Molecular aptamers for real-time protein-protein interaction study
Molecular aptamers for real-time protein-protein interaction study
复制标题
DOI：
10.1002/chem.200400983
复制
发表时间：
2005-07-18
影响因子：
4.3
通讯作者：
Tan, WH
中科院分区：
文献类型：
--
作者：
Cao, ZH;Tan, WH
蛋白质-蛋白质相互作用在细胞功能中起着关键作用，但目前实时研究这些相互作用的技术有限。我们报道了对蛋白质-蛋白质相互作用的实时监测，而无需标记两个相互作用的蛋白质中的任何一个；该过程对蛋白质的结合性质的影响最小。我们的策略使用蛋白质/适体复合体来探测竞争试验中的相互作用，其中适体与其目标蛋白的结合被与目标蛋白相互作用的第二种蛋白质改变。设计了两种信号转导策略，即荧光共振能量转移(FRET)和荧光各向异性，以研究人cc-凝血酶与不同蛋白质之间的相互作用。我们的方法被证明是简单和有效的，不需要标记蛋白质，利用容易获得的适体，提供详细的蛋白质-蛋白质相互作用信息，并且对于蛋白质检测和蛋白质-蛋白质相互作用研究具有良好的灵敏度。FRET和荧光各向异性方法在提供对相互作用蛋白质的动力学、机制、结合位置和结合动力学的洞察方面相互补充。
Protein-protein interactions play critical roles in cellular functions, but current techniques for real-time study of these interactions are limited. We report the real-time monitoring of protein-protein interactions without labeling either of the two interacting proteins; this procedure poses minimum effects on the binding properties of the proteins. Our strategy uses a protein/aptamer complex to probe the interactions in a competitive assay where the binding of an aptamer to its target protein is altered by a second protein that interacts with the target protein. Two signal transduction strategies, fluorescence resonance energy transfer (FRET) and fluorescence anisotropy, have been designed to study the interactions of human cc-thrombin with different proteins by using two aptamers specific for two binding sites on alpha-thrombin. Our method has been shown to be simple and effective, does not require labeling of proteins, makes use of easily obtainable aptamers, provides detailed protein-protein interaction information and has excellent sensitivity for protein detection and protein-protein interaction studies. The FRET and the fluorescent anisotropy approaches complement each other in providing insight into the kinetics, mechanisms, binding sites and binding dynamics of the interacting proteins.