The long-term goal of this proposal is to elucidate the molecular mechanism of troponin-linked calcium regulation of skeletal muscle contraction. Several experimental approaches will be used to gain a detailed understanding of Ca2+-dependent interactions among the following thin filament proteins: the Ca2+-binding troponin C (TnC), the inhibitory troponin I (TnI), actin, and the tropomyosin-binding protein troponin T (TnT).
The specific aims are to elucidate: (1) the mechanism whereby Ca2+ binding to TnC changes its interaction with TnI, thus triggering the series of events leading to muscle contraction; (2) the mechanism whereby Ca2+- dependent changes in TnC-Tnl interactions are transmitted to actin in the thin filament; (3) the vital but little-characterized interactions of TnT with TnC and TnI. The principal experimental methods to be used are: (1) site-directed mutagenesis of the three Tn subunits in order to provide functionally interesting sites for spectroscopic probes and chemical crosslinkers; (2) covalent crosslinking to identify sites of contact among the proteins: (3) frequency domain measurements of fluorescence resonance energy transfer to measure distance distributions between specific sites in thin filament protein complexes: (5) crystallization studies with the goal of preparing troponin subunits and complexes for three-dimensional structure determination by X-ray diffraction. The results of these studies may help to diagnose, prevent, and perhaps ultimately cure, congenital muscle diseases, some resulting from mutations of single amino acids, in which crucial aspects of Ca2+ regulation are not functioning normally.
