Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures
Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures
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DOI：
10.1021/ac048577c
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发表时间：
2005-03-01
影响因子：
7.4
通讯作者：
Krylov, SN
中科院分区：
文献类型：
--
作者：
Berezovski, M;Krylov, SN
我们介绍了平衡混合物的温控非平衡毛细管电泳（NECEEM），并演示了其用于研究蛋白质- dna相互作用的热化学。作为一种均相动力学方法，温度控制的NECEEM独特地允许发现平衡和复杂形成的动力学参数的温度依赖关系，而无需将相互作用的分子固定在固体基质表面。在这项工作中，我们应用温控NECEEM研究了两种蛋白质-DNA对的热化学性质：(i) Taq DNA聚合酶及其DNA适体和（ii）大肠杆菌单链DNA结合蛋白与20碱基长的单链DNA。我们确定了三个参数的温度依赖性：平衡结合常数（k -b）、络合物解离速率常数（k(off)）和络合物形成速率常数（k(on)）。蛋白质- dna对的Kb(T)函数都有类似相变的点，这表明相互作用的大分子结构的构象变化依赖于温度。k（on）和koff的温度依赖性提供了构象变化如何影响两个相反的过程的见解：结合和解离。最后，求出了不同构象下复杂构象的热力学参数δ tah和δ tas。凭借其独特的功能和对其他大分子相互作用的潜在适用性，温控NECEEM为用于研究动态分子复合物的分析方法库建立了一个有价值的补充。
We introduce temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and demonstrate its use to study thermochemistry of protein-DNA interactions. Being a homogeneous kinetic method, temperature-controlled NECEEM uniquely allows finding temperature dependencies of equilibrium and kinetic parameters of complex formation without the immobilization of the interacting molecules on the surface of a solid substrate. In this work, we applied temperature controlled NECEEM to study the thermochemistry of two protein-DNA pairs: (i) Taq DNA polymerase with its DNA aptamer and (ii) E. coli single-stranded DNA binding protein with a 20-base-long single-stranded DNA. We determined temperature dependencies of three parameters: the equilibrium binding constant (K-b), the rate constant of complex dissociation (k(off)), and the rate constant of complex formation (k(on)). The Kb(T) functions for both protein-DNA pairs bad phase-transition-like points suggesting temperature-dependent conformational changes in structures of the interacting macromolecules. Temperature dependencies of k(on) and koff provided insights into how the conformational changes affected two opposite processes: binding and dissociation. Finally, thermodynamic parameters, DeltaH and DeltaS, for complex formation were found for different conformations. With its unique features and potential applicability to other macromolecular interactions, temperature-controlled NECEEM establishes a valuable addition to the arsenal of analytical methods used to study dynamic molecular complexes.