Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations
Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations
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DOI：
10.1021/ja0351847
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发表时间：
2003-08-27
影响因子：
15
通讯作者：
Imperiali, B
中科院分区：
文献类型：
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作者：
Vázquez, ME;Nitz, M;Imperiali, B
开发化学探针来研究调节生物事件的复杂磷酸化信号级联对理解这些过程至关重要。我们在此描述了一种双功能探针，其能够在空间和时间上释放生物活性配体，同时允许同时监测其与感兴趣的蛋白质的结合。用Tyr（-2）取代已知与14-3-3蛋白结合的序列RLYRpSLPA中的环境敏感性荧光氨基酸DANA不会对结合亲和力产生不利影响，并允许监测结合过程。肽与14-3-3的结合将荧光报告单元置于疏水口袋中，这改变了荧光最大发射强度和波长。与此同时，新开发的光不稳定的1-（2-硝基苯基）乙基笼状磷酸丝氨酸允许控制释放的生物活性配体通过暴露的关键磷酸丝氨酸功能后，紫外线照射。
The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.