Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site
Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site
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DOI：
10.1016/j.pep.2004.10.017
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发表时间：
2005-02-01
影响因子：
1.6
通讯作者：
Kneale, GG
中科院分区：
文献类型：
--
作者：
Chant, A;Kraemer-Pecore, CM;Kneale, GG
组氨酸 (His) 标签是通过金属亲和层析分离蛋白质最常用的融合标签之一。进行实验时，融合标签通常会附着在蛋白质上，并假设添加对结构或功能没有影响。在本研究中，我们制备了来自构巢曲霉的基因调节蛋白AreA的四种蛋白用于结晶实验：包含最小DNA结合区的91个氨基酸肽，有和没有His标签（分别是HZFB和ZFB），以及之前提出的AreA的整个DNA结合域的155个氨基酸蛋白，有和没有 His 标签（分别为 HG 1 b 和 G 1 b）。为了测试四种 AreA 蛋白的完整性，使用荧光光谱法进行了尿素变性实验和 DNA 结合研究。 DNA 结合数据显示所有蛋白质的解离常数相似，K-d 值在纳摩尔范围内。然而，尿素变性数据清楚地表明，与 ZFB、HG1b 和 G1b 蛋白相比，HZFB 蛋白表现出完全不同的变性特征。 HZFB 蛋白质的轮廓表明唯一色氨酸周围存在构象改变，而其他蛋白质则显示 3 至 4 M 尿素浓度之间的转变点。这些数据表明，虽然所研究的任何蛋白质的功能都没有改变，但其中一种带有组氨酸标签的蛋白质的结构与该蛋白质的天然形式不同。 (C) 2004 Elsevier Inc. 保留所有权利。
Histidine (His) tags are one of the most popular fusion tags for the isolation of proteins via metal affinity chromatography. The fusion tag is routinely left attached to the protein when carrying out experiments, with the assumption that the addition has no effect on structure or function. In the present study, we have prepared four proteins of the gene regulatory protein AreA from Aspergillis nidulans for crystallization experiments: a 91-amino acid peptide encompassing the minimal DNA-binding region, both with and without the His-tag (HZFB and ZFB, respectively), and a 155-amino acid protein previously proposed to be the entire DNA-binding domain for AreA, both with and without the His-tag (HG 1 b and G 1 b, respectively). To test the integrity of the four AreA proteins, urea denaturation experiments and DNA-binding studies were performed using fluorescence spectroscopy. The DNA-binding data showed similar dissociation constants for all proteins, with K-d values in the nanomolar range. The urea denaturation data, however, clearly indicated that the HZFB protein exhibited a completely different denaturation profile when compared to the ZFB, HG1b, and G1b proteins. The HZFB protein showed a profile indicative of the presence of an altered conformation around the sole tryptophan, whereas the other proteins showed a transition point between 3 and 4 M urea concentration. These data show that, although function was not altered for any of the proteins studied, the Structure of one of the His-tagged proteins was different from the native form of that protein. (C) 2004 Elsevier Inc. All rights reserved.