Fluorescence Energy Transfer of Complexes of Skeletal Muscle Troponin C and Melittin Derivatives

Abstract

We studied Ca²⁺-dependent structural change of rabbit skeletal troponin C (TnC)-melittin (ME) complex as a model of TnC-troponin I complex. In previous study, we found that the distance between Met-25 and Cys-98 of TnC in TnC-ME complex increased upon binding of Ca²⁺ to TnC [H. Sano and T. Iio (1995) J. Biochem. 118, 996-1000]. In this study, we used a fluorescence energy transfer method. As a fluorescent donor, we used the tryptophan residue in four melittin derivatives, in which residue 2, 5, 8, or 13 was replaced with tryptophan. As acceptor, we used dansylaziridine (DANZ) bound to Met-25 of TnC, or N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1, 5-I-AEDANS) bound to Cys-98 of TnC. For all TnC^DANZ-ME complexes, the donor-acceptor distance (11.9-17.7 Å) did not remarkably depend on Mg²⁺ or Ca²⁺ binding of TnC or on the position of tryptophan in ME derivatives. The same results were obtained for TnC^AEDANS-ME complexes in the absence of Ca²⁺ (distance 15.2-21.7 Å). But in the presence of Ca²⁺, tryptophan residues in the central region of ME were near to Cys-98 of TnC (distance much less than 10.4 Å). Based on these results, we conclude that ME is enfolded by the N- and C-lobes of TnC, and the ME rod is almost perpendicular to a line connecting Met-25 and Cys-98 of TnC. The position of the ME rod shifts upon binding of Ca²⁺ to TnC.