Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
Biochemistry. 2012 Jan 10;51(1):565-72. doi: 10.1021/bi201642p. Epub 2011 Dec 16.
The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.
孔雀石绿适体与两种密切相关的配体，孔雀石绿（MG）和四甲基罗丹明（TMR），具有几乎相同的亲和力。MG 配体由三个从中心碳原子伸出的苯环组成，而 TMR 则有三个环中的两个通过醚键连接。适体中 MG 和 TMR 的结合口袋，从高分辨率结构可知，仅在几个核苷酸的构象上有所不同。在此，我们应用等温滴定量热法（ITC）来比较 MG 和 TMR 与适体结合的热力学。通过 ITC 滴定在 15-60°C 的温度范围内获得结合热容量。发现 MG 结合有两个温度区：一个从 15 到 45°C，MG 以大的负热容量和表观计量比（n）0.4 结合；另一个从 50 到 60°C，MG 以正热容量和 n1.1 结合。另一方面，TMR 的结合仅揭示了一个结合温度区，具有较小的负热容量和 n~1.2。两种配体之间的热容量差异很大，这表明在 MG 结合时发生了明显更多的构象重排，这与计算得到的可用配体结合结构的溶剂可及表面积差异一致。最后，我们注意到 MG 的结合计量比不仅通过提高温度，而且通过降低 Mg（2+）浓度或增加 ITC 注射之间的时间而得到改善。这些研究表明，动态配体与功能 RNA 的结合需要 RNA 本身具有显著的动力学。
