-protein interactions induced by Ca2+ and myosin binding or dissociation events. In the second study, we successfully implemented our steady-state Förster resonance energy methods in the in situ molecular environment of skinned cardiac muscle fibers. We determined that strong actomyosin interactions associated with cycling cross-bridges enhance the Ca2+ sensitivity of the thin filament by exerting a stabilizing effect on N-cTnC opening that is likely indirect and mediated through cTnI. We also discovered that while this Ca2+ sensitizing effect of strong actomyosin interactions is allosterically transmitted between thin filament regulatory units, cooperativity itself is intrinsic to the thin filament ultrastructure such that strong actomyosin interactions are not needed to achieve cooperative Ca2+-dependent thin filament activation. Finally, in the third study, which generated preliminary data for a grant proposal, by preparing a truncation mutant of cTnI wherein the cTnI-Md had been removed and then incorporating this mutant into skinned muscle fibers, we determined that cTnI-Md plays important roles in thin filament cooperativity and the basal inhibition of force generation at diastolic Ca2+ levels. Additionally, we used time-resolved FRET measurements to study the relationship between thin filament activation, cross-bridge binding state, and sarcomere length. We found compelling evidence that myofilament length dependent activation involves the modulation of cross-bridge feedback activation of the thin filament by changes in sarcomere length.
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Details
- Title
- In vitro and in situ fluorescence spectroscopic investigations of the structural basis of the thin filament regulatory switching activity of cardiac troponin
- Creators
- Daniel Rieck
- Contributors
- Wen-ji Dong (Advisor)Murali Chandra (Committee Member)Buel D Rodgers (Committee Member)Bernard J Van Wie (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 262
- Identifiers
- 99900581844801842
- Language
- English
- Resource Type
- Dissertation