Asensio J L, Lane A N, Dhesi J, Bergqvist S, Brown T
Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, London, NW7 1AA, UK.
J Mol Biol. 1998 Feb 6;275(5):811-22. doi: 10.1006/jmbi.1997.1520.
The influence of the position of the CG.C+ triplet and the contribution of protonation at the N3 of the Hoogsteen cytosine residue on the stability of various sequences of parallel triple helices having the general composition d[(A5G)-x-(T5C)-x-(T5C)] and d[(A4G2)-x-(T4C2)-x-(T4C2)], where x is the hexaethylene glycol linker, has been determined by NMR, ultraviolet melting and absorbance spectrophotometry. The apparent pK value, i.e. the pH at which the observable has changed by 50% of its range, was typically in the range 6 to 7. However, the NMR spectra unequivocally showed that the pK of the protonated cytosine residue must be at least 9.5 for internal positions. This is five units above the pK of the free nucleotide, and represents a free energy of stabilisation from protonation of >11.5 RT. The pK of terminal cytosine residues is much lower, in the range 6.2 to 7.2, accounting for a free energy of stabilisation from protonation of 3.6 to 6 RT. The van't Hoff enthalpies were determined for the dissociation of the protonated triplex into the duplex+strand, and for the duplex to strand transition. The mean value for the duplexes were 23 to 27 kJ mol-1 base-pair, and 25 to 30 kJ mol-1 for the triplexes containing internal CG.C+ triplets. Good agreement was obtained for the thermodynamic parameters by the different methods. Free energy differences for the transition between the protonated triplex and the duplex+protonated strand were calculated at 298 K. The DeltaG of stabilisation of an internal CG.C triplet compared with a terminal CG.C triplet was about 6 kJ mol-1 ; a similar stabilisation was observed for the triplexes containing two CG.C triplets compared with those containing a single CG.C triplet. The very large stabilisation from protonation is too large to be accounted for by a single hydrogen bond, and is likely to include contributions from electrostatic interactions of the positive charge with the phosphate backbone, and more favourable interactions between neighbouring bases owing to the very different electronic properties of the protonated C.
通过核磁共振（NMR）、紫外熔解和吸光分光光度法，已确定了CG.C+三联体位置的影响以及Hoogsteen胞嘧啶残基N3位质子化对具有一般组成d[(A5G)-x-(T5C)-x-(T5C)]和d[(A4G2)-x-(T4C2)-x-(T4C2)]的各种平行三链螺旋序列稳定性的贡献，其中x为六甘醇连接基。表观pK值，即观测值在其变化范围内改变50%时的pH值，通常在6至7范围内。然而，NMR光谱明确显示，对于内部位置，质子化胞嘧啶残基的pK值必须至少为9.5。这比游离核苷酸的pK值高五个单位，代表质子化产生的稳定自由能>11.5 RT。末端胞嘧啶残基的pK值低得多，在6.2至7.2范围内，质子化产生的稳定自由能为3.6至6 RT。测定了质子化三链体解离成双链+单链以及双链到单链转变的范特霍夫焓。双链体的平均值为23至27 kJ mol-1碱基对，含内部CG.C+三联体的三链体为25至30 kJ mol-1。通过不同方法获得的热力学参数吻合良好。计算了298 K时质子化三链体与双链+质子化单链之间转变的自由能差。与末端CG.C三联体相比，内部CG.C三联体的稳定化ΔG约为6 kJ mol-1；与含单个CG.C三联体的三链体相比，含两个CG.C三联体的三链体也观察到类似的稳定化。质子化产生的非常大的稳定化程度太大，无法用单个氢键来解释，可能包括正电荷与磷酸主链的静电相互作用以及由于质子化C的电子性质差异很大而导致的相邻碱基之间更有利的相互作用。
