Melittin-binding of troponin C.
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)
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Department of Physics, Faculty of Science, Nagoya University, Aichi.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008555:Melitten / Q000737:chemistry*; D009132:Muscles / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012685:Sepharose / Q000031:analogs & derivatives; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D008555:Melitten; C033452:Phenyl-Sepharose CL-4B; D012685:Sepharose