Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments.
Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments.
复制标题
肌钙蛋白 T 和 Ca2 依赖于三元肌钙蛋白复合物和重构细丝中肌钙蛋白 I 的 Cys48 和 Cys133 之间的距离。
DOI：
10.1021/bi962461w
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Tao,T
中科院分区：
文献类型：
--
作者：
Luo,Y;Wu,JL;Gergely,J;Tao,T
脊椎动物横纹肌的收缩受Ca 2+与异源三聚体蛋白质肌钙蛋白（Tn）的相互作用调节，所述异源三聚体蛋白质肌钙蛋白（Tn）由肌钙蛋白-C（TnC）、肌钙蛋白-I（TnI）和肌钙蛋白-T（TnT）组成。虽然对Ca ~（2+）诱导的TnC（Tn的Ca ~（2+）结合亚基）的构象变化了解很多，但对TnI（抑制亚基）如何响应Ca ~（2+）与TnC的结合知之甚少。在这项工作中，我们使用共振能量转移来测量在Cys 48和Cys 133的N-和C-末端结构域，分别，TnI连接的探针之间的距离。通过将Cys 64转化为Ser，构建了一个突变的兔骨骼肌肌钙蛋白I（TnI 48/133，C64 S），用荧光供体1，5-IAEDANS和非荧光受体DAB-Mal标记Cys 48和Cys 133处的两个巯基，发现未复合的TnI和与TnC的二元复合物中的TnI的探针间距离均为1.41 μ m。在具有TnT的三元Tn复合物中，该距离增加到51 μ m。这些距离并没有改变显着的Ca 2+结合的TnC。在重构的细丝中，在饱和Ca 2+存在下，该距离保持为50 μ m，但在Mg 2+存在下用EGTA去除Ca 2+后，该距离增加至166 μ m。我们的研究结果表明，首先，虽然TnC只有很小的影响，TnI的全球构象，TnT的存在下，在三元Tn复合物引起了一个明显的伸长TnI。其次，而没有检测到的钙离子依赖的变化，在全球的构象TnI在Tn复合物中的自由在溶液中，去除钙离子引起的N-和C-末端TnI区域的重构细丝的实质性分离，由于TnI和肌动蛋白的C-末端区域之间的相互作用在放松状态。
Contraction of vertebrate striated muscle is regulated by the interaction of Ca2+with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibitory subunit, responds to the binding of Ca2+to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N- and C-terminal domains, respectively, of TnI. A mutant rabbit skeletal TnI, TnI48/133(C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of ∼41 Å for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 Å in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca2+to TnC. In the reconstituted thin filament, this distance remained to be 50 Å in the presence of saturating Ca2+, but increased to ∼66 Å on removing Ca2+with EGTA in the presence of Mg2+. Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca2+-dependent change in the global conformation of TnI in the Tn complex free in solution, the removal of Ca2+caused a substantial separation of the N- and C-terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the relaxed state.