Stepwise C-Terminal Truncation of Cardiac Troponin T Alters Function at Low and Saturating Ca.

Activation of striated muscle contraction occurs in response to Ca binding to troponin C. The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin-catalyzed ATP hydrolysis. It now appears that the C-terminal 14 amino acids of cardiac troponin T (TnT) control the level of activity at both low and high Ca. We made a series of C-terminal truncation mutants of human cardiac troponin T, isoform 2, to determine if the same residues of TnT are involved in the low and high Ca effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca. Changes in acrylodan tropomyosin fluorescence and the degree of Ca stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca. These measurements define the distribution of actin-tropomyosin-troponin among the three regulatory states. Residues SKTR and GRWK of TnT were required for the functioning of TnT at both low and high Ca. Thus, the effects on forming the inactive B-state and in retarding formation of the active M-state require the same regions of TnT. We also observed that the rate of binding of rigor subfragment 1 to pyrene-labeled regulated actin at saturating Ca was higher for the truncation mutants than for wild-type TnT. This violated an assumption necessary for determining the B-state population by this kinetic method.