Quantitative protein stability measurement in vivo
Quantitative protein stability measurement in vivo
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DOI：
10.1038/nsb1001-879
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发表时间：
2001-10-01
期刊：
影响因子：
--
通讯作者：
Oas, TG
中科院分区：
文献类型：
--
作者：
Ghaemmaghami, S;Oas, TG
蛋白质的天然状态和变性状态之间的平衡可能是其功能和调节的关键。传统上，折叠平衡常数已在体外使用纯化的蛋白质和简单的缓冲液测量。然而，蛋白质的生物环境可能与这些体外条件不同，可能会显著干扰稳定性。在这里，我们提出了第一个定量比较蛋白质的稳定性在体外和在细胞质中的土方大肠杆菌使用酰胺氢交换检测MALDI质谱（SUPREX）。结果表明，单体λ阻遏物在细胞内的热力学稳定性与其在体外简单缓冲液中的稳定性相同。然而，当E.将大肠杆菌置于高渗环境中，体内稳定性大大增强。体内SUPREX方法提供了一种通用的定量方法来测量细胞中的蛋白质稳定性，并将用于细胞内稳定性信息提供重要生物学见解的应用。
The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these Invitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Eschericia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.