Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH
Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH
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DOI：
10.1038/nchembio.2007.49
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发表时间：
2007-12-01
影响因子：
14.8
通讯作者：
Schepartz, Alanna
中科院分区：
文献类型：
--
作者：
Luedtke, Nathan W.;Dexter, Rachel J.;Schepartz, Alanna
使用荧光双砷试剂 FlAsH-EDT2 和 ReAsH-EDT2 在体外和哺乳动物细胞中选择性标记包含两个半胱氨酸对的重组多肽和蛋白质结构域，这些半胱氨酸对在一级序列中位于远端，但在天然折叠或组装状态中位于近端。这种策略被称为二分四半胱氨酸展示，能够检测活细胞中蛋白质-蛋白质相互作用和替代蛋白质构象。作为原理证明，我们证明多肽-双砷复合物的平衡稳定性和荧光强度与蛋白质折叠或组装的热力学稳定性相关。不稳定的蛋白质变体形成不太稳定和不太明亮的双砷复合物，这使得能够将表达折叠多肽和蛋白质结构域的活细胞与含有破坏性点突变的活细胞区分开来。二分四半胱氨酸展示可能提供一种检测与阿尔茨海默病、帕金森病和囊性纤维化相关的早期蛋白质错误折叠事件的方法；它还可以实现稳定离散蛋白质折叠的化合物的高通量筛选。
Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide - biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.