Wang Z Y, Sarkar S, Gergely J, Tao T
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts General Hospital 02114.
J Biol Chem. 1990 Mar 25;265(9):4953-7.
We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.
我们利用体外诱变技术在大肠杆菌中合成了一种重组兔骨骼肌肌钙蛋白C（命名为TnC57），其中Cys-98被亮氨酸取代，N端结构域C螺旋中的Ala-57被半胱氨酸取代。用双功能光交联剂二苯甲酮-4-马来酰亚胺标记的TnC57，在与肌钙蛋白I的二元复合物以及与肌钙蛋白I和肌钙蛋白T的三元复合物中，均可与肌钙蛋白I发生光交联。当Ca2+结合到低亲和力触发位点时，用探针N-碘乙酰-N'-(5-磺基-1-萘基)乙二胺标记的TnC57的荧光寿命从13.2±0.1纳秒降至11.8±0.1纳秒。与肌钙蛋白I或肌钙蛋白I和肌钙蛋白T两者形成复合物，在有无Ca2+的情况下，该寿命均显著增加。在二元或三元复合物中，当Ca2+结合到低亲和力位点时，该寿命从15.5纳秒增加到18.0纳秒。互补的丙烯酰胺猝灭研究得出的结果与荧光寿命结果一致。我们的结果表明，肌钙蛋白C的C螺旋与肌钙蛋白I相互作用，这证实了最近的零长度交联结果（Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299 - 304）。此外，这些结果支持了一个模型（Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638 - 2644），即Ca2+与肌钙蛋白C N端结构域中的触发位点结合导致B螺旋和C螺旋远离中央螺旋移动，从而暴露出一个假定的肌钙蛋白I疏水结合位点。
