Molecular Assembly and the role in infection of Tail-lysozyme from bacteriophage T4.

分子组装及其在噬菌体 T4 尾部溶菌酶感染中的作用。

基本信息

批准号：
07680712
负责人：
ARISAKA Fumio
金额：
$ 1.41万
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：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Tail-lysozyme, gp5^<**>, is a 42 kD structural protein of the baseplate which has a lysozyme activity and is located under the baseplate. Upon adsorption of the phage to Escherichia coli, it penetrates into the outer membrane together with the tail tube and digs a whole on the peptidoglycan layr so that the tail tube can reach the inner membrane. We have previously isolated the tail-lysozyme and shown that the tail-lysozyme has the same substrate specificity as that of T4 lysozyme, gp e, namely N-acetyl muramidase (1). The expected molecular weight from the nucleotide sequence was 62 kD and in fact it is expressed as a 62 kD protein from the cloned gene 5, but the lysozyme activity is absent in the precurser protein (2). In the present study, we have determined the N-and C-terminal sequence of the mature protein. The N-terminal sequence coincided with that expected from the nucleotide sequence and the the C-terminus was Val390. A region with high homology (44% identical) to gp e, is located towards the C-terminus of the mature gp 5. N-terminal region is likely to assume a domain which constitutes the integral part of the baseplate. From the result, it was concluded that the 20 kD fragment is cleaved off from the precurser protein to become mature 42 kD tail-lysozyme.On the other hand, we have mapped the mutation sites of 12 mutants which have a mutation in gene 5. These include five ts (temperature sensitive), two hs (heat sensitive) and five amber mutants. All the mutation sites were mapped upstream of Val390. Among the ts mutants, 5ts1 (3) is a bypass e mutant wich does not require gp e to lyse from within. This was Gly322Asp mapped in the lysozyme domain. Another ts mutant, 5DH6318, is a bypass 63 mutant which does not require gp63 for efficient attachment of the tail fibers. This was Ala65Thr mapped in the "structural" domain. Including these mutations, other mutations will be discussed in respect to the structure-function relation of the tail-lysozyme.

Tail-Lysozyme，GP5^<**>是底板的42 kD结构蛋白，具有溶菌酶活性，位于底板下。在将噬菌体吸附到大肠杆菌上后，它与尾管一起渗透到外膜中，并在肽聚糖层上挖整个整体，以便尾管可以到达内膜。我们先前已经分离了尾囊肿，并表明尾囊肿具有与T4溶菌酶，GP E的底物特异性相同的底物特异性，即n-乙酰基杂种酶（1）。来自核苷酸序列的预期分子量为62 kD，实际上它是克隆基因5的62 kD蛋白，但在前体蛋白中不存在溶菌酶活性（2）。在本研究中，我们确定了成熟蛋白的N和C末端序列。 N末端序列与核苷酸序列的预期和C末端为Val390。与GP E的高同源性区域（44％相同）的区域位于成熟GP 5的C末端。N末端区域可能假设构成碱基整体部分的结构域。从结果得出的结论是，将20 kD片段从前体蛋白切割成成熟的42 kD尾囊肿。另一方面，我们映射了12个突变体的突变位点，这些突变体的突变位点在基因5中具有突变。这些突变体在基因5中。这些突变体包括五个TS（温度敏感），两个hs（热敏感）（热敏感）和五个amber突变体。所有突变位点均在Val390的上游映射。在TS突变体中，5TS1（3）是一个旁路突变体，不需要GP e从内部进行裂解。这是Gly322ASP在溶菌酶结构域中映射的。另一个TS突变体5DH6318是一个旁路63个突变体，不需要GP63即可有效地附着尾纤维。这是在“结构”域中映射的ALA65THR。包括这些突变，将讨论其他突变，以与尾lysozyme的结构功能关系进行讨论。