INTERACTION OF TROPONIN-C AND TROPONIN-C FRAGMENTS WITH TROPONIN-I AND THE TROPONIN-I INHIBITORY PEPTIDE

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1: 1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 X 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments. The tryptophan-labeled troponin I inhibitory peptide binds to troponin C and to the N- and C-terminal fragments of troponin C in the presence of calcium with a stoichiometry of 1:1 and association constants of 5.3 x 10(5), 2.5 x 10(5), and 6.5 X 10(4) M-1, respectively. The calcium dependencies of the association constants are largest for troponin C (approximately 10-fold) with smaller values for the N-terminal (approximately 3-fold) and C-terminal (approximately 2-fold) fragments. These data are most easily understood in terms of a solution structure for troponin C which is compact relative to the crystal. The large calcium dependence in the troponin C-troponin I interaction (> 50-fold) might arise from the coupling of a conformational change in the N-terminal domain of troponin C with the release of a positive free energy of interaction between the calcium binding domains.