Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations.
Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations.
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DOI：
10.1021/bi00452a020
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发表时间：
1989-12
期刊：
影响因子：
2.9
通讯作者：
W. Nerdal;D. Hare;B. Reid
中科院分区：
文献类型：
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作者：
W. Nerdal;D. Hare;B. Reid
利用二维核磁共振波谱技术研究了中心含有EcoRI限制性内切位点序列GAATTC的自互补DNA双链[d(CGCGAATTCGCG)]2的溶液结构。利用随时间变化的核Overhauser效应谱计算了155对质子间的初始交叉弛豫速率。这些初始交叉弛豫速率被转换成质子间距离，并进入距离（界）矩阵。使用距离几何算法（DSPACE）创建嵌入式起始结构，并对这些结构进行细化，直到它们与距离矩阵表现出良好的一致性；在改进过程中加入了对称约束，使得改进后的距离几何结构中的两条链几乎完全相同，并显著提高了与距离矩阵的一致性。然后通过数值积分在指定半径内相邻质子之间的所有z-磁化转移路径，从显式坐标计算出其中一个距离几何结构的NOESY谱。对该距离几何结构中与实验NOESY时间轨迹不一致的距离进行相应调整。这一过程反复进行，直到计算谱与实验谱达到很好的一致性。最终的结构与实验noesi光谱产生良好的一致性，在C3-G4碱基步骤和A6-T7碱基步骤显示扭结，似乎与报道的EcoRI限制性内切酶DNA结合的扭结相似。溶液结构与DNA双链的晶体结构不同。
The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.