Hartel A J, Lankhorst P P, Altona C
Eur J Biochem. 1982 Dec 15;129(2):343-57. doi: 10.1111/j.1432-1033.1982.tb07057.x.
Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate N6-dimethyladenylyl(3'-5')uridine (m2(6)A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 degrees C to 105 degrees C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of m2(6)A-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions: (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules: (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the least-squares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are: delta H degrees x = -28.8 kJ mol-1, delta S degrees x = -93 J mol-1 K-1. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are: delta H degrees A = -35 kJ mol-1 and delta S degrees A = -95 J mol-1 K-1.
在水溶液中，于100兆赫、360兆赫和400兆赫下测量了修饰的单磷酸核糖二核苷N6 - 二甲基腺苷酰基(3'-5')尿苷（m2(6)A - U）中碱基和糖质子的化学位移。使用了七个不同的样品，浓度范围为0.28毫摩尔/升至32.7毫摩尔/升。温度在-5摄氏度至105摄氏度之间变化，并采用了内部温度校准。通过一种新方法：差分浓度/温度曲线（DCTP），定量分离了分子间自缔合和分子内堆积对化学位移的影响。测试了几种计算模型，该分析使我们能更深入地了解m2(6)A - U在分子水平上的行为。对于自缔合和堆积，简单的双态方法已经比完全忽略缔合的模型有了显著改进。对每个质子的化学位移行为进行计算机最小二乘法分析，得出了自缔合和堆积的热力学参数。然而，双态模型不足以准确再现所有观测结果。要得到令人满意的拟合需要另外两个假设：（a）芳香族质子在堆积和未堆积分子中经历不同的缔合位移；（b）在未堆积微态组之间存在温度依赖性构象平衡。堆积态被视为代表单一构象物种。在最小二乘法优化中实施这个扩展模型，能够在实验误差范围内再现一千多个化学位移观测值。推导出的分子内堆积的热力学平衡参数为：ΔH°x = -28.8千焦/摩尔，ΔS°x = -93焦/摩尔·开尔文。这些数值与我们早期从圆二色光谱获得的结果吻合良好。推导出的缔合过程的平衡焓和熵值为：ΔH°A = -35千焦/摩尔和ΔS°A = -95焦/摩尔·开尔文。
