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Studies on the structure and function of a biomotor dynein

生物运动动力蛋白的结构和功能研究

基本信息

批准号：
10640673
负责人：
OGAWA Kazuo
金额：
$ 1.79万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Dynein is a very huge motor protein in contrast to myosin and kinesin. There are two isoforms of dyneins; axonemal and cytoplasmic ones. However, dyneins so far cloned have four P-loop (ATP-binding) motives in the midregion of the molecules. In the present work, I have determined the true ATPase activity site of outer arm dynein from sea urchin sperm axonemes by the recombinant protein expression.In an expression of a series of deleted mutants in Baculo virus system, products were not extracted under the mild condition possibly because of an abortive folding of expressed mutants. To get soluble mutants, we have tried the dual expression of mutants and the other protein component contained in outer arm dynein. In situ dynein (heavy chain of outer arm dynein) associates with intermediate chain (IC1). Thus, we expressed simultaneously a series of mutants and IC1 in Baculo virus system.ICI was flagged with 6xHis. Taking the in situ binding of dynein heavy chain and IC1 into consideration, … More mutants could be bound to the Ni-affinity resin via the interaction of them with IC1-6xHis. I confirmed that mutants were bound to the resin, although the percentages of bound and unbound ones were very low.Mutant ranged in the four P-Loop motives showed an ATPase activity, though to greatly Lesser extents than in situ dynein. So, I prepared a series of smaller mutants containing only one P-Loop motif in the molecule to identify the ATPase site of dynein heavy chain and the significance of four P-Loop motives. Dual expression of them and ICI-6xIlis and ATPase assay demonstrated that only the mutant containing N-terminal first P-Loop (PI) showed ATPase activity. The other P-loop motif itself didn't show any ATPase activity.From the present work, the first P-loop motif of dynein molecule seems responsible for ATPase activity of dynein. Although the solubility of mutants was improved by the dual expression, Low specific activity of mutants suggest the occurrence of still abortive folding of them. Less

动力蛋白是相对于肌球蛋白和驱动蛋白而言的一种非常巨大的运动蛋白。动力蛋白有两种亚型：轴丝型和细胞质型。然而，迄今为止克隆的动力蛋白在分子的中间区域有四个P-环（ATP结合）基序。本研究利用重组蛋白表达的方法，确定了海胆精子外臂动力蛋白的ATP酶活性位点，在杆状病毒系统中表达了一系列缺失突变体，在温和条件下没有提取到产物，可能是表达突变体折叠失败所致。为了获得可溶性突变体，我们尝试了突变体和外臂动力蛋白所含的其他蛋白组分的双重表达。原位动力蛋白（外臂动力蛋白的重链）与中间链（IC 1）缔合。因此，我们在Baculo病毒系统中同时表达了一系列突变体和IC 1。考虑到动力蛋白重链与IC 1的原位结合， ...更多信息突变体可以通过它们与IC 1 -6xHis的相互作用结合到Ni亲和树脂上。我证实了突变体与树脂结合，尽管结合和未结合的百分比很低。在四个P-Loop基序范围内的突变体显示出ATP酶活性，尽管其程度大大低于原位动力蛋白。因此，我制备了一系列分子中仅含有一个P-Loop基序的较小突变体，以确定动力蛋白重链的ATP酶位点和四个P-Loop基序的意义。双表达、ICI-6xIlis和ATP酶活性测定表明，只有含有N端第一个P-Loop（PI）的突变体具有ATP酶活性。动力蛋白分子的第一个P环基序可能与动力蛋白的ATP酶活性有关。虽然突变体的可溶性通过双重表达得到改善，但突变体的低比活性表明它们仍然发生了失败的折叠。少