STRUCTURAL STUDIES ON THE CONTRACTILE TAIL SHEATH PROTEIN OF BACTERIOPHAGE T4

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的分子量为71,160，有658个氨基酸残基。1)用TNM（四硝基甲烷）硝化gp18的酪氨酸残基，发现31个酪氨酸残基中有10个或11个（Tyr63/73、254,270,455,460,493,523,535,569和590）以单体状态或尾鞘扩展形式被修饰，但只有5个残基（Tyr254,270,455,460和493）以鞘收缩形式被修饰。特别是Tyr270和455的硝化过程与sp18的结合状态无关。另一方面，用巯基特异性试剂ABD-F（4-氨基磺酰基）-7-氟-2,1,3-苯并二唑)对Cys残基进行修饰，发现gp18的5个半胱氨酸残基中，Cys377、Cys477和Cys607含有一个巯基。Cys402和406很可能是由二硫键连接的。Cys607可以在单体状态和相关状态下被修饰，而Cys477只能在gp18的单体状态下被修饰。2)有限的蛋白水解、免疫印迹和免疫电镜显示，抗胰蛋白酶片段是Ala82-Lys316，当结合上面和下面描述的数据时，Amos和Klug（1975）的三维图像重建显示，残基250到410的区域似乎形成了尾鞘的突出部分。3)铜绿假单胞菌噬菌体PS17尾鞘基因序列测定显示，尾鞘蛋白有385个氨基酸，T4噬菌体gp18中相应的序列被分成两部分，分别存在于初级结构的两个位置。

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.