Spielmann H P, Dwyer T J, Hearst J E, Wemmer D E
Structural Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
Biochemistry. 1995 Oct 10;34(40):12937-53.
We have used two-dimensional 1H NMR spectroscopy to determine the solution structures of the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furanside monoadducted (MAf) and the photoisomeric HMT interstrand cross-linked (XL) DNA oligonucleotide d(5'-GCGTACGC-3')2. The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for both the MAf and XL from both A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviations of the coordinates for the 40 structures for the MAf and XL were 1.12 and 1.10 A, respectively. The rmsd of the MAf with respect to the XL is 2.20 A. The local DNA structure is distorted in both adducts, with the helix unwound by 34 degrees and 25 degrees for the MAf and XL, respectively, and an overall helical repeat of 11 base pairs, caused by intercalation of the HMT. The MAf is a photochemical intermediate on the path to interstrand XL. Considerable local structural distortion is induced by both adducts, but the DNA returns to B-form structure within three base pairs of the damage site. There is no significant bend in the helix axis of either the MAf or the XL. We have evaluated the accuracy of the two major methods of converting NOESY data into interproton distances, the isolated spin-pair approximation (ISPA) and the complete relaxation rate matrix analysis (RMA). Both methods were evaluated by comparing the resulting calculated interproton distances generated to known covalently fixed distances in the HMT. The overall structures were evaluated by checking their agreement with biophysical evidence from non-NMR techniques. Only the modified RMA method gave correct interproton distances.
我们使用二维¹H NMR光谱法来确定4'-(羟甲基)-4,5',8-三甲基补骨脂素(HMT)呋喃糖苷单加合物(MAf)以及光异构化的HMT链间交联(XL)DNA寡核苷酸d(5'-GCGTACGC-3')₂的溶液结构。结构的确定基于使用MARDIGRAS程序对NOESY交叉峰强度进行的全弛豫矩阵分析。在这些计算过程中，采用了改进的程序来考虑NOESY光谱中的实验“噪声”。由NOE得出的距离约束被应用于受限分子动力学计算。从A-型和B-型双链DNA起始结构出发，分别为MAf和XL生成了20个最终结构。MAf和XL的40个结构的坐标的均方根(rms)偏差分别为1.12 Å和1.10 Å。MAf相对于XL的rmsd为2.20 Å。两种加合物中的局部DNA结构均发生扭曲，MAf和XL的螺旋分别解开34度和25度，并且由于HMT的嵌入导致整体螺旋重复为11个碱基对。MAf是链间交联途径上的光化学中间体。两种加合物均引起相当大的局部结构扭曲，但DNA在损伤位点的三个碱基对内恢复为B-型结构。MAf或XL的螺旋轴均无明显弯曲。我们评估了将NOESY数据转换为质子间距离的两种主要方法的准确性，即孤立自旋对近似法(ISPA)和完整弛豫速率矩阵分析法(RMA)。通过将所得计算出的质子间距离与HMT中已知的共价固定距离进行比较，对这两种方法进行了评估。通过检查它们与来自非NMR技术的生物物理证据的一致性来评估整体结构。只有改进的RMA方法给出了正确的质子间距离。
