Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation

Mild proteolytic cleavage of the troponin complex yields TnT(1), the N-terminal fragment of troponin T, and TnT(2)IC, a complex of the C-terminal fragment of troponin T (TnT(2)) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT(1) and TnT2IC bind tightly to the tropomyosin actin (Tm . actin) thin filament and influence the interaction of myosin subfragment 1 (S1) with Tm . actin. TnT(1) does not affect the rate of S1 binding to Tm . actin but does increase the cooperativity with which S1 ''turns on'' Tm . actin, monitored by the excimer fluorescence of a pyrene label attached to Cys 190 of Tm [Geeves, M. A., & Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. The apparent cooperative unit size of Tm . actin is increased from 6 to 9 by TnT(1) and to 12 by whole troponin. In contrast, TnT(2)IC has no effect on the cooperativity of Tm . actin but does make the apparent S1-binding rate constant, k(app), Ca2+-sensitive; i.e., in the absence of Ca2+, k(app) is reduced 2-3-fold by both TnT(2)IC and whole troponin. Thus, the N- and C-terminal regions of TnT appear to act independently in modulating effects of S1 binding to the Tm . actin thin filament that are important in regulation.