Velcro-binding by Cardiac Troponin-I Traps Tropomyosin on Actin in a Low-energy Relaxed State

During muscle relaxation at low sarcoplasmic Ca-concentration, the 40-nm long tropomyosin coiled coil is attracted by the C-terminal regulatory domain of troponin subunit-I to a "steric-blocking" B-state position on actin subunits of cardiac and skeletal muscle thin filaments. Tropomyosin located in this B-state position obstructs myosin-binding sites on actin, limiting access of myosin-crossbridge heads on actin. In turn, the steric-hindrance imposed on myosin-binding diminishes actomyosin ATPase, crossbridge movement along actin, and contractility, thus causing relaxation. In contrast, during muscle activation, at high sarcoplasmic Ca levels, the troponin-induced tropomyosin interference is relieved, the tropomyosin coiled coil returns to its default C-state position on actin, and contractility proceeds. In the current study, we examined the energetics associated with tropomyosin's shift in position from its C-state to its B-state on actin and the influence of troponin-I on this relaxed state transition. Control studies showed that in the absence of troponin, the free energy difference between B- and C-state positions of tropomyosin on actin is negligible, i.e. neither B- nor C-state is obviously preferred on troponin-free actin. In contrast, widely separated sites along the C-terminal regulatory domain of troponin-I are responsible for a favorable free energy change of about -0.75 kcal/mol, driving the tropomyosin C-state to B-state shift. Corresponding truncation and point mutations along C-terminal region of TnI lead to a less favorable regulatory transition and are linked to cardiac muscle dysfunction.