Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via G. Moruzzi, 1, 56124 Pisa, Italy.
Biophys Chem. 2011 Dec;159(2-3):287-93. doi: 10.1016/j.bpc.2011.07.008. Epub 2011 Aug 10.
The bacterial redox protein azurin has been shown to be able to enter into cancer cells and induce apoptosis by stabilizing p53. Although the formation of a complex between the two proteins has been demonstrated, little is known about their binding features. We investigated the interaction between the transcription activation domain of p53 (p53(1-63)) and Pseudomonas aeruginosa azurin using fluorescence and phosphorescence spectroscopic techniques. Trp phosphorescence lifetime measurements revealed conformational changes in azurin induced by the interaction with p53(1-63). Acrylamide quenching of Trp phosphorescence also indicated a significant increase in the overall flexibility of azurin upon binding to p53(1-63). We show that azurin binds to the N-terminal region of p53 with a dissociation constant in the 5-10 μM range. No change in the fluorescence and phosphorescence emission of p53(1-63) was detected in the presence of azurin. This result indicated that no Trp residue of p53(1-63) is located in the interaction site with azurin and therefore suggested that the azurin binding site does not overlap that of MDM2, the protein that plays a crucial role in the p53 regulation. The present results may assist in the design of novel cancer treatments based on p53 stabilization by azurin.
细菌氧化还原蛋白蓝铜蛋白已被证明能够进入癌细胞，并通过稳定 p53 诱导细胞凋亡。尽管已经证明两种蛋白质形成复合物，但它们的结合特征知之甚少。我们使用荧光和磷光光谱技术研究了铜绿假单胞菌蓝铜蛋白与 p53 转录激活结构域（p53(1-63)）之间的相互作用。色氨酸磷光寿命测量结果表明，p53(1-63)与 azurin 相互作用会引起 azurin 的构象变化。丙烯酰胺猝灭色氨酸磷光也表明，azurin 与 p53(1-63)结合后整体灵活性显著增加。我们表明，azurin 与 p53 的 N 端区域结合，解离常数在 5-10 μM 范围内。在 azurin 存在下，未检测到 p53(1-63)的荧光和磷光发射变化。这一结果表明，p53(1-63)的色氨酸残基没有位于与 azurin 相互作用的位点，因此表明 azurin 的结合位点与在 p53 调节中起关键作用的蛋白质 MDM2 不重叠。目前的结果可能有助于设计基于 azurin 稳定 p53 的新型癌症治疗方法。
