SYSTEMATIC MUTATION OF BACTERIOPHAGE-T4 LYSOZYME
SYSTEMATIC MUTATION OF BACTERIOPHAGE-T4 LYSOZYME
复制标题
DOI：
10.1016/0022-2836(91)90738-r
复制
发表时间：
1991-11-05
影响因子：
5.6
通讯作者：
POTEETE, AR
中科院分区：
文献类型：
--
作者：
RENNELL, D;BOUVIER, SE;POTEETE, AR
将琥珀突变引入噬菌体T4溶菌酶基因的每个密码子（除了起始AUG）。琥珀等位基因被引入一个称为P22 e416的噬菌体P22杂交体中，其中正常的P22溶菌酶基因被其T4同源物取代，因此依赖于T4溶菌酶形成噬斑的能力。在鼠伤寒沙门氏菌的琥珀抑制菌株上测试所得琥珀突变体的空斑形成。用其他杂交菌株进行的实验表明，为了评价为有害的，突变必须将溶菌酶活性降低到野生型P22 e416产生的溶菌酶活性的3%以下。将覆盖T4溶菌酶的164个密码子中的163个的琥珀突变体的集合接种在13个抑制菌株上，每个抑制菌株响应琥珀密码子插入不同的氨基酸残基，测试蛋白质中每个位置（除了第一个）处的多个单个氨基酸取代的影响。在T4溶菌酶中产生的2015个单一氨基酸取代中，发现328个足以抑制噬斑形成。超过一半（55%）的蛋白质中的位置耐受所有取代检查。在N端琥珀片段中，只有161个或更多残基的琥珀片段是有活性的，许多有害取代的影响可以根据T4溶菌酶的已知结构来解释。分子中难以置换的残基通常具有溶剂不可及的侧链;催化Glu 11和Asp 20残基是明显的例外。特别敏感的位点包括参与催化位点附近的埋藏盐桥的残基（Asp 10、Arg 145和Arg 148）和其他一些可能具有关键结构作用的残基（Gly 30、Trp 138和Tyr 161）。
Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains ofSalmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid residue in response to the amber codon, tests the effects of multiple single amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active.The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).