Nonin S, Leroy J L, Guéron M
Groupe de Biophysique de l'Ecole Polytechnique et de l'URA D1254 du CNRS, Palaiseau, France.
Biochemistry. 1995 Aug 22;34(33):10652-9. doi: 10.1021/bi00033a041.
We have estimated the dissociation constant of the terminal base pairs of the B-DNA duplexes formed by 5'-d(CGCGATCGCG) and 5'-d(TAGCGCTA) by two methods, one based on the change in imino proton chemical shift with temperature and the other on the apparent pK shift of the imino proton, as monitored by the change in chemical shift of aromatic protons. These methods do not rely on imino proton exchange, whose rate was also measured. (1) The effect of ammonia on the imino proton exchange rate of the terminal pair of the 5'-d(CGCGATCGCG) duplex is 67 times less than on the isolated nucleoside. This provides an upper limit on the exchange rate from the closed pair. In fact, the effect is just as predicted from the dissociation constant, assuming that there is no exchange at all from the closed pair and that, as has been argued previously, external catalysts act on the open state as they do on the isolated nucleoside. The inhibition of catalyzed proton exchange in the closed pair, despite exposure of one face of the pair to solvent, is a new feature of the exchange process. It will allow determination of the dissociation constant of terminal pairs from the exchange rate. (2) Intrinsic catalysis of proton exchange is less efficient for the terminal pair than for an internal one. A possible explanation is that proton transfer across the water bridge responsible for intrinsic catalysis is slower, as expected if the open-state separation of the bases is larger in a terminal pair. This observation may lead to a direct method for the study of fraying. (3) At 0 degrees C, the dissociation constant of the second pair of the 5'-d(CGCGATCGCG) duplex is close to the square of the constant for the terminal pair, as predicted from a simple model of fraying. The enthalpy and entropy of opening of the terminal pairs may be compared with those of nearest neighbor interactions derived from calorimetry [Breslauer, K. J., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750].
我们通过两种方法估算了由5'-d(CGCGATCGCG)和5'-d(TAGCGCTA)形成的B-DNA双链体末端碱基对的解离常数，一种方法基于亚氨基质子化学位移随温度的变化，另一种方法基于亚氨基质子的表观pK位移，通过芳香族质子化学位移的变化进行监测。这些方法不依赖于亚氨基质子交换，我们也测量了其速率。(1)氨对5'-d(CGCGATCGCG)双链体末端碱基对亚氨基质子交换速率的影响比对分离核苷的影响小67倍。这为封闭碱基对的交换速率提供了一个上限。实际上，该影响正如根据解离常数所预测的那样，假设封闭碱基对根本不发生交换，并且如先前所述，外部催化剂对开放状态的作用与对分离核苷的作用相同。尽管碱基对的一个面暴露于溶剂中，但封闭碱基对中催化质子交换受到抑制是交换过程的一个新特征。这将允许从交换速率确定末端碱基对的解离常数。(2)末端碱基对质子交换的内在催化效率低于内部碱基对。一种可能的解释是，负责内在催化的水桥的质子转移较慢，如果末端碱基对中碱基的开放状态间距更大，这是可以预期的。这一观察结果可能会导致一种研究碱基对解链的直接方法。(3)在0摄氏度时，5'-d(CGCGATCGCG)双链体第二对碱基对的解离常数接近末端碱基对常数的平方，这是根据一个简单的碱基对解链模型预测的。末端碱基对打开的焓和熵可以与量热法得出的最近邻相互作用的焓和熵进行比较[布雷斯劳尔，K. J.等人(1986年)《美国国家科学院院刊》83，3746 - 3750]。
