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STRUCTURAL STUDIES ON THE CONTRACTILE TAIL SHEATH PROTEIN OF BACTERIOPHAGE T4

噬菌体T4收缩尾鞘蛋白的结构研究

基本信息

批准号：
62580203
负责人：
FUMIO Arisaka
金额：
$ 0.96万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

In order to elucidate the molecular mechanism of assembly and contraction of the tail sheath of bacteriophage T4, we have studieed the structure of the tail sheath protein, gp18. Gp18 has a molecular weight of 71,160 with 658 amino acid residues. 1)Nitration of tyrosine residues of gp18 by TNM (tetranitromethane) has revealed that 10 or 11 tyrosine residues (Tyr63/73, 254,270,455,460,493,523,535,569 and 590) out of 31 tyrosines were modifiable either in the monomeric state or the extended form of the tail sheath, but only five of these residues (Tyr254,270,455,460 and 493) were modified in the contracted form of the sheath. Especially, the nitration of Tyr270 and 455 proceeded independent of the association state of sp18. On the other hand, modification of Cys residues by a sulfhydryl group-specific reagent, ABD-F (4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole), has revealed that Cys377, Cys477 and Cys607, among 5 cysteine residues in gp18, have a sulfhydryl group. Cys402 and 406 are very likely connected by a disulfide bond.Cys607 can be modified n monomeric and associated states, whereas Cys477 is modifiable only in the monomeric state of gp18. 2) Limited proteolysis, immunoblotting and immuno-electron microscopy have shown that the trypsin-resistant fragment is Ala82-Lys316 and that, when combined with the data described above and below, the region of residues 250 through 410 appears to form the protruding part of the tail sheath as revealed by three-dimensional image reconstruction by Amos and Klug (1975). 3) Sequence determination of the tail sheath gene of Pseudomonas aeruginosa phage PS17 revealed that the tail sheath protein has 385 amino acids and that the corresponding sequence in T4 phage gp18 was split into two parts and present in two separate places in the primary structure.

为了阐明T4噬菌体尾鞘组装和收缩的分子机制，我们对尾鞘蛋白gp18的结构进行了研究。Gp18的相对分子质量为71,160，含658个氨基酸残基。1)用四硝基甲烷硝化gp18的酪氨酸残基，发现31个酪氨酸中有10个或11个酪氨酸残基(Tyr63/73,254,270,455,460,493,523,535,569和590)在尾鞘的单体状态或扩展形式下是可修饰的，但只有5个残基(Tyr254,270,455,460和493)是以收缩形式修饰的。特别是，Tyr270和455的硝化过程与SP18的缔合状态无关。另一方面，用一种巯基特异的试剂ABD-F(4-aminosulfonyl)-7-fluoro-2，1，3-benzoxadiazole)，修饰半胱氨酸残基，发现在gp18的5个半胱氨酸残基中，Cys377、Cys477和Cys607都有一个巯基。Cys402和406很可能以二硫键连接，Cys607可以被单体和缔合状态修饰，而Cys477只能在单体gp18状态下被修饰。2)有限的蛋白分解、免疫印迹和免疫电子显微镜显示，胰酶抗性片段是Ala82-Lys316，当结合上面和下面描述的数据时，根据Amos和Klug(1975)的三维图像重建，残基250到410似乎形成了尾鞘的突出部分。3)对铜绿假单胞菌噬菌体PS17的尾鞘基因进行了序列测定，发现其尾鞘蛋白由385个氨基酸组成，T4噬菌体gp18的相应序列被分成两部分，存在于一级结构中的两个不同位置。