Kaul Malvika, Pilch Daniel S
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA.
Biochemistry. 2002 Jun 18;41(24):7695-706. doi: 10.1021/bi020130f.
We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.
我们使用光谱学和量热技术来表征氨基糖苷类抗生素新霉素、巴龙霉素和核糖霉素与一种模拟大肠杆菌16S rRNA A位点的RNA寡核苷酸的结合。我们的结果揭示了以下显著特征：（i）氨基糖苷类结合增强了A位点RNA双链体的热稳定性，这种热增强程度随着pH值和/或Na(+)浓度的增加而降低。（ii）随着pH值升高，氨基糖苷类的RNA结合焓变得更放热（有利），这一观察结果与一个或多个药物氨基的结合相关质子化一致。（iii）作为缓冲液函数进行的等温滴定量热法（ITC）研究表明，氨基糖苷类与宿主RNA的结合与质子摄取相关，结合的质子数取决于pH值。具体而言，pH值升高导致结合的质子数相应增加。（iv）在25和37摄氏度下进行的ITC研究表明，氨基糖苷类-RNA络合与负的热容变化（ΔC(p)）相关，其大小随着pH值升高而变大。（v）观察到的氨基糖苷类对RNA的结合亲和力随着pH值和/或Na(+)浓度的增加而降低。此外，这些RNA结合亲和力背后的热力学力也随pH值变化。具体而言，随着pH值升高，对观察到的RNA结合亲和力的焓贡献增加，而对结合的相应熵贡献降低。（vi）氨基糖苷类对宿主RNA的亲和力遵循新霉素>巴龙霉素>核糖霉素的顺序。新霉素相对于巴龙霉素或核糖霉素的增强亲和力主要（如果不是完全）源于焓。（vii）新霉素和巴龙霉素的RNA结合亲和力的盐依赖性与至少三个药物NH(3)(+)基团参与与宿主RNA的静电相互作用一致。总体而言，我们的结果揭示了氨基糖苷类结构的特定改变对与A位点模型RNA寡核苷酸结合热力学的影响。这种系统的比较研究是建立增强我们对决定和控制氨基糖苷类对RNA识别的分子力理解所需的热力学数据库的关键第一步。
