Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments
Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments
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DOI：
10.1002/pro.2520
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发表时间：
2014-10-01
期刊：
影响因子：
8
通讯作者：
Minor, Wladek
中科院分区：
文献类型：
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作者：
Majorek, Karolina A.;Kuhn, Misty L.;Minor, Wladek
获得纯化的活性蛋白是大多数体外生物医学、生化和药物发现实验的起点。多组氨酸亲和标签的使用大大提高了蛋白质纯化过程的效率，但可能对结构和/或活性测量产生负面影响。同样，缓冲分子也可能扰乱蛋白质的构象稳定性或其活性。在测定铜绿假单胞菌（PA4794） gcn5相关n -乙酰转移酶（GNAT）的结构时，我们发现HEPES和多组氨酸亲和标签在底物结合位点（分别）结合。在HEPES的情况下，分子诱导活性位点的构象变化，但不会显著影响酶的活性。相比之下，未裂解的his标签不会引起主要的构象变化，而是作为肽底物的弱竞争性抑制剂。在另外两种GNAT酶中，我们观察到his标签的存在对这些蛋白的活性有很强的影响。蛋白质制备对功能研究的影响可能会影响其他实验室实验的可重复性，即使方案之间的变化乍一看似乎微不足道。此外，本文的结果表明，调整每种蛋白质或蛋白质家族的实验条件，并研究这些因素对蛋白质活性和结构的影响是多么重要，因为它们可能会显著改变功能表征和筛选方法的有效性。因此，我们发现多组氨酸标签和缓冲分子HEPES结合在底物结合位点，并影响活性位点的构象和GNAT乙酰转移酶的活性。我们认为，这种差异可能会影响某些实验的可重复性，因此可能对后续研究产生重大的“连锁反应”。
The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant "ripple effect" on subsequent studies.