Calcium Binding to the Low Affinity Sites in Troponin C Induces Conformational Changes in the High Affinity Domain

Residues 89-100 of troponin C (C8s-loo) and 96-116 of troponin 1 (196-116) interact with each other in the troponin complex (Dalgarno, D. C., Grand, R. J. A., Levine, B. A. Moir, A,, J. G., Scott, G. M. M., and Perry, S . V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Caa+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J. M., Grand, R. J. A., and Perry, S. V. (1976) Bioclrem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P. C., Rosenfeld, S . S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We havestudied Ca2+-induced changes in the region C88-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethy1)aminonaphthalenel-sulfonic acid. Equilibrium titration of the labeled TnC with Ca” indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg,-TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca” binding to the unoccupied sites I and I1 followed by a slower increase ( I t = 9.9 s-l) that represents Mg2+-Ca2+ exchange at sites I11 and IV. In the TnCeTnI complex, the fast phase is much larger and the Mg2+-Caa+ exchange at sites I11 and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Caz+ binding to sites I and I1 may be instrumental in triggering activation of the thin filament by facilitating a contact between Cs9-100 and Ist”s.