Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells
Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells
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DOI：
10.1002/cbic.200700404
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发表时间：
2007-11-23
期刊：
影响因子：
3.2
通讯作者：
Muira, Tom W.
中科院分区：
文献类型：
--
作者：
Hahn, Michael E.;Pellois, Jean-Philippe;Muira, Tom W.
翻译后修饰蛋白质直接成像的理想技术是将荧光信号的出现与依赖于修饰的蛋白质激活事件联系起来。在这里，我们利用蛋白质半合成技术，表达蛋白连接(EPL)，制备信号蛋白Smad2的笼状类似物；然后以相关的方式光控制类似物的功能和荧光。我们表明，这一策略允许滴定活性磷酸化Smad2的细胞水平，以其生物学相关的全长形式。我们还制备了一个用正交荧光团标记的非磷酸化的笼状全长Smad2类似物，并同时在同一细胞中对该蛋白的磷酸化和非磷酸化形式进行了成像。这一策略应该能够通过直接比较去功能化后修饰和未修饰形式的蛋白质的行为来剖析翻译后修饰(PTM)的细胞后果。
An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.