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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Mutagenesis-based definitions and probes of residue burial in proteins.
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Mutagenesis-based definitions and probes of residue burial in proteins.

机译：基于诱变的定义和蛋白质中残留残基的探针。

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摘要

Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis datacan be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small proteins/domains are quite feasible.
机译：通过使用定点诱变，将101-aa大肠杆菌毒素CcdB的每个残基替换为Ala，Asp，Glu，Lys和Arg。每个突变体的体内活性被表征为细胞分裂或死亡B蛋白（CcdB）转录水平的控制器。突变数据表明，可访问性值为5％是定义掩埋残基的适当临界值。在所有掩埋位置，Asp的引入都会在所有CcdB转录水平上导致无活性的表型。掩埋位置上取代时的去稳定的平均量以Asp> Glu> Lys> Arg> Ala的顺序降低。麦芽糖结合蛋白和硫氧还蛋白两种其他蛋白在掩埋位点的Asp取代也显示出严重的不稳定作用。 Ala和Asp扫描诱变与剂量依赖性表达表型相结合，显示出有关蛋白质结构和活性的重要信息。这些结果还表明，这种扫描诱变数据可用于对顺序序列比对及其相应的同源性模型进行排序，以及区分正确和不正确的结构比对。随着寡核苷酸成本的不断降低和定点诱变程序的日益高效，针对小蛋白/结构域的全面扫描诱变实验是十分可行的。

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