The long-term goal of my research is to elucidate the molecular mechanism of troponin-linked calcium regulation of skeletal and cardiac muscle contraction. Several experimental approaches are being used to gain a detailed understanding of calcium-dependent interactions among the following thin filament proteins: actin, tropomyosin (Tm) the Ca2+-binding troponin C (TnC), the inhibitory troponin I (TnI), and the Tm-binding protein troponin T (TnT). Specific aims are to determine: (1) how Ca2+ binding to TnC affects its interactions with TnT and TnI; (2) how troponin regulates actomyosin ATPase activity; (3) the three-dimensional structure of TnT and its interaction sites with TnC and TnI. The principal experimental methods being used are: (1) site-directed mutagenesis to create mutants of the three troponin subunits in order to create functionally interesting sites for extrinsic and intrinsic spectroscopic probes; (2) fluorescence spectroscopy for various purposes, including: fluorescence quenching and lifetime measurements to examine changes in the environments of intrinsic or extrinsic probes; steady-state fluorescence to determine protein-protein and protein-ligand binding constants; the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific protein sites; (3) capillary electrophoresis (CE) to obtain qualitative and quantitative data on protein-protein and protein-ligand interactions; (4) Nuclear magnetic resonance (NMR) spectroscopy to determine troponin subunit three-dimensional structures and interaction sites in solution; (5) other methods such as circular dichroism and HPLC for protein purification and characterization.
Kobayashi, T., Zhao, X., Wade, R., and Collins, J.H.: Ca2+-Dependent Interaction of the Inhibitory Region of Troponin I with Acidic Residues in the N-terminal Domain of Troponin C. Biochimica et Biophysica Acta 1430: 214-221, 1999.
Kobayashi, T., Zhao, X., Wade, R., and Collins, J.H.: Involvement of Conserved, Acidic Residues in the N-Terminal Domain of Troponin C in Calcium-Dependent Regulation. Biochemistry 38: 5386-5391, 1999.
Khaitlina, S, Antropova, O., Kuznetsova, I., Turoverov, K., and Collins, J.H.: Correlation Between Polymerizability and Conformation in Scallop -like Actin and Rabbit Skeletal Muscle -Actin. Arch. Biochem. Biophys. 368: 105-111, 1999.
Kobayashi, T., Kobayashi, M., Gryczynski, Z., Lakowicz, J.R., and Collins, J.H.: Inhibitory Region of Troponin I: Ca2+-dependent Structural and Environmental Changes in the Troponin-Tropomyosin Complex and in Reconstituted Thin Filaments. Biochemistry 39: 86-91, 2000.
Zhao, X., Kobayashi, T., Gryczynski, Z., Gryczynski, I., Lakowicz, Wade, and Collins, J.H.: Calcium-Induced Flexibility Changes in the Troponin C-Troponin I Complex. Biochem. Biophys. Acta, in press.