Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.
J Phys Chem B. 2010 Sep 2;114(34):11155-63. doi: 10.1021/jp1000545.
RNA-peptide interactions have been intensively studied at the structural level; however, in the absence of thermodynamic studies, the molecular forces that dictate the binding specificities and affinities remain elusive. Here we evaluate the thermodynamics (DeltaG, DeltaH, DeltaS) of HIV-1 TAR RNA hairpin and Tat peptide interaction as well as the hydration changes that accompany these interactions, through a series of calorimetric, spectroscopic, and osmotic stress studies. Tat peptide binding enhances the thermal stability of the TAR RNA hairpin; however, the thermal enhancement decreases with increasing Na(+) concentration. The equilibrium association constant (K(a)) is determined by fluorescence titrations and examined as a function of Na(+) concentration and temperature. The binding constant (K(a)) decreases with increasing Na(+) concentration. The binding free energy (DeltaG) is found to have a large nonpolyelectrolyte component with release of a single counterion upon binding. The ITC profiles showed two independent sites binding, indicating specific as well as nonspecific interactions. The enthalpy changes associated with both the binding sites are found to be more negative for the binding process at lower salt concentration of 20 mM Na(+). Our binding studies under osmotic stress conditions show that there is a release of 28 (+/-4) and 21 (+/-3) water molecules upon complex formation at 20 and 80 mM Na(+) concentration supporting the observed positive entropy contributions and accounting for discrepancies between DeltaH(cal) and DeltaH(vH). In aggregate, our results suggest that the hydrogen bonding of arginine to TAR RNA dictates the binding interaction.
RNA-肽相互作用在结构水平上得到了深入研究；然而，在缺乏热力学研究的情况下，决定结合特异性和亲和力的分子力仍然难以捉摸。在这里，我们通过一系列量热、光谱和渗透压应激研究，评估了 HIV-1 TAR RNA 发夹和 Tat 肽相互作用的热力学（ΔG、ΔH、ΔS）以及伴随这些相互作用的水合变化。Tat 肽结合增强了 TAR RNA 发夹的热稳定性；然而，随着 Na(+)浓度的增加，热增强作用减小。平衡结合常数（K(a)）通过荧光滴定确定，并作为 Na(+)浓度和温度的函数进行检查。结合常数（K(a)）随 Na(+)浓度的增加而减小。发现结合自由能（ΔG）具有较大的非聚电解质成分，结合时释放单个抗衡离子。ITC 图谱显示了两个独立的结合位点，表明存在特异性和非特异性相互作用。在较低盐浓度 20 mM Na(+)下，与两个结合位点相关的焓变都发现为负值，这表明结合过程的焓变更负。我们在渗透压应激条件下的结合研究表明，在 20 和 80 mM Na(+)浓度下形成复合物时，分别释放 28（+/-4）和 21（+/-3）个水分子，这支持了观察到的正熵贡献，并解释了 ΔH(cal)和 ΔH(vH)之间的差异。总的来说，我们的结果表明，精氨酸与 TAR RNA 的氢键决定了结合相互作用。
