Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.
Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.
复制标题
果蝇驱动蛋白重链茎的茎是α-螺旋盘绕的线圈。
DOI：
10.1083/jcb.116.4.957
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
Goldstein LS
中科院分区：
文献类型：
--
作者：
de Cuevas M;Tao T;Goldstein LS
Kinesin是一种机械力化学酶，由三个不同的结构域组成：球头结构域、杆状结构域和小球尾结构域。茎结构域具有阿尔法螺旋螺旋线圈的序列特征。为了深入了解Kinesin茎的结构，我们从果蝇kinogaster kinesin重链基因的一段中表达了它，并从大肠杆菌中纯化了它。当用EM观察时，这种蛋白质形成一个40-55 nm长的棒状结构，偶尔在分子中间附近的铰链状区域弯曲。另一项EM研究和化学交联研究表明，这种蛋白质形成一个平行的二聚体，并且两条链是注册的。最后，利用圆二色谱，我们证明该蛋白质在25℃的生理水溶液中约为55-60%的α-螺旋，在4℃时约为85-90%的α-螺旋。根据这些结果，我们得出结论：Kinesin重链的柄形成α-螺旋的盘绕结构。圆二向色性信号的温度依赖性有两个主要的转变，在25-30℃和45-50℃，这表明茎中的部分α-螺旋结构比其他结构更不稳定。通过在大肠杆菌中分别产生茎的氨基末端(COIL 1)和羧基末端(COIL 2)的一半，我们发现在30℃以下熔化的区域位于COIL 1内，而COIL 2的大部分熔化在45℃以上。我们认为这种稳定性的差异可能在Kinesin的力产生机制或调节中发挥作用。
Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40- 55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross- linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha- helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha- helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha- helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.