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

尾溶菌酶（Tail-lysozyme, gp5^<**>）是一个42 kD的基板结构蛋白，位于基板下方，具有溶菌酶活性。噬菌体被大肠杆菌吸附后，与尾管一起进入外膜，在肽聚糖层上挖出一个整体，使尾管到达内膜。我们之前已经分离出尾溶菌酶，并证明尾溶菌酶与T4溶菌酶gp e，即n -乙酰化酶具有相同的底物特异性(1)。核苷酸序列的预期分子量为62 kD，实际上它以克隆基因5的62 kD蛋白表达，但在前体蛋白中缺乏溶菌酶活性(2)。在本研究中，我们确定了成熟蛋白的n端和c端序列。n端序列与核苷酸序列吻合，c端为Val390。一个与gp e高度同源（44%相同）的区域位于成熟的gp 5的c端。n端区域可能呈现构成基板不可分割部分的区域。结果表明，20 kD片段从前体蛋白上断裂，成为成熟的42 kD尾溶菌酶。另一方面，我们绘制了12个突变体的突变位点，其中基因5发生突变。其中包括5个ts（温度敏感型），2个hs（热敏型）和5个琥珀色突变体。所有突变位点都定位在Val390上游。在这些突变体中，5ts1(3)是一个旁路突变体，不需要gp e从内部裂解。这是Gly322Asp在溶菌酶结构域中的映射。另一个ts突变体5DH6318是一个旁路63突变体，它不需要gp63来有效地附着尾部纤维。这是Ala65Thr在“结构”域的映射。包括这些突变，其他突变将讨论有关尾溶菌酶的结构-功能关系。

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.