Structural Correlates of the Temperature Sensitive Phenotype Derived from Saturation Mutagenesis Studies of CcdB
Structural Correlates of the Temperature Sensitive Phenotype Derived from Saturation Mutagenesis Studies of CcdB
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DOI：
10.1021/bi8014345
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发表时间：
2008-12-09
期刊：
影响因子：
2.9
通讯作者：
Varadarajan, Raghavan
中科院分区：
文献类型：
--
作者：
Bajaj, Kanika;Dewan, Pooja C.;Varadarajan, Raghavan
温度敏感型(Ts)突变体被广泛用于可逆调节体内蛋白质功能和了解必需基因的功能。尽管如此，人们对产生ts表型的蛋白质结构特征和机制知之甚少。此外，这样的突变体通常很难分离，限制了它们的使用。在这项研究中，构建了一个包含101个残基的同源二聚体大肠杆菌毒素CcdB所有可能的单点突变的75%的文库。根据突变体在两个不同温度和六个不同表达水平下的活性来表征突变体。在筛选的1430个单点突变中，231个(16%)突变体表现为ts表型。其中大部分包括在所有22个掩埋位点发现的120个ts突变体和在所有7个与DNA旋转酶结合的活性位点残基上发现的34个ts突变体。在其余的ts突变体中，有16个位于Vander Waals与活性中心残基接触的残基，36个位于部分埋藏的残基，30个是由Pro导入的。因此，几乎所有ts突变体都可以在不了解折叠途径的情况下，根据天然蛋白质的结构进行合理化。对数据进行分析，以获得对ts表型的分子特征的洞察，并概述基于结构和序列的标准，以设计任何球形蛋白的ts突变体。通过对TBP、T4溶菌酶和Ga14这三种无关蛋白的ts突变体的成功预测，验证了该标准的有效性。
Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in vander Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Ga14.