Characterization of [4Fe-4S]-containing and cluster-free forms of Streptomyces WhiD.
Characterization of [4Fe-4S]-containing and cluster-free forms of Streptomyces WhiD.
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DOI：
10.1021/bi901498v
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发表时间：
2009-12-29
期刊：
影响因子：
2.9
通讯作者：
Le Brun, Nick E.
中科院分区：
文献类型：
--
作者：
Crack, Jason C.;den Hengst, Chris D.;Jakimowicz, Piotr;Subramanian, Sowmya;Johnson, Michael K.;Buttner, Mark J.;Thomson, Andrew J.;Le Brun, Nick E.
WhiD是放线菌中特有的类WhiB（Wbl）铁硫蛋白家族的成员，是天蓝色链霉菌孢子形成后期所必需的。与所有其他Wbl蛋白一样，WhiD迄今尚未以含有大量簇的可溶形式纯化，并且表征依赖于簇重构蛋白。因此，Wbl研究的主要目标是获得和表征含有铁-硫簇的天然蛋白质。在这里，我们报告的分析S。coelicolorWhiD是从大肠杆菌中厌氧纯化得到的。大肠杆菌中的可溶性蛋白含有一个由四个半胱氨酸连接的[4Fe-4S]2+簇。暴露于氧气后，与[4Fe-4S]簇相关的光谱特征在缓慢的反应中丢失，该反应不寻常地直接产生apo-WhiD，而没有显著浓度的簇中间体。发现该过程高度依赖于pH，在pH 7.0和8.0之间观察到最佳稳定性。低分子量硫醇，包括真菌硫醇类似物和硫氧还蛋白，对WhiD簇损失产生了小但显著的保护作用，这是一种可能具有生理重要性的活性。发现[4Fe-4S]2+ WhiD与超氧化物的反应比与氧或过氧化氢的反应快得多，这也可能具有生理意义。损失的[4Fe-4S]簇形成脱辅基蛋白不稳定的蛋白质折叠显着，但没有导致完全展开。最后，apo-WhiD在基于胰岛素的二硫键还原酶测定中表现出可忽略的活性，表明其不作为一般蛋白质二硫键还原酶发挥作用。
WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from E. coli as a soluble protein containing a single [4Fe-4S]2+ cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. [4Fe-4S]2+ WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apo-protein destabilized the protein fold significantly, but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay demonstrating that it does not function as a general protein disulfide reductase.
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影响因子：
2.4
作者：
Bennis, S;Chami, F;Remmal, A
通讯作者：
Remmal, A
DOI：
10.1073/pnas.0609514104
发表时间：
2007-02-13
影响因子：
11.1
作者：
Crack, Jason C.;Green, Jeffrey;Thomson, Andrew J.
通讯作者：
Thomson, Andrew J.
影响因子：
13.8
作者：
Combet, C;Blanchet, C;Deléage, G
通讯作者：
Deléage, G
影响因子：
2.9
作者：
Brereton, PS;Duderstadt, RE;Adams, MWW
通讯作者：
Adams, MWW
影响因子：
3.9
作者：
Crack, Jason C.;Jervis, Adrian J.;Le Brun, Nick E.
通讯作者：
Le Brun, Nick E.