Microscale fluorescent thermal stability assay for membrane proteins
Microscale fluorescent thermal stability assay for membrane proteins
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DOI：
10.1016/j.str.2008.02.004
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发表时间：
2008-03-01
期刊：
影响因子：
5.7
通讯作者：
Stevens, Raymond C.
中科院分区：
文献类型：
--
作者：
Alexandrov, Alexander I.;Mileni, Mauro;Stevens, Raymond C.
系统地了解膜蛋白在各种不同溶液条件下的稳定性并没有广泛应用于膜蛋白，主要是由于保持蛋白质处于溶解状态所需的洗涤剂的存在以及这些洗涤剂在生物物理表征过程中通常产生的背景所引起的技术问题。在本报告中，我们介绍了一种使用巯基特异性荧光染料N-[4-（7-二乙基氨基-4-甲基-3-香豆素基）苯基]马来酰亚胺（CPM）的高效微尺度荧光稳定性筛选，用于在不同溶液和配体条件下对膜蛋白进行稳定性分析。该屏幕利用嵌入蛋白质内部的天然半胱氨酸的化学反应性作为折叠状态整体完整性的传感器。通过深入研究蛋白质稳定性景观获得的热信息可以有效地用于指导，纯化和生物物理表征工作，包括结晶。为了评估该方法，我们分析了包括Apelin G蛋白偶联受体（APJ）在内的三个不同的蛋白家族。
Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide, purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).