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Studies on Molecular Mechanism of Force Generation by Recombinant Dynein

重组动力蛋白产生力的分子机制研究

基本信息

批准号：
15370063
负责人：
SUTOH Kazuo
金额：
$ 9.6万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We have succeeded to express recombinant fragment of Dictyostelium cytoplasmic dynein in Dictyostelium cells (Nishiura et al. J.Biol.Chem (2004)). The expressed dynein fragment with molecular mass of 380kDa is consisted of the C-terminal heavy chain fragment, and maintains full motor activities : the fragment, a single-headed dynein motor, has high microtubule-activated ATPase activity (120 s^<-1>), and drives microtubule sliding at 3 μm/sec. By microtubule-landing assays, the sliding is shown to be non-processive, and the duty ratio is determined as 0.37. By combining these values, the step size is expected to be 8 nm. Thus, this active recombinant cytoplasmic dynein fragment behaves like the single-headed kinesin, another microtubule-based motor. By using this unique expression system, we have examined functional roles of four potential ATPase sites in the AAA+-ring of the dynein motor domain. We introduced point mutations in the highly conserved Walker A and Walker B sequences in these four potential ATPase sites, and examined their basal and microtubule-activated ATPase activities as well as their motility. We have found that the first AAA+ module has the primary ATPase site responsible for the force generation, and the third AAA+ module has the ATPase site tightly coupled with the former (Kon et al., Biochemistry (2004)). We have then developed the GFP-based FRET system to detect ATP hydrolysis cycle-dependent structural transitions of dynein, which may correspond to power stroke (Kon et al., Nature Structural and Molecular Biology (2005)). We identified two distinct structural states that have distinct fluorescence spectra due to FRET between GFP and BFP attached to two locations on the active dynein motor. We also correlated these two states to intermediate states of cyclic ATP hydrolysis at the dynein primary ATPase site.

我们已成功地在Dictyostelials细胞中表达了胞质动力蛋白的重组片段(Nishiura et al.J.Biol.Chem(2004))。表达的动力蛋白片段由C端重链片段组成，并保持了完整的运动活性：该片段为单头动力蛋白马达，具有较高的微管激活的ATP酶活性(120 S)，并驱动微管以3μm/s的速度滑动。微管着陆分析表明，滑动是非过程性的，占空比为0.37。通过组合这些值，步长预计为8 nm。因此，这种活跃的重组胞浆动力蛋白片段的行为类似于另一种基于微管的发动机--单头动力蛋白。通过使用这个独特的表达系统，我们研究了动力蛋白运动域的AAA+环上四个潜在的ATPase位点的功能作用。我们在这四个潜在的ATPase位点上引入了高度保守的Walker A和Walker B序列的点突变，并检测了它们的基础和微管激活的ATPase活性以及它们的运动性。我们已经发现，第一个AAA+模块具有负责产生力的主要ATPase位点，而第三个AAA+模块具有与前者紧密耦合的ATPase位点(Kon等人，生物化学(2004))。然后，我们开发了基于GFP的FRET系统来检测动力蛋白的ATP水解周期依赖的结构转变，这可能对应于功率中风(Kon等人，自然结构和分子生物学(2005))。我们鉴定了两种不同的结构状态，它们具有不同的荧光光谱，这是由于GFP和BFP之间的FRET连接到活性动力蛋白马达上的两个位置。我们还将这两种状态与动力蛋白初级ATPase位点上的循环ATP水解的中间状态相关联。