Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core
Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core
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DOI：
10.1074/jbc.272.30.18686
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发表时间：
1997-07-25
影响因子：
4.8
通讯作者：
Kanaya, S
中科院分区：
文献类型：
--
作者：
Akasako, A;Haruki, M;Kanaya, S
大肠杆菌核糖核酸酶HI的疏水核内有一个空腔，空腔的两端有两个核心残基Ala（52）和瓦尔（74）。我们构建了一系列Ala（52）单突变蛋白和Ala（52）被Gly、瓦尔、Ile、Leu或Phe取代，瓦尔（74）被Ala或Leu取代的双突变蛋白，除A52 W、A52 R和A52 G/V74 A外，所有这些突变蛋白都被过量生产和纯化。通过CD测量蛋白在pH 3.2下的热变性表明，空腔足够大以容纳三个甲基或亚甲基而不产生严重的菌株，蛋白质的稳定性与取代残基的疏水性之间存在相关性，因此，许多突变体蛋白质比野生型蛋白质更稳定，在空腔处具有带电荷或极其庞大的残基的突变蛋白的稳定性低于从取代残基的疏水性预期的那些，表明在这些突变蛋白中的突变位点处产生了相当多的菌株。然而，远紫外和近紫外GD光谱和酶活性的检测表明，所有突变蛋白具有与野生型蛋白相似的结构，这些结果表明疏水核心中的空腔关闭。coli RNase HI的构象相当稳定，这可能是空腔填充突变有效提高该蛋白热稳定性的原因。
Escherichia coli ribonuclease HI has a cavity within the hydrophobic core, Two core residues, Ala(52) and Val(74), resided at both ends of this cavity, We have constructed a series of single mutant proteins at Ala(52), and double mutant proteins, in which Ala(52) was replaced by Gly, Val, Ile, Leu, or Phe, and Val(74) was replaced by Ala or Leu, All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified, Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains, A correlation was observed between the protein stability and the hydrophobicity of the substituted residue, As a result, a number of the mutant proteins were more stable than the wild-type protein, The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins, However, examination of the far- and near-UV GD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein, These results suggest that the cavity in the hydrophobic core off. coli RNase HI is conformationally fairly stable, This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein.