DEOXYRIBOSE CONFORMATION IN [D(GTATATAC)]2 - EVALUATION OF SUGAR PUCKER BY SIMULATION OF DOUBLE-QUANTUM-FILTERED COSY CROSS-PEAKS
DEOXYRIBOSE CONFORMATION IN [D(GTATATAC)]2 - EVALUATION OF SUGAR PUCKER BY SIMULATION OF DOUBLE-QUANTUM-FILTERED COSY CROSS-PEAKS
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DOI：
10.1021/bi00461a021
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发表时间：
1990-03-06
期刊：
影响因子：
2.9
通讯作者：
JAMES, TL
中科院分区：
文献类型：
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作者：
SCHMITZ, U;ZON, G;JAMES, TL
通过使用质子二维核欧沃豪塞效应 (2D NOE) 光谱、同核质子双量子过滤 COSY (2QF-COSY) 光谱、质子自旋晶格弛豫时间测量和 31P1H 异核位移相关光谱，确定了水溶液中 [d(GTATATAC)]2 的可交换和不可交换质子和磷共振 (11.75 T)。由于线宽较大，无法从包括 ECOSY 或 2QF-COSY 在内的任何光谱中直接提取邻位质子耦合常数值。然而，定量 2QF-COSY 光谱模拟与实验光谱的比较可以阐明耦合常数。通过计算和实验数据探索了这种方法的范围和局限性。研究发现，质子线宽从一个残基到下一个残基以及从一个质子到下一个残基内表现出一定的可变性，并且精确的线宽对于准确评估耦合常数至关重要。实验 2QF-COSY 光谱与任何核苷酸残基的刚性脱氧核糖构象都不一致。经典的双态模型在 C2''-endo（伪旋转角 P = 162°）和 C3''-endo（P = 9°）构象之间快速跳跃，能够解释末端残基糖的光谱特征：60% C2''-endo 和 40% C3''-endo。然而，只有改变经典的二态模型以使主要构象异构体的伪旋转角为 144° 时，才能模拟来自 -TATATA-core 的 2QF-COSY 交叉峰。而不是 162%。在这种情况下，主要构象异构体达到80-85%。或者，光谱数据与三态模型一致，其中C2''-endo和C3''-endo构象分别具有最大和最小的群体，但存在对应于C1''-exo（P = 126°）的第三构象异构体，这与最近的分子动力学计算一致。这种替代方案产生了 50% (P = 162°)、35% (P = 126°) 和 15% (P = 9°) 的 -TATATA-糖群体。光谱结果表明 T 和 A 之间的糖包装变化不大。然而，交叉峰成分强度和特征光谱失真的微小差异确实表明存在一些未量化的变化。 31P1H 异核化学位移相关光谱表现出交替的化学位移和耦合常数，表明 TA 和 AT 连接之间的磷酸二酯主链构象差异。
Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps betwen C2''-endo (pseudorotation angle P = 162.degree.) and C3''-endo (P = 9.degree.) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2''-endo and 40% C3''-endo. However, the 2QF-COSY cross-peaks from the -TATATA-core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144.degree. instead of 162%. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with three-state model in which C2''-endo and C3''-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1''-exo (P = 126.degree.) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162.degree.), 35% (P = 126.degree.), and 15% (P = 9.degree.) for the -TATATA-sugars. The spectral results indicate little variation of sugar packed between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.