Nikonowicz E P, Gorenstein D G
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Biochemistry. 1990 Sep 18;29(37):8845-58. doi: 10.1021/bi00489a048.
Assignment of the 1H and 31P NMR spectra of a tandem G.A mismatched base pair decamer oligodeoxyribonucleotide duplex, d(CCAAGATTGG)2, has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. Unusual downfield 31P resonances have been assigned by a pure absorption phase constant-time heteronuclear 31P-1H correlated spectrum to be associated with the phosphates on the 5'- and 3'-sides of the mismatched guanosine residue. JH3'-P coupling constants for each of the phosphates of the decamer were obtained from the 1H-31P J-resolved selective proton-flip 2D spectrum. The two most downfield-shifted 31P resonances each appear to consist of two overlapping signals that can be resolved into two distinct doublets with different coupling constants in the J-resolved spectrum. This as well as the temperature dependence of the 31P spectra demonstrates that two distinct conformations exist at lower temperatures. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. A linear correlation between 31P chemical shifts and the measured coupling constants is quite good (only when the larger set of coupling constants of the two most downfield 31P signals is included). The 31P chemical shifts as well as the measured coupling constants tend to follow the positional variation seen in other duplexes of interior phosphates resonating more upfield than terminal residues and of interior phosphates exhibiting smaller coupling constants; however, this pattern is disrupted at the site of the mismatch. Modeling and initial NOESY distance restrained molecular mechanics energy minimization and restrained molecular dynamics support previous observations that the mismatched guanine and adenine bases are both in anti conformations. Most significantly, the epsilon backbone torsional angle variaions calculated from the NOESY distance restrained structures are in agreement with both the crystal structure values and the measured JH3'-P coupling constants.
通过二维1H-1H和异核31P-1H相关光谱对串联G·A错配碱基对十聚体寡脱氧核糖核苷酸双链体d(CCAAGATTGG)2的1H和31P NMR光谱进行了归属。通过纯吸收相恒时异核31P-1H相关光谱将异常的低场31P共振归属于错配鸟苷残基5'和3'侧的磷酸酯。从1H-31P J分辨选择性质子翻转二维光谱中获得十聚体各磷酸酯的JH3'-P耦合常数。两个最向低场移动的31P共振峰似乎各自由两个重叠信号组成，在J分辨光谱中可解析为具有不同耦合常数的两个不同的双峰。这以及31P光谱的温度依赖性表明在较低温度下存在两种不同的构象。利用修正的Karplus关系获得了C4'-C3'-O3'-P扭转角(ε)。31P化学位移与测量的耦合常数之间的线性相关性相当好(仅当包含两个最向低场的31P信号中较大的一组耦合常数时)。31P化学位移以及测量的耦合常数倾向于遵循在其他双链体中观察到的位置变化，即内部磷酸酯的共振比末端残基更偏向高场，并且内部磷酸酯的耦合常数较小；然而，这种模式在错配位点被破坏。建模以及初始NOESY距离约束分子力学能量最小化和约束分子动力学支持了先前的观察结果，即错配的鸟嘌呤和腺嘌呤碱基均处于反式构象。最显著的是，从NOESY距离约束结构计算出的ε主链扭转角变化与晶体结构值和测量的JH3'-P耦合常数均一致。
