Melittin-binding of troponin C.

Abstract

Ca(2+)-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 KCl, melittin was removed from apo troponin C but still bound to Ca(2+)-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 Ca(2+)-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 Ca(2+)-binding to the low affinity Ca(2+)-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-flow experiments revealed that the time course of Trp-19 fluorescence change induced by the Ca(2+)-release from the low affinity Ca(2+)-binding sites of troponin C in the complex was biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)