Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552 -: Reciprocal mutation analysis
Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552 -: Reciprocal mutation analysis
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DOI：
10.1074/jbc.m412392200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Sambongi, Y
中科院分区：
文献类型：
--
作者：
Oikawa, K;Nakamura, S;Sambongi, Y
细胞色素c(552)中有5个氨基酸残基具有极高的稳定性。(HTc(552.))来自一种嗜热菌，嗜热氢杆菌。用铜绿假单胞菌同源但不太稳定的细胞色素C-551(PA c(551))中的相应残基替换了分布在HTC-552三个区域的5个残基。五元组Htc(552年)。突变体(A7F/M13V/Y34F/Y43E/178V)对盐酸胍的稳定性与PA C-511相同，表明HTC-552中的5个残基对整体稳定性有必要和充分的贡献。在三个HTC551变异体中，Y34F/Y43E突变导致的不稳定程度最大，为-13.3kJ摩尔(-1)，其次是A7F/M13V(-3.3W摩尔(-1))，然后是178V(-1.5W摩尔(-1))。突变顺序为F34Y/E43Y、F7A/V13WL和V78I(分别为13.4、10.3和0.3W·mol~(-1))。盐酸胍的变性结果与相同变异体的热变性结果一致。本研究建立了一种相互突变分析方法。通过交换两个稳定性不同的同源蛋白之间的残基来实验评估侧链接触的效果。对这两种蛋白质的比较研究是评估氨基酸对整体稳定性的贡献的有用工具。
Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552). (HT c(552.)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT C-552 were replaced with the corresponding residues in the homologous but less stable cytochrome C-551 (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552). variant (A7F/M13V/Y34F/Y43E/ 178V) showed the same stability against guanidine hydrochloride denaturation as that of PA C-511, suggesting that the five residues in HT C-552 necessarily and sufficiently contribute to the overall stability. In the three HT C551 variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 W mol(-1)) and then 178V (-1.5 W mol(-1)). The order of destabilization in HT C-552 was the same as that of stabilization in PA C-551 with reverse mutations such as F34Y/ E43Y, F7A/V13WL and V78I (13.4, 10.3, and 0.3 W mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the nmino acid contribution to the overall stability.