Abstract
Activation of striated muscle contraction occurs in response to Ca2+ 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 Ca2+. 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 Ca2+ effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+. Changes in acrylodan tropomyosin fluorescence and the degree of Ca2+ stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca2+. 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 Ca2+. 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 Ca2+ 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.