Deep quench: An expanded dynamic range for protein kinase sensors
Deep quench: An expanded dynamic range for protein kinase sensors
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DOI：
10.1021/ja068280r
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发表时间：
2007-03-14
影响因子：
15
通讯作者：
Lawrence, David S.
中科院分区：
文献类型：
--
作者：
Sharma, Vyas;Agnes, Richard S.;Lawrence, David S.
提供蛋白激酶活性的荧光读数的测定提供了鉴定和表征抑制剂、评估结构-功能关系以及将酶活性与细胞行为相关联的手段。虽然在文献中已经描述了几种蛋白激酶传感器，但是它们对磷酸化的荧光响应通常是适度的到中等的（1.1-8倍）。我们开发了一种“深度猝灭”策略，可以引起cAMP依赖性蛋白激酶底物的荧光显着放大（> 60倍）。我们描述了传感器的设计，通过库的合成和筛选相结合的检测开发，表征的检测组件，和两个抑制物种的评估。
Assays that furnish a fluorescent readout of protein kinase activity provide a means to identify and characterize inhibitory agents, assess structure-function relationships, and correlate enzyme activity with cellular behavior. Although several protein kinase sensors have been described in the literature, their fluorescent response to phosphorylation is generally modest to moderate (1.1-8-fold). We have developed a "deep quench" strategy that elicits a dramatic amplification of fluorescence (> 60-fold) in cAMP-dependent protein kinase substrates. We describe sensor design, assay development via a combination of library synthesis and screening, characterization of the assay components, and an assessment of two inhibitory species.