Kean James, Cleverley Robert M, O'Ryan Liam, Ford Robert C, Prince Stephen M, Derrick Jeremy P
University of Manchester, Manchester, UK.
Mol Membr Biol. 2008 Dec;25(8):653-63. doi: 10.1080/09687680802541169.
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.
热荧光分析在结构基因组学中是一项宝贵的技术，为评估蛋白质的结晶性提供了一种高通量方法。该技术对于可溶性蛋白质已经有了很好的表征，但对于膜蛋白的描述较少。在这里，我们展示了基于热荧光的稳定性分析在詹氏甲烷球菌离子通道CorA上的成功应用。分析中游离去污剂浓度的优化很重要，因为过高的浓度会掩盖与蛋白质热解折叠相关的荧光变化。结果表明，CorA在低pH条件下稳定，但对盐浓度相对不敏感。二价金属阳离子也能够稳定该蛋白质，其稳定能力顺序为Co2+>Ni2+>Mn2+>Mg2+>Ca2+。最后，去除寡聚组氨酸标签也被证明可以提高CorA的热稳定性。从这项详细研究中得出了关于该技术对其他膜蛋白的普遍适用性的结论。
