Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Poster Abstracts

3-POS Board 3 THE UNSTRUCTURED C-TERMINAL REGION OF TROPONIN T RETARDS CALCIUM ACTIVATION OF STRIATED MUSCLE Joseph M Chalovich 1 ; Li Zhu 1 ; 1 Brody School of Medicine ECU, Biochem & Mol Biol, Greenville, NC 27834, NC, USA Calcium binding to TnC alters contacts among troponin subunits leading to reorientation of tropomyosin on actin and a large increase in activity. Eisenberg and Weihing (10.1038/2281092a0) were the first of many to show that myosin lacking bound ATP or ADP-Pi (activating states) can further increase activity even at saturating calcium. That is, something in the thin filament retards full calcium activation. That limitation of activation requires basic residues within the C-terminal 14-16 residues of TnT. Removal of those residues from TnT doubles the ATPase activity. Negative charges introduced at some positions in that region of TnT are also activating. Fluorescence and kinetics measurements show that the unstructured C terminal basic region of TnT stabilizes the inactive state of regulated actin formed in the absence of calcium and “activating” species of myosin (see 10.1016/S0006-3495(81)84777-7 for activating species) and destabilizes the active state at high calcium levels. Furthermore, the C terminal basic region of TnT increases the calcium level required for activation of skeletal (10.1021/acs.biochem.0c00499) and cardiac (10.1021/acs.biochem.0c00430) fibers. A FRET analysis showed that calcium binding positioned the C-terminal region of TnT, and the inhibitory and switch regions of TnI away from actin-tropomyosin (10.1021/acs.biochem.2c00090). Unlike those regions of TnI, the C-terminal region of TnT did not appear to contact TnC, although it moved toward it. Removal of the charged residues from C-terminal TnT mimicked the calcium induced changes in the position of C-terminal TnT and the inhibitory region of TnI. However, the switch region of TnI was not near TnC as it was in the calcium state. Conclusion: The disordered, basic, C-terminal region of TnT is necessary for full inactivation, it inhibits activation by Ca 2+ and permits full activation upon binding activating forms of myosin to actin. It does all of this in concert with the other known elements of troponin.

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