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首页> 外文期刊>Molecular membrane biology >Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay.
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Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay.

机译：使用基于Thermofluor的稳定性测定法对詹氏甲烷球菌的CorA Mg2 +传输通道进行表征。

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摘要

The Thermofluor assay has been a valuable asset in structural genomics, providing a high-throughput method for assessing the crystallizability of proteins. The technique has been well characterized for soluble proteins but has been less extensively described for membrane proteins. Here we show the successful application of a Thermofluor-based stability assay to an ion channel, CorA from Methanococcus jannaschii. Optimization of the concentration of free detergent within the assay was important, as excessive concentrations mask the fluorescence change associated with thermal unfolding of the protein. CorA was shown to be stabilized by low pH, but relatively insensitive to salt concentration. Divalent metal cations were also capable of stabilizing the protein, in the order Co2+>Ni2+>Mn2+>Mg2+>Ca2+. Finally, removal of the oligohistidine tag was also shown to improve the thermal stability of CorA. Conclusions are drawn from this detailed study about the general applicability of this technique to other membrane proteins.
机译：Thermofluor分析是结构基因组学中的宝贵资产，它提供了一种高通量方法来评估蛋白质的结晶性。该技术已针对可溶性蛋白进行了很好的表征，但对膜蛋白的描述却较少。在这里，我们显示了基于Thermofluor的稳定性测定法成功应用到离子通道，即来自詹氏甲烷球菌的CorA。测定中游离洗涤剂浓度的优化很重要，因为过量浓度会掩盖与蛋白质热解折叠有关的荧光变化。低pH值可稳定CorA，但对盐浓度相对不敏感。二价金属阳离子也能够稳定蛋白质，顺序为Co2 +> Ni2 +> Mn2 +> Mg2 +> Ca2 +。最后，还显示除去寡组氨酸标签可改善CorA的热稳定性。从这项详细的研究得出有关该技术对其他膜蛋白的一般适用性的结论。

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