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

尾溶菌酶（Tail-lysozyme），gp 5 ^**>，是具有溶菌酶活性并位于基板下方的基板的42 kD结构蛋白。当噬菌体吸附到大肠杆菌上时，它与尾管一起渗透到外膜中，并在肽聚糖层上挖一个整体，使得尾管可以到达内膜。我们以前已经分离了尾溶菌酶，并表明尾溶菌酶具有与T4溶菌酶gp e相同的底物特异性，即N-乙酰胞壁酶（1）。核苷酸序列的预期分子量为62 kD，事实上，克隆的基因5将其表达为62 kD的蛋白质，但溶菌酶活性不存在于溶菌酶蛋白质中（2）。在本研究中，我们已经确定了成熟蛋白的N-和C-末端序列。N-末端序列与核苷酸序列的预期序列一致，C-末端为Val 390。与gp e具有高度同源性（44%相同）的区域位于成熟gp 5的C-末端。N-末端区域可能呈现构成基板的组成部分的结构域。结果表明，20 kD的片段是从溶菌酶蛋白上切割下来的，形成了成熟的42 kD的尾溶菌酶。另一方面，我们对12个突变体的突变位点进行了定位，这些突变体在基因5上有突变。这些突变体包括5个温度敏感突变体、2个热敏感突变体和5个琥珀突变体。所有的突变位点都定位在Val 390的上游。在ts突变体中，5 ts 1（3）是一个旁路突变体，它不需要gp e从内部裂解。这是定位在溶菌酶结构域中的Gly 322 Asp。另一个ts突变体5DH 6318是旁路63突变体，其不需要gp 63来有效附着尾纤维。这是Ala 65 Thr定位在“结构”域中。包括这些突变，其他的突变将被讨论的结构-功能关系的尾部溶菌酶。

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.