Quantitation of Affinity, Avidity, and Binding Kinetics of Protein Analytes with a Dynamically Switchable Biosurface
Quantitation of Affinity, Avidity, and Binding Kinetics of Protein Analytes with a Dynamically Switchable Biosurface
复制标题
DOI：
10.1021/ja3061276
复制
发表时间：
2012-09-19
影响因子：
15
通讯作者：
Rant, Ulrich
中科院分区：
文献类型：
--
作者：
Knezevic, Jelena;Langer, Andreas;Rant, Ulrich
介绍了一种用于分析蛋白质与表面束缚配体相互作用的无标记方法。短 DNA 杠杆通过交流电势在微电极上进行电驱动，并且通过荧光能量转移实时测量它们的开关动态。通过对 DNA 杠杆动态运动的时间分辨测量，可以检测到蛋白质与 DNA 杠杆顶部配体的结合。我们演示了结合动力学（k(on)、k(off) 速率常数）、解离常数（pM 体系中的 K-D）以及竞争性结合剂的影响（EC50 值）的定量。此外，“switchSENSE”方法揭示了亲合力效应，并允许区分具有一个或多个结合位点的分析物。在一项比较研究中，对六种六组氨酸标签蛋白与三次氮基三乙酸 (NTA(3)) 配体的相互作用进行了定量。发现它们的结合动力学和亲和力变化高达 2 个数量级，这证明蛋白质各自的化学环境显着影响 His(6)-NTA(3) 相互作用。
A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K-D in the pM regime), and the influence of competitive binders (EC50 values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.