Hennecke J, Sebbel P, Glockshuber R
Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Hönggerberg, Zürich, CH-8093, Switzerland.
J Mol Biol. 1999 Mar 5;286(4):1197-215. doi: 10.1006/jmbi.1998.2531.
One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.
蛋白质折叠的关键问题之一是多肽链是否需要独特的成核位点才能折叠成天然状态。为了确定折叠过程中可能必不可少的多肽片段，我们对一种天然末端彼此靠近的蛋白质进行了完整的环形置换分析。作为模型系统，我们使用了来自大肠杆菌的二硫键氧化还原酶DsbA，它是一种由189个氨基酸残基组成的单体蛋白。为了在其多肽链的所有可能位置引入新的末端，我们构建了一个随机环形置换的dsbA基因文库，并在体内筛选有活性的环形置换变体。总共鉴定出51种不同的活性变体。新的末端分布在约70%的多肽链上，其中大多数出现在规则的二级结构内。在DsbA序列约30%的区域中未发现新的末端，这些区域基本上对应于DsbA的四个α螺旋。通过定向诱变将新的末端引入这些“禁区片段”会产生整体折叠改变且催化活性大幅降低的蛋白质。相比之下，到目前为止分析的所有活性变体都显示出与DsbA野生型相当的结构和催化特性。我们认为随机环形置换能够识别蛋白质中对折叠和稳定性至关重要的连续结构元件。
