Vertebrate muscle contraction is regulated by the binding and unbinding of Ca2+ to the thin-filament protein troponin (TN). Upon Ca2+ binding, structural changes occur within troponin, which are transmitted to the thin- filament protein tropomyosin (Tm), which runs along the helical groove of actin. Tm, in turn, is believed to mediate the interaction of myosin and actin, which is responsible for muscle contraction. We propose to use neutron scattering, as well as an NMR experiment, to elucidate the in situ structures and organization of Tn subunits within whole Tn with and without regulating Ca2+ bound to TnC. The spatial organization of Tn subunits will be studied with respect to Tm and the complete thin filament. Deuteration of a member of a complex will allow member(s) to be rendered """"""""invisible"""""""" to neutrons. Structures of the visible components will be thus investigated in situ without ambiguity. Specifically, we will study the in situ structures of the Tn subunits TnC, TnI and TnT+/- regulatory Ca2+. We will determine the organization of Tn subunits +/-Ca2+ in whole troponin as well as in thin filaments. We will determine the radial position of Tn subunits relative to Tm +/- Ca2+. A search for changes in the radial and azimuthal positions of Tn subunits +/-Ca2+ in solutions and oriented thin filaments will help us to further understand how troponin subunits control Tm movement during regulation. We will also investigate the changes in the in situ structures of F- actin as a """"""""control"""""""" for these experiments. We propose implementing a novel NMR method for determining the high-resolution in situ structure of TnC. This proposal is directed at fundamental basic science questions concerning molecular aspects of the regulation of skeletal muscle contraction. However, this structural information may give insight into disease processes such as hypertrophic cardiomyopathies which are associated with missence mutations in cardiac troponins I and T and tropomyosin. It is anticipated that these same techniques may in the future be applied to the study of cardiac regulatory proteins and may provide a structural basis for understanding the role of phosphorylation of cardiac TnI, the unique features of the interaction of cardiac TnI with cardiac TnC (which possesses only a single regulatory Ca2+-binding site), and the effect of ischemia and calcium-sensitizing drugs on TnC.
| Blumenschein, Tharin M A; Stone, Deborah B; Fletterick, Robert J et al. (2006) Dynamics of the C-terminal region of TnI in the troponin complex in solution. Biophys J 90:2436-44 |
| Pirani, Alnoor; Vinogradova, Maia V; Curmi, Paul M G et al. (2006) An atomic model of the thin filament in the relaxed and Ca2+-activated states. J Mol Biol 357:707-17 |
| Blumenschein, Tharin M A; Stone, Deborah B; Fletterick, Robert J et al. (2005) Calcium-dependent changes in the flexibility of the regulatory domain of troponin C in the troponin complex. J Biol Chem 280:21924-32 |
| Vinogradova, Maia V; Stone, Deborah B; Malanina, Galina G et al. (2005) Ca(2+)-regulated structural changes in troponin. Proc Natl Acad Sci U S A 102:5038-43 |
| King, William A; Stone, Deborah B; Timmins, Peter A et al. (2005) Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering. J Mol Biol 345:797-815 |
