Simultaneous triggering of protein activity, and fluorescence
Simultaneous triggering of protein activity, and fluorescence
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DOI：
10.1021/ja0499142
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发表时间：
2004-06-16
影响因子：
15
通讯作者：
Muir, TW
中科院分区：
文献类型：
--
作者：
Pellois, JP;Hahn, ME;Muir, TW
许多生物学领域可以从空间和时间上控制蛋白质活性的方法中受益匪浅。在这里，我们描述了一种允许同时光触发蛋白质的活性和荧光的方法。Smad2是转化生长因子-β(转化生长因子-β)信号转导途径的核心蛋白，它被一个荧光团和一个可光裂解的部分修饰，该部分既是一个笼状基团，又是一个荧光猝灭基团。在笼子状态下，该蛋白质与蛋白质SarA形成非荧光杂二聚体。紫外光照射和光切笼组产生荧光同源三聚体。这些体外实验表明，可以获得模拟转化生长因子-β信号通路中丝氨酸磷酸化的关键生化事件的光化学触发，并且荧光可以作为蛋白质活性的读出。事实证明，这种方法对于监测蛋白质在活细胞内的活性和位置特别有用。
Many areas of biology can benefit greatly from methods to spatially and temporally control protein activity. Here, we describe an approach that allows the simultaneous photo-triggering of the activity and the fluorescence of a protein. Smad2, a protein central to the transforming growth factor-β (TGF-β) signal transduction pathway, was modified with a fluorophore and a photocleavable moiety that acted as both a caging and a fluorescence quenching group. In its caged state, the protein formed a non-fluorescent heterodimer with the protein SARA. Irradiation with UV light and photocleavage of the caging group produced a fluorescent homotrimer. These in vitro experiments demonstrated that a photochemical trigger mimicking the critical biochemical event of serine phosphorylation involved in the TGF-β signaling pathway could be obtained and that fluorescence could be used as a read-out of protein activity. This approach should prove particularly useful for the monitoring of a protein's activity and location inside of living cells.