Structure Formation of the Neck which Links the Head and the Tail of Bacteriophage

连接噬菌体头部和尾部的颈部结构形成

基本信息

批准号：
18570147
负责人：
ARISAKA Fumio
金额：
$ 2.63万
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 (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

During the assembly of phage T4, the neck is formed to block the DNA leakage and to prepare for the tail association after DNA packaging into the head is completed. The neck is composed of gp13 (gp: gene product) and gp14 each consists of 309 and 256 amino acid residues with the molecular weight of 41,226 and 32,868, respectively. In order to elucidate the structure and the subunit arrangement of the neck, genes coding for the two proteins were cloned by PCR method, an overexpression system was constructed and the genes were induced by IPTG. The two proteins were purified by ammonium sulfate fractionation, ion exchange chromatography, gen filtration as a single band in SDS-PAGE. Far-ultraviolet circular dichroism spectra of the two proteins indicated that gp13 is rich in α-helix, whereas gp13 is rich in β-sheet. On the other hand, sedimentation velocity indicated that both proteins are monomers with the sedimentation coefficient of 2.80S and 2.00S, respectively and that the frictional ratios were 1.22 and 1.73, respectively, which indicated that gp14 is more elongated than gp13. The two proteins did not interact each other under normal physiological conditions, but it was found that the two proteins formed a specific complex with the sedimentation coefficient of 16.6S. The complex had the molecular weight of 497,000 which together with the molar ratio of 2:1 based on SDS-PAGE indicated that the complex is a hetero 15-mer, (gp 13)10(gp 14)5. This complex is considered to be identical with the complex which was observed when the two proteins were co-expressed. Electron microscopic observations revealed that the complex formed a ring with the diameter of 156 Å which coincided with the electron density of the three-dimensional image of the neck reconstructed from electron micrograph.

在噬菌体T4组装过程中，形成颈部以阻断DNA泄漏并为DNA包装完成后尾巴关联做准备。颈部由GP13（GP：基因产物）组成，GP14分别由309和256氨基酸组成，分子量分别为41,226和32,868。为了阐明颈部的结构和亚基排列，通过PCR方法克隆了两种蛋白质编码的基因，构建了一个过表达系统，并由IPTG诱导基因。通过硫酸铵分馏，离子交换色谱法，Gen过滤作为SDS-PAGE中的单个条带来纯化这两种蛋白质。两种蛋白质的远硫酸酯圆形二分法表明GP13富含α-螺旋，而GP13富含β-折叠。另一方面，沉积速度表明，这两种蛋白质都是单体，分别为2.80和2.00，摩擦比分别为1.22和1.73，这表明GP14比GP13更长。两种蛋白质在正常的物理条件下没有相互相互作用，但是发现这两种蛋白质形成了一种特定的络合物，沉积系数为16.6s。该络合物的分子量为497,000，基于SDS-PAGE的摩尔比为2：1，表明该复合物是杂音15-Mer，（GP 13）10（GP 14）5。该复合物被认为与当两种蛋白共表达时观察到的复合物相同。电子显微镜观测表明，该复合物形成了一个环，直径为156Å，这与从电子显微照片重建的颈部三维图像的电子密度相吻合。