MELITTIN-BINDING OF TROPONIN-C

Ca2+-dependent interaction between skeletal muscle troponin C and a bee venom melittin, which can be regarded as a mimic of the troponin C-binding peptide of troponin I, was investigated. Sephadex gel chromatography revealed that melittin bound to troponin C irrespective of the presence or absence of Ca2+ in 50 mM KCl and 50 mM Tris-HCl, pH 7.5. At high salt concentration, 0.5-1.2 M KCI, melittin was removed from apo troponin C but still bound to Ca2+-loaded troponin C. Hydrophobic affinity chromatography revealed that hydrophobic region(s) appeared on the surface of troponin C upon Ca2+ binding but vanished upon Ca2+ release. Therefore, at physiological salt concentration, melittin binds to Ca2+-loaded troponin C by both hydrophobic and electrostatic interactions, and it binds to apo troponin C by electrostatic interaction. The midpoint of the fluorescence titration curve of troponin C-melittin complex, observed by Trp-19 fluorescence, scarcely depended on the concentration of MgCl2. This means Trp-19 of melittin detects only the conformational change of troponin C induced by Ca2+-binding to the low affinity Ca2+-binding sites (sites I and II) of troponin C; that is, the C-terminal of melittin binds to the N-terminal of troponin C. Fluorescence stopped-how experiments revealed that the time course of Trp-19 fluorescence change induced by the Ca2+-release from the low affinity Ca2+-binding sites of troponin C in the complex was biphasic. An analysis of the rate constants of the reactions revealed that the fast phase reflected the Ca2+-release reaction from the low affinity sites, and the slow phase reflected the resultant structural change of the complex.