Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.
Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.
复制标题
对于硫氧还蛋白折叠重要的氨基酸残基只能通过研究该蛋白质的生理相关还原形式来揭示。
DOI：
10.1021/bi100784h
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Beckwith,Jon
中科院分区：
文献类型：
--
作者：
Huber,Damon;Chaffotte,Alain;Eser,Markus;Planson,Anne-Gaelle;Beckwith,Jon
来自大肠杆菌的硫氧还蛋白-1 经常被用作蛋白质折叠研究中的模型底物。然而，为了方便起见，这些研究主要集中在氧化型硫氧还蛋白上，而不是还原型硫氧还蛋白（生理学上更相关的物种）。在这里，我们描述了还原硫氧还蛋白的重折叠动力学和构象热力学的首次广泛表征。我们之前描述过一种基因筛选，该筛选产生了突变型硫氧还蛋白，其在氧化和还原形式下折叠得更慢。在这项研究中，我们将减少硫氧还蛋白折叠的更详细分析应用于大量折叠突变体，其中包括从继续遗传筛选中获得的突变体。我们已经鉴定出在还原态而非氧化态下表现出折叠缺陷的突变蛋白。其中一些取代代表了不寻常的折叠突变体，因为它们导致折叠构象中暴露于溶剂的位置处的半保守取代，并且似乎不影响蛋白质的构象稳定性。此外，遗传选择仅在有限数量的位点产生突变体，这可能指向折叠途径中最关键的氨基酸，并特别强调了羧基末端氨基酸在硫氧还蛋白折叠中的作用。我们的结果证明了研究生理相关折叠种类的重要性。
Thioredoxin-1 fromEscherichia colihas frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin proteins that fold more slowly in both the oxidized and reduced forms. In this study, we apply our more detailed analysis of reduced thioredoxin folding to a larger number of folding mutants that includes those obtained from continuation of the genetic screen. We have identified mutant proteins that display folding defects specifically in the reduced state but not the oxidized state. Some of these substitutions represent unusual folding mutants in that they result in semiconservative substitutions at solvent-exposed positions in the folded conformation and do not appear to affect the conformational stability of the protein. Further, the genetic selection yields mutants at only a limited number of sites, pointing to perhaps the most critical amino acids in the folding pathway and underscoring, in particular, the role of the carboxy-terminal amino acids in the folding of thioredoxin. Our results demonstrate the importance of studying the physiologically relevant folding species.
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DOI：
10.1073/pnas.90.3.1043
发表时间：
1993-02-01
影响因子：
11.1
作者：
DAILEY, FE;BERG, HC
通讯作者：
BERG, HC
影响因子：
5.7
作者：
JENG, MF;CAMPBELL, AP;DYSON, HJ
通讯作者：
DYSON, HJ
DOI：
10.1093/protein/10.12.1425
发表时间：
1997-12-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
de Lamotte-Guéry, F;Pruvost, C;Decottignies, P
通讯作者：
Decottignies, P
DOI：
10.1073/pnas.0509583102
发表时间：
2005-12-27
影响因子：
11.1
作者：
Huber, D;Cha, MI;Beckwith, J
通讯作者：
Beckwith, J
影响因子：
2.9
作者：
Georgescu, RE;Li, JH;Chaffotte, AF
通讯作者：
Chaffotte, AF