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X-ray Crystallographic Analysis of Bacteriophage Precursor Structures

噬菌体前体结构的 X 射线晶体学分析

基本信息

批准号：
10044070
负责人：
ARISAKA Fumio
金额：
$ 1.54万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
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

As a step toward determination of the structure of the baseplate of bacteriophage T4 in atomic resolution, three precursor structures were chosen for crystallization, One of them was the initiation complex of the wedge of the baseplate, gp10-gp11 complex. It is considered to constitute the tail pins at the vertices of the hexagonal baseplate, which protrude from the bottom of the baseplate. Based on SDS-PAGE, Edman degradation of the complex and analytical ultracentrifugation, it was shown that the-complex is a hetero-hexamer with the subunit composition of (gp10)ィイD23ィエD2 (gp11)ィイD23ィエD2. Crystal of the complex suitable for X-ray analysis has not been obtained although small crystalls have been found. On the other hand, the second target for X-ray crystal analysis, i.e., gp5-gp27 complex, has been crystallized. Based on the results of sedimentation equilibrium and SDS-PAGE, the subunit structure of the complex was concluded to be hetero-nanomer, (gp5)ィイD23ィエD2(gp27)ィイD26ィエD2. The crystal yielded the resolution of approximately 2.5 A. At present, efforts are made to obatain isomorphous crystal containing selenomethionine. Gp5 is a structurally essential component, yet possesses lysozyme domain which locally digests peptidoglycan of the host E. coli upon infection so that the tail tube can reach the inner membrane. Concerning the third target protein, crystal has not been obtained, but we have recently succeeded in expressing a mutant protein which lacks the N-terminal seven residues that contain five negative charges. The mutant protein was shown to retain the native secondary and oligomeric structure. We plan to examine the possibility of crystallization of the mutant protein.

作为在原子分辨率上确定噬菌体T4的基板结构的一个步骤，选择了三种前体结构进行结晶，其中之一是基板楔形的起始复合物，gp 10-gp 11复合物。它被认为是构成六角底板顶点的尾销，从底板底部突出。SDS-PAGE、Edman降解和超离心分析表明，该复合物为异源六聚体，亚基组成为（gp 10）β-D23 β-D2（gp 11）β-D23 β-D2。尽管已经发现了小晶体，但尚未获得适合于X射线分析的配合物的晶体。另一方面，X射线晶体分析的第二目标，即，GP 5-GP 27复合物，已结晶。根据沉降平衡和SDS-PAGE结果，该复合物的亚基结构为异源纳米聚体，（gp 5）β-D23 β-D2（gp 27）β-D26 β-D2。该晶体产生约2.5 A的分辨率。目前，人们正努力获得含硒代蛋氨酸的类质同象晶体。Gp 5是一种结构上必需的组分，但具有溶菌酶结构域，其局部地取代宿主E的肽聚糖。大肠杆菌感染后，使尾管可以达到内膜。关于第三种靶蛋白，尚未获得晶体，但我们最近成功地表达了一种突变蛋白，该蛋白缺少含有5个负电荷的N-末端7个残基。突变蛋白质被证明保留了天然的二级和寡聚体结构。我们计划研究突变蛋白质结晶的可能性。