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X-ray Crystal Structure Analysis of Chaperonins (hsp60)

伴侣蛋白 (hsp60) 的 X 射线晶体结构分析

基本信息

批准号：
04454619
负责人：
MIKI Kunio
金额：
$ 4.42万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We have purified and crystallized chaperonin (hsp60), a molecular chaperon protein, from a thermophilic bacterium, Thermus thermophilus, in order to determine its three-dimensional structure by X-ray crystallography.We obtained two forms of chaperonin crystals, plates and hexagonal prisms by the use of polyethyleneglycol as a precipitant. The diffraction studies on the macromolecular Weissenberg camera by synchrotron radiation at Photon Factory showed that the former and latter crystals diffracted X-rays up to 10 and 7 A resolution, respectively. The plate and hexagonal prism crystals belong to the monoclinic space group P2 or P2_1 and hexagonal space group P6_322, respectively. The unit-cell parameters of both crystals were also determined.The SDS and native polyacrylamide gel electrophoreses of these crystals revealed that the plate crystals contain the holo-chaperonin complex composed of cpn60 14mer and cpn10 7mer. On the other hand, it was found that hexagonal crystals are composed of the monomeric 50KDa fragments which were thought to be occurred by natural proteolysis of cpn60.As resolution limit for X-ray diffraction of the 50kDa fragment was not so high, the crystallization was performed for the following two modified samples ; 1) the purified 50KDa fragment and 2) the 50KDa fragment treated by trypsin. Crystals with different unit-cell dimensions were obtained but their resolution limit does not reach atomic resolution.The further improvement of resolution limits of these crystals is still underway.

我们从嗜热细菌Thermus thermophilus中纯化并结晶了分子伴侣蛋白chaperonin (hsp60)，以便通过x射线晶体学确定其三维结构。我们用聚乙二醇作为沉淀剂，得到了两种形式的伴侣蛋白晶体，板状和六方棱镜状。在光子工厂用同步辐射对大分子Weissenberg照相机进行了衍射研究，结果表明前者和后者晶体的x射线衍射分辨率分别高达10和7 A。平板晶体和六角形棱镜晶体分别属于单斜空间群P2或P2_1和六角形空间群P6_322。测定了两种晶体的单胞参数。SDS和天然聚丙烯酰胺凝胶电泳结果表明，平板晶体含有由cpn60 - 14mer和cpn10 - 7mer组成的全息伴侣蛋白复合物。另一方面，发现六方晶体是由50KDa的单体片段组成的，这些片段被认为是由cpn60的自然蛋白水解产生的。由于50kDa片段的x射线衍射分辨率限制不是很高，所以对以下两个修饰后的样品进行结晶；1)纯化的50KDa片段和2)经胰蛋白酶处理的50KDa片段。得到了不同尺寸的单晶，但其分辨率极限均达不到原子分辨率。进一步提高这些晶体的分辨极限仍在进行中。