Sket Primoz, Crnugelj Martin, Plavec Janez
Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, PO Box 660, SI-1001 Ljubljana, Slovenia.
Nucleic Acids Res. 2005 Jun 28;33(11):3691-7. doi: 10.1093/nar/gki690. Print 2005.
Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.
多核核磁共振研究表明，d(G3T4G4)形成的G-四链体在其三对G-四重体之间有两个阳离子结合位点。在15NH4+离子存在下折叠的d(G3T4G4)2溶液中滴定结合更紧密的K+离子，发现了一种混合的单K+-单15NH4+形式，它代表了二15NH4+向二K+形式转化的中间体。类似地，发现15NH4+离子取代了d(G3T4G4)2四链体内的Na+离子。15NH4+对两个结合位点的偏好性比Na+离子小得多，比K+对15NH4+离子的偏好性小得多。G-四链体核心内的两个阳离子结合位点差异很大，以至于在滴定过程中，与靠近边缘型环的位点结合的15NH4+离子总是首先被K+离子取代。直到K+离子已经占据第一个结合位点，第二个结合位点才会被K+离子占据。在12个K+离子浓度下，对在核磁共振时间尺度上缓慢交换的三种二阳离子形式的浓度进行定量分析，得到了平衡结合常数。K+离子与d(G3T4G4)2内的位点U和L结合相对于15NH4+离子更有利，吉布斯自由能分别约为-24和-18 kJ mol(-1)，这分别包括阳离子脱水能的差异。
