EVIDENCE FOR 2-SITE BINDING OF TROPONIN-I INHIBITORY PEPTIDES TO THE N-DOMAIN AND C-DOMAIN OF TROPONIN-C

The interactions of two troponin I peptides, Ip1 (residues 96-116) and Ip2 (residues 104-116), with spectral probe mutants F29W and F105W of intact troponin C (TnC) and of isolated N (residues 1-90) and C (resi dues 88-162) domains of TnC have been examined. Ip-induced fluorescenc e emission spectral changes were observed with all four proteins in th e presence of Ca2+. Different dependencies of these spectral changes o n Ip concentration for intact F29W and F105W are interpreted in terms of two binding sites on TnC. The binding of Ip1 to the C domain (K-D1 = 0.50 mu M) is 20-40-fold stronger than to the N domain. The binding affinity of Ip1 to both the N and C domains is, greater than that of I p2. The binding strengths of Ip1 to the N domain of intact F29W and is olated F29W/ND are the same within experimental error; that to isolate d F105W/CD is weakened by 5-6-fold relative to the C domain of intact F105W. Ip-induced fluorescence changes are dependent on the presence o f Ca2+ and are not seen in the presence of Mg2+ alone nor in the absen ce of divalent cations. This is true even though Ip2 binds to TnC unde r all three conditions, as demonstrated by affinity chromatography. Th e accumulated evidence indicates that the F --> W mutations have not s ignificantly affected the binding of Ip peptides to TnC. The binding o f Ipl to the C domain of intact TnC increases its Ca2+ affinity withou t significantly affecting that of the N domain; only at high molar rat ios of Ip1:TnC is the Ca2+ affinity of the N domain increased.