Determination of the Free-Energy Coupling for Binding of Calcium Ions and Troponin I to Calmodulin

Regulation of a wide variety of biological systems by Ca²⁺ is now known to be mediated through calmodulin, a Ca²⁺-binding protein. Calmodulin forms Ca²⁺-dependent complexes with several proteins, including troponin I. We have determined the free-energy coupling (∆G_CT) for binding of Ca²⁺ and troponin I to calmodulin by measuring Ca²⁺ binding to calmodulin and to the 1:1 calmodulin-troponin I complex by equilibrium dialysis. The dissociation constant for the Ca²⁺₄.calmodulin and troponin I complex was also determined directly by monitoring fluorescence intensity changes accompanying complex formation between Ca²⁺₄.CaM and Ndansylaziridine- troponin I, a fluorescent derivative of troponin I. Calmodulin displayed four Ca²⁺-binding sites of similar affinity with a geometric mean dissociation constant of 14 µM. In the presence of saturating troponin I, the geometric mean dissociation constant for the four Ca²⁺-binding sites was shifted to 1.7 µM. ∆G°_ct was therefore -1.25 kcal/mol of Ca²⁺. Saturation of calmodulin with Ca²⁺ would therefore be expected to increase its affinity for troponin I about 4500-fold. A dissociation constant of 20 nM was determined for the Ca²⁺₄.calmodulin-N-dansylaziridine-troponin I complex. The dissociation constant of the calmodulin-N-dansylaziridinetroponin I complex in the absence of Ca²⁺ should therefore be about 90 µ. A prediction of this data is that binding of Ca²⁺ to calmodulin will show strong positive cooperativity when Ca²⁺ binding to a substoichiometric number of sites is sufficient to promote calmodulin-troponin I complex formation.