Leijon M, Zdunek J, Fritzsche H, Sklenar H, Gräslund A
Department of Biophysics, Stockholm University, Sweden.
Eur J Biochem. 1995 Dec 15;234(3):832-42. doi: 10.1111/j.1432-1033.1995.832_a.x.
The nonamer duplex d(GCAAAAACG).d(CGTTTTTGC) was studied by 1H-NMR at 500 MHz. With the exception of the H5' and H5" sugar protons, all protons were assigned by two-dimensional NMR experiments [NOE spectroscopy (NOESY), double-quantum-filtered J-correlated spectroscopy (DQF-COSY) and total correlation spectroscopy (TOCSY)]. The exchange kinetics of the imino protons of the Watson-Crick base pairing were studied at 15 degrees C by measuring inversion-recovery rates under conditions of extensive ammonia base catalysis. Extrapolation to infinite base concentration gave anomalous long lifetimes for the A-tract in accordance with previous results [Leroy, J.-L., Charettier, E., Kochoyan, M. & Guéron, M. (1988) Biochemistry 27, 8894-8898]. On average, 11 NOESY distance constraints/nucleotide were evaluated using the complete relaxation matrix approach. Deoxyribose coupling constants were obtained from simulations of the DQF-COSY cross-peaks, assuming a rapid two-state equilibrium between a C2'-endo and C3'-endo conformer. The sugars were found to be predominantly in the C2'-endo conformation. The NMR-derived distance and torsion constraints were implemented into three different restrained-molecular-dynamics (rMD) protocols, two in vacuo, with different charges on the phosphate group and the third with the solvent explicitly included. All protocols displayed good convergence from different starting structures. The structures derived from the three protocols satisfied experimental restraints equally well and had similar final energies. Although the overall pattern of sequence dependence of helical parameters shows some resemblance in all structures, we find that the absolute amplitudes of the parameters are largely dependent on the rMD protocols, particularly the twist parameters. The minor groove distance P(n + 2)-P(m + 2) varies from 0.7 nm to 1.2 nm in the three protocols. Still the NOESY-derived anomalously short distances AH2(n)-H1'(m + 1) and AH2(n)-H1'(n + 1), n and m denote complementary residues, which are assumed to be indicative of a compressed minor groove, are kept in all calculated structures.
通过500 MHz的1H-NMR对九聚体双链体d(GCAAAAACG).d(CGTTTTTGC)进行了研究。除了H5'和H5"糖质子外，所有质子均通过二维NMR实验[核Overhauser效应光谱法（NOESY）、双量子滤波J相关光谱法（DQF-COSY）和全相关光谱法（TOCSY）]进行了归属。在15℃下，通过测量在大量氨碱催化条件下的反转恢复速率，研究了沃森-克里克碱基对中亚氨基质子的交换动力学。外推至无限碱基浓度时，A序列显示出异常长的寿命，这与之前的结果一致[勒鲁瓦，J.-L.，沙雷蒂耶，E.，科乔扬，M. & 盖龙，M.（1988年）《生物化学》27，8894 - 8898]。平均而言，使用完整弛豫矩阵方法评估了每个核苷酸11个NOESY距离约束。通过对DQF-COSY交叉峰的模拟获得脱氧核糖耦合常数，假设C2'-内式和C3'-内式构象之间存在快速的双态平衡。发现糖主要处于C2'-内式构象。将NMR得出的距离和扭转约束应用于三种不同的受限分子动力学（rMD）方案，其中两种是在真空中，对磷酸基团采用不同的电荷，第三种明确包含了溶剂。所有方案从不同的起始结构都显示出良好的收敛性。从这三种方案得出的结构同样很好地满足了实验约束，并且具有相似的最终能量。尽管所有结构中螺旋参数的序列依赖性总体模式显示出一些相似性，但我们发现参数的绝对幅度在很大程度上取决于rMD方案，特别是扭转参数。在这三种方案中，小沟距离P(n + 2)-P(m + 2)在0.7纳米至1.2纳米之间变化。不过，在所有计算结构中，由NOESY得出的异常短距离AH2(n)-H1'(m + 1)和AH2(n)-H1'(n + 1)（n和m表示互补残基，假定其指示小沟压缩）仍然存在。
