Mutagenesis-based definitions and probes of residue burial in proteins
Mutagenesis-based definitions and probes of residue burial in proteins
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DOI：
10.1073/pnas.0505089102
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发表时间：
2005-11-08
影响因子：
11.1
通讯作者：
Varadarajan, R
中科院分区：
文献类型：
--
作者：
Bajaj, K;Chakrabarti, P;Varadarajan, R
通过使用定点诱变，将 101-aa 埃希氏菌、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控制器或死亡 B 蛋白 (CcdB) 转录水平的函数。突变数据表明，5% 的可及性值是定义埋藏残基的适当截止值。在所有埋藏位置，天冬氨酸的引入导致所有 CcdB 转录水平的非活性表型。埋藏位置取代时的平均失稳量按 Asp>Glu>Lys>Arg>Ala 的顺序降低。另外两种蛋白质（麦芽糖结合蛋白和硫氧还蛋白）中埋藏位点的天冬氨酸取代也被证明会严重破坏稳定性。丙氨酸和天冬氨酸扫描诱变与剂量依赖性表达表型相结合，可产生有关蛋白质结构和活性的重要信息。这些结果还表明，此类扫描诱变数据可用于对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，针对小蛋白质/结构域的全面扫描诱变实验是相当可行的。
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 data can 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.