Conformational detection of p53's oligomeric state by FlAsH Fluorescence.
Conformational detection of p53's oligomeric state by FlAsH Fluorescence.
复制标题
通过 FlaAsH 荧光对 p53 寡聚状态进行构象检测。
DOI：
10.1016/j.bbrc.2009.04.073
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发表时间：
2009
影响因子：
3.1
通讯作者：
Gage,MatthewJ
中科院分区：
文献类型：
--
作者：
Webber,TawnyaM;Allen,AndrewC;Ma,WaiKit;Molloy,RhettG;Kettelkamp,CharisseN;Dow,CaitlinA;Gage,MatthewJ
p53肿瘤抑制蛋白是预防肿瘤形成的关键检查点，并且p53的功能依赖于活性四聚体的适当形成。体外研究表明，p53以四聚体的形式最有效地结合DNA，尽管预测失活的p53在体内是单体的。我们证明FlAsH结合可用于区分p53的寡聚状态，为探索体内p53寡聚化提供了潜在的工具。FlAsH四-半胱氨酸结合基序已被沿着p53四聚化结构域中的二聚体和四聚体界面掺入，以产生p53的二聚体和四聚体状态的报告分子，尽管四个半胱氨酸的几何形状对于有效的FlAsH结合是关键的。此外，我们证明FlAsH结合可用于实时监测四聚体的形成。这些结果证明了使用FlAsH荧光监测体内蛋白质-蛋白质相互作用的潜力。
The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein–protein interactions in vivo.