Scott E V, Zon G, Marzilli L G, Wilson W D
Department of Chemistry, Emory University, Atlanta, Georgia 30322.
Biochemistry. 1988 Oct 4;27(20):7940-51. doi: 10.1021/bi00420a053.
One- and two-dimensional NMR studies on the oligomer dA1T2G3C4G5C6A7T8, with and without actinomycin D (ActD), were conducted. Analysis of the NMR data, particularly 2D NOE intensities, revealed that the free oligonucleotide is a duplex in a standard right-handed B form. At the ratio of 1 ActD/duplex (R = 1), 1D NMR studies indicate that two 1:1 unsymmetric complexes form in unequal proportions with the phenoxazone ring intercalated at a GpC site, in agreement with previous studies [Scott, E.V., Jones, R.L., Banville, D.L., Zon, G., Marzilli, L.G., & Wilson, W.D. (1988) Biochemistry 27, 915-923]. The 2D COSY data also confirm this interpretation since eight cytosine H6 to H5 and two ActD H8 to H7 cross-peaks are observed. At R = 2, both COSY and NOESY spectra confirm the formation of a unique 2:1 species with C2 symmetry. The oligomer remains in a right-handed duplex but undergoes extreme conformational changes both at and adjacent to the binding site. The deoxyribose conformation of T2, C4, and C6 shifts from primarily C2'-endo in the free duplex to an increased amount of C3'-endo in the 2:1 complex as revealed by the greater intensity of the base H6 to 3' NOE cross-peak relative to the intensity of the H6 to H2' NOE cross-peak. This conformational change widens the minor groove and should help alleviate the steric crowding of the ActD peptides. The orientation of the ActD molecules at R = 2 has the quinoid portion of the phenoxazone ring at the G3pC4 site and the benzenoid portion of the phenoxazone ring at the G5pC6 site on the basis of NOE cross-peaks from ActD H7 and H8 to G5H8 and C6H6. All base pairs retain Watson-Crick type H-bonding, unlike echinomycin complexes [e.g., Gao, X., & Patel, D.J. (1988) Biochemistry 27, 1744-1751] where Hoogsteen base pairs have been observed. In contrast to previous studies on ActD, we were able to distinguish the two peptide chains.(ABSTRACT TRUNCATED AT 400 WORDS)
我们对寡聚物dA1T2G3C4G5C6A7T8在有和没有放线菌素D（ActD）的情况下进行了一维和二维核磁共振研究。对核磁共振数据的分析，特别是二维核Overhauser效应（NOE）强度分析，表明游离的寡核苷酸是标准右手B型双链体。在ActD与双链体的比例为1（R = 1）时，一维核磁共振研究表明形成了两种1:1不对称复合物，其比例不等，吩恶嗪环插入在一个GpC位点，这与之前的研究结果一致[斯科特，E.V.，琼斯，R.L.，班维尔，D.L.，宗，G.，马尔齐利，L.G.，&威尔逊，W.D.（1988年）《生物化学》27卷，915 - 923页]。二维化学位移相关谱（COSY）数据也证实了这一解释，因为观察到了八个胞嘧啶H6到H5以及两个ActD H8到H7的交叉峰。在R = 2时，COSY和核Overhauser效应光谱（NOESY）都证实形成了具有C2对称性的独特2:1物种。该寡聚物仍保持右手双链体结构，但在结合位点及其相邻位置发生了极端的构象变化。T2、C4和C6的脱氧核糖构象从游离双链体中的主要C2'-内型转变为2:1复合物中增加的C3'-内型，这是由碱基H6到3'的NOE交叉峰强度相对于H6到H2'的NOE交叉峰强度更大所揭示的。这种构象变化拓宽了小沟，应该有助于缓解ActD肽的空间拥挤。根据ActD H7和H8到G5H8和C6H6的NOE交叉峰，在R = 2时ActD分子的取向是吩恶嗪环的醌型部分在G3pC4位点，吩恶嗪环的苯型部分在G5pC6位点。所有碱基对都保持沃森 - 克里克型氢键，这与棘霉素复合物不同[例如，高，X.，&帕特尔，D.J.（1988年）《生物化学》27卷，1744 - 1751页]，在棘霉素复合物中观察到了Hoogsteen碱基对。与之前对ActD的研究不同，我们能够区分两条肽链。（摘要截断于400字）
