The long term goal of this research program is to achieve deep and clinically practical insights into the control of the heart, by molecular level investigation of the thin filament. This will be advanced by investigating the dynamic properties of the key regulatory proteins, cardiac troponin and tropomyosin, to reveal their modes of action and functions. The troponin- tropomyosin complex very tightly controls striated muscle contraction by conferring near- absolute calcium-dependence upon myosin's productive interactions with the thin filament. This regulation requires both flexibility and stiffness as important properties of troponin and tropomyosin. They must both move while attached to the thin filament in critical patterns, and also resist movement in other ways. Thus, the detailed dynamic properties of troponin and tropomyosin are particularly important for the regulation of cardiac contraction as well as having general significance for the function of these proteins. We have developed the means to study these dynamics with a very powerful approach: hydrogen/deuterium exchange with ultra high resolution mass spectrometry (HDX). The method holds great promise for understanding contractile regulation, and is uniquely available in our laboratory for this work. We will use HDX, strategically supplemented when valuable by electron microscopy, to ascertain critical aspects of troponin's regulatory switch mechanism. In one approach, we will investigate this mechanism by delineation of the effects of the troponin residues most essential for switching muscle contraction either on or off. Also, we will investigate troponin functions involving action at a distance by studies of troponin containing targeted mutations. Finally, we will determine and map the variable dynamics of tropomyosin that are vital for its function, using the same HDX approach. The overall aim of the work is to advance greatly our understandings of the basic molecular mechanism that directly controls the contraction of the heart and other striated muscles.
This research will provide molecular information on the control of muscle contraction generally, and the control of the heart's contraction in particular, by studying the intra-cellular protein machinery responsible for activating and inactivating each contraction. This subject is of fundamental importance for the causes and potential treatments of heart failure, coronary insufficiency, hypertension, and disorders of skeletal muscles.
|Mun, Ji Young; Previs, Michael J; Yu, Hope Y et al. (2014) Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci U S A 111:2170-5|
|Sousa, Duncan R; Stagg, Scott M; Stroupe, M Elizabeth (2013) Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state. J Mol Biol 425:4544-55|
|Li, Xiaochuan Edward; Tobacman, Larry S; Mun, Ji Young et al. (2011) Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry. Biophys J 100:1005-13|
|Sousa, Duncan; Cammarato, Anthony; Jang, Ken et al. (2010) Electron microscopy and persistence length analysis of semi-rigid smooth muscle tropomyosin strands. Biophys J 99:862-8|
|Kowlessur, Devanand; Tobacman, Larry S (2010) Troponin regulatory function and dynamics revealed by H/D exchange-mass spectrometry. J Biol Chem 285:2686-94|
|Kozaili, Julie Mouannes; Leek, Daniel; Tobacman, Larry S (2010) Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker. J Biol Chem 285:38034-41|
|Ali, Laith F; Cohen, Joshua M; Tobacman, Larry S (2010) Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study. Biochemistry 49:10873-80|
|Kowlessur, Devanand; Tobacman, Larry S (2010) Low temperature dynamic mapping reveals unexpected order and disorder in troponin. J Biol Chem 285:38978-86|
|Lehman, William; Galinska-Rakoczy, Agnieszka; Hatch, Victoria et al. (2009) Structural basis for the activation of muscle contraction by troponin and tropomyosin. J Mol Biol 388:673-81|
|Siththanandan, V B; Tobacman, L S; Van Gorder, N et al. (2009) Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils. Pflugers Arch 458:761-76|
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