Facilitation of Crystallization of Proteins by Trimming

通过修剪促进蛋白质结晶

基本信息

批准号：
03454564
负责人：
SHIRAKIHARA Yasuo
金额：
$ 4.1万
依托单位：
HYOGO UNIVERSITY OF TEACHER EDUCATION
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454564/
关键词：
X-ray crystallography Three-dimensional Structure of Proteins Crystallization Proteolytic Enzyme

项目摘要

In order to accelerate to get the structure of the proteins by x-ray protein crystallography, a trial has been made to facilitate crystal growth by trimming the target protein appropriately. This trial is in accordance with calls for more independent 3-dimensional structure information.The target proteins were:F1-ATPase from a thermophilic bacterium, its alpha3beta3 complex, rat DNA polymerase beta, Env Z protein, PhoB protein, Band 3 protein, CamR repressor, Asp racemase and flagellin. Those proteins were subjected to a limited proteolysis by a number of proteases to look for appropriately trimmed molecules. The trimmed molecules were then purified and subjected to crystallization experiment.The results are: 1.In case of rat DNA polymerase beta, a large C-terminal fragment (representing 80 % of the molecule) was found to crystallize to a suitable form. The intact molecule resisted to crystallization. The crystals diffracted to at least to 2.5 A^^ﾟ resolution, and the data collection and derivative search is now in progress. This indicates that the trimmed molecule does form good crystals. 2.41 k fragment of flagellin formed crystals. The intact flagellin formed helical aggregates under most of crystallization conditions. Although crystals were formed, the conditions are to be refined to get better crystals. 3.Appropriate fragments of PhoB protein and CamR repressor were found. They are now subjected to purification.

为了加速通过X射线蛋白质晶体学获得蛋白质的结构，进行了通过适当修剪目标蛋白质来促进晶体生长的尝试，该尝试符合对更独立的3维结构信息的要求。目标蛋白是：来自嗜热细菌的F1-ATP酶、其α3β3复合物、大鼠DNA聚合酶β、Env Z蛋白、PhoB蛋白、Band 3蛋白、CamR阻遏物、Asp消旋酶和这些蛋白质被多种蛋白酶进行有限的蛋白水解，以寻找适当修剪的分子，然后纯化修剪的分子并进行结晶实验。结果是： 1.在大鼠DNA聚合酶β的情况下，有一个大的DNA聚合酶。发现 C 端片段（代表分子的 80%）结晶成合适的形式。完整的分子抵抗结晶。 2.5 A^^ﾟ分辨率，数据收集和导数搜索正在进行中，这表明修剪后的分子确实形成了良好的鞭毛蛋白晶体片段，完整的鞭毛蛋白在大多数结晶条件下形成了螺旋聚集体。虽然形成了晶体，但仍需改进条件以获得更好的晶体。 3.目前已找到合适的PhoB蛋白和CamR阻遏物片段。纯化。