Meyer-Almes F J, Porschke D
Max Planck Institut für biophysikalische Chemie, Göttingen, Germany.
J Mol Biol. 1997 Jun 27;269(5):842-50. doi: 10.1006/jmbi.1997.1086.
The complexes formed between the cyclic AMP receptor and three different promoter DNA fragments, including a synthetic 30 bp fragment with the sequence used for determination of the crystal structure, have been analysed in solution by measurements of the electric dichroism (ED) at an ionic strength of 105 mM, using a special instrument based on cable discharge. The ED of the protein is negligible and, thus, the ED of the complexes is determined by the DNA and its orientation relative to the protein. The complex formed between the cyclic AMP receptor with the 30 bp fragment is characterized by a positive ED, indicating that the electric dipole is perpendicular relative to the direction of the helix; moreover, the dipole changes its nature from an induced one for the free DNA to a permanent one of 3.0 x 10(-27) Cm for the complex; both the limiting value of the ED +0.3 and the dichroism decay time constant of 62 ns found for the complex (free DNA: 52 ns; 20 degrees C) demonstrate bending of the DNA double helix. All these parameters are calculated quantitatively from the crystal structure: bead model simulations are used to derive the coefficients of rotational diffusion and to define the center of diffusion, which is the reference for calculation of the dipole vector; the dipole vector is then the basis for calculation of the limit value of the dichroism; the time constants are derived from the diffusion coefficients of the bead model The calculated parameters are in very satisfactory agreement with the experimental ones, demonstrating agreement of the structures in the crystal and in solution with respect to their essential features. These results also demonstrate the utility of electrooptical procedures for a quantitative comparison of crystal or model structures with structures in solution. While the crystal structure has been determined only for the complex with the 30 bp promoter fragment, it has been relatively simple to extend measurements of the ED to complexes formed with a 40 bp and a 203 bp promoter fragment: the data obtained for a 40 bp fragment with the consensus binding sequence are quite similar to those obtained for the 30 bp promoter, whereas the data obtained for the 203 bp promoter clearly show a much higher degree of protein induced bending with a bending angle of approximately 180 degrees.
环磷酸腺苷（cAMP）受体与三个不同的启动子DNA片段形成的复合物，包括一个用于确定晶体结构的含30个碱基对（bp）的合成片段，已在溶液中进行分析。分析方法是在105 mM的离子强度下，使用基于电缆放电的特殊仪器测量电二色性（ED）。蛋白质的ED可忽略不计，因此，复合物的ED由DNA及其相对于蛋白质的方向决定。环磷酸腺苷受体与30 bp片段形成的复合物的特征是具有正的ED，这表明电偶极垂直于螺旋方向；此外，偶极的性质从游离DNA的诱导偶极变为复合物的3.0×10⁻²⁷ Cm的永久偶极；复合物的ED极限值为 +0.3以及二色性衰减时间常数为62 ns（游离DNA：52 ns；20℃），这些都表明DNA双螺旋发生了弯曲。所有这些参数都是根据晶体结构定量计算得出的：珠子模型模拟用于推导旋转扩散系数并确定扩散中心，扩散中心是计算偶极向量的参考；偶极向量进而成为计算二色性极限值的基础；时间常数由珠子模型的扩散系数得出。计算得到的参数与实验参数非常吻合，表明晶体和溶液中的结构在基本特征方面是一致的。这些结果还证明了电光程序在晶体或模型结构与溶液中结构的定量比较中的实用性。虽然仅确定了与30 bp启动子片段形成的复合物的晶体结构，但将ED测量扩展到与40 bp和203 bp启动子片段形成的复合物相对简单：具有共有结合序列的40 bp片段获得的数据与30 bp启动子获得的数据非常相似，而203 bp启动子获得的数据清楚地表明蛋白质诱导的弯曲程度要高得多，弯曲角度约为180度。
