Chou S H, Cheng J W, Reid B R
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
J Mol Biol. 1992 Nov 5;228(1):138-55. doi: 10.1016/0022-2836(92)90497-8.
The solution structure of a rather unusual B-form duplex [d(ATGAGCGAATA)]2 has been determined using two-dimensional nuclear magnetic resonance (2D-NMR) and distance geometry methods. This sequence forms a stable ten base-pair B-form duplex with 3' overhangs and two pairs of adjacent G:A mismatches paired via a sheared hydrogen-bonding scheme. All non-exchangeable protons, including the stereo-specific H-5'S/H-5'R of the 3G and 7G residues, were assigned by 2D-NMR. The phosphorus spectrum was assigned using heteronuclear correlation with H-3' and H-4' reasonances. The complete assignments reveal several unusual nuclear Overhauser enhancements (NOEs) and unusual chemical shifts for the neighboring G:A mismatch pairs and their adjacent nucleotides. Inter-proton distances were derived from time-dependent NOEs and used to generate initial structures, which were further refined by iterative back-calculation of the two-dimensional nuclear Overhauser enhancement spectra; 22 final structures were calculated from the refined distance bounds. All these final structures exhibit fully wound helical structures with small penalty values against the refined distance bounds and small pair-wise root-mean-square deviation values (typically 0.5 A to 0.9 A). The two helical strands exchange base stacking at both of the two G:A mismatch sites, resulting in base stacking down each side rather than down each strand of the twisted duplex. Very large twist angles (77 degrees) were found at the G:A mismatch steps. All the final structures were found to have BII phosphate conformations at the adjacent G:A mismatch sites, consistent with observed downfield 31P chemical shifts and Monte-Carlo conformational search results. Our results support the hypothesis that 31P chemical shifts are related to backbone torsion angles. These BII phosphate conformations in the adjacent G:A mismatch step suggest that hydrogen bonding of the G:A pair G-NH2 to a nearby phosphate oxygen atom is unlikely. The unusual structure of the duplex may be stabilized by strong interstrand base stacking as well as intrastrand stacking, as indicated by excellent base overlap within the mismatch stacks.
已使用二维核磁共振（2D-NMR）和距离几何方法确定了一种相当不寻常的B型双链体[d(ATGAGCGAATA)]2的溶液结构。该序列形成了一个稳定的十碱基对B型双链体，带有3'突出端以及通过剪切氢键模式配对的两对相邻G:A错配。通过2D-NMR对所有不可交换质子进行了归属，包括3G和7G残基的立体特异性H-5'S/H-5'R。利用与H-3'和H-4'共振的异核相关性对磷谱进行了归属。完整的归属揭示了相邻G:A错配及其相邻核苷酸的几个不寻常的核Overhauser增强（NOE）和不寻常的化学位移。质子间距离从时间依赖的NOE中导出，并用于生成初始结构，通过对二维核Overhauser增强谱的迭代反计算对其进行进一步优化；根据优化后的距离限制计算出了22个最终结构。所有这些最终结构均呈现出完全缠绕的螺旋结构，相对于优化后的距离限制具有较小的惩罚值，且成对的均方根偏差值较小（通常为0.5 Å至0.9 Å）。两条螺旋链在两个G:A错配位点处都进行了碱基堆积的交换，导致碱基堆积沿双链体的每一侧而非每一条链向下。在G:A错配步骤处发现了非常大的扭转角（77度）。发现所有最终结构在相邻的G:A错配位点处具有BII磷酸构象，这与观察到的31P化学位移向低场移动以及蒙特卡罗构象搜索结果一致。我们的结果支持31P化学位移与主链扭转角相关的假设。相邻G:A错配步骤中的这些BII磷酸构象表明，G:A对中G-NH2与附近磷酸氧原子形成氢键的可能性不大。双链体的不寻常结构可能通过强链间碱基堆积以及链内堆积得以稳定，这由错配堆积内出色的碱基重叠所表明。
