Dong Wen-Ji, Robinson John M, Xing Jun, Cheung Herbert C
Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, AL 35294-0005, USA.
J Biol Chem. 2003 Oct 24;278(43):42394-402. doi: 10.1074/jbc.M304858200. Epub 2003 Aug 9.
Upon Ca2+ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Förster resonance energy transfer in Ca2+ titration and stopped-flow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca2+ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca2+ from cTnC occurs in one step (t1/2 approximately 5 ms), and this transition is not affected by Ca2+ release from the C-domain. The other two transitions triggered by Ca2+ dissociation are biphasic with the fast phase (t1/2 approximately 5 ms) correlated with Ca2+ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca2+ (t1/2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca2+ switching between activation and deactivation of cardiac muscle contraction.
心肌在钙离子激活后，肌钙蛋白亚基会发生几种结构变化。这些变化包括心肌肌钙蛋白C（cTnC）N结构域的开放、心肌肌钙蛋白I（cTnI）抑制区域二级结构的改变，以及这两种蛋白质在cTnC - cTnI界面处间距的变化。我们在钙离子滴定和停流实验中使用福斯特共振能量转移，通过重组心肌肌钙蛋白来描绘这些转变。对能量转移结果进行量化，以得出钙离子解离过程中位点间距离变化的时间依赖性曲线。由cTnC释放调节性钙离子引起的cTnC N结构域的关闭是一步完成的（半衰期约为5毫秒），并且这种转变不受C结构域释放钙离子的影响。由钙离子解离触发的其他两种转变是双相的，快速相（半衰期约为5毫秒）与cTnC N结构域释放钙离子相关。这些转变比结合的调节性钙离子的释放（半衰期3.6毫秒）要慢，并且在恢复其失活状态的构象时以协同方式相互耦合。动力学结果确定了与心肌收缩激活和失活过程中钙离子转换相关的结构变化幅度。
