Studies on the structure and function of a biomotor dynein

与肌凝蛋白和运动蛋白不同，动力蛋白是一种巨大的运动蛋白。动力蛋白有两种同工型；轴突和细胞质。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）动机。在本工作中，我通过重组蛋白的表达，确定了海胆精子轴突中外臂动力蛋白的真正atp酶活性位点。在Baculo病毒系统中表达一系列缺失突变体时，在温和条件下未能提取产物，可能是由于表达的突变体折叠失败。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中其他蛋白质成分的双重表达。原位动力蛋白（外臂动力蛋白重链）与中间链（IC1）相结合。因此，我们在Baculo病毒系统中同时表达了一系列突变体和IC1。ICI被标记为6xHis。考虑到动力蛋白重链与IC1的原位结合，更多的突变体可以通过与IC1- 6xhis的相互作用结合到ni亲和树脂上。我确认突变体与树脂结合，尽管结合和未结合的百分比非常低。在四个P-Loop动机中的突变体显示atp酶活性，尽管其程度远低于原位动力蛋白。因此，我准备了一系列分子中只含有一个P-Loop基序的较小突变体，以鉴定动力蛋白重链的atp酶位点和四个P-Loop动机的意义。双表达、ci - 6xilis和atp酶实验表明，只有含有n端第一p环（PI）的突变体具有atp酶活性。另一个P-loop基序本身没有显示任何atp酶活性。从目前的工作来看，动力蛋白分子的第一个p环基序似乎与动力蛋白的atp酶活性有关。虽然双表达提高了突变体的溶解度，但突变体的低特异性活性表明它们仍然存在流产折叠。少

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