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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的组装过程中，形成颈部以阻止DNA泄漏，并为DNA包装到头部完成后的尾部缔合做准备。颈部由gp 13（gp：基因产物）和gp 14组成，gp 13和gp 14分别由309和256个氨基酸残基组成，分子量分别为41，226和32，868。为了进一步阐明颈的结构和亚基排列，我们采用PCR方法克隆了这两个蛋白的编码基因，构建了过表达系统，并通过IPTG诱导表达。经硫酸铵分级沉淀、离子交换层析、凝胶过滤等纯化后，SDS-PAGE显示单一条带。远紫外圆二色性光谱分析表明，gp 13富含α-螺旋，而gp 13富含β-折叠。另一方面，沉降速度表明，两种蛋白质都是单体，沉降系数分别为2.80 S和2.00 S，摩擦比分别为1.22和1.73，这表明gp 14比gp 13更长。两种蛋白在正常生理条件下不相互作用，但发现两种蛋白形成了一种特异性复合物，沉降系数为16.6S。该复合物具有497，000的分子量，其与基于SDS-PAGE的2：1的摩尔比一起表明该复合物是杂15聚体，（gp 13）10（gp 14）5。该复合物被认为与当两种蛋白质共表达时观察到的复合物相同。电子显微镜观察表明，该复合物形成了一个直径为156 nm的环状结构，与电子显微镜重建的颈部三维图像的电子密度相吻合。