Lee M, Chang D K, Hartley J A, Pon R T, Krowicki K, Lown J W
Department of Chemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1988 Jan 12;27(1):445-55. doi: 10.1021/bi00401a066.
Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.
利用高分辨率1H NMR技术推导了自互补十脱氧核糖核苷酸d(CGCAATTGCG)2的结构和动力学性质，以及原型左旋肌动蛋白（即信息读取寡肽）与该十脱氧核糖核苷酸之间的相互作用。通过二维NMR研究已完全归属了d(CGCAATTGCG)2的非交换质子和亚氨基质子共振。该十脱氧核糖核苷酸以右手B-DNA形式存在。在1:1复合物的1H NMR谱中，由于配体杂环部分的各向异性效应，AH2(4)、AH2(5)、T-CH3(6)和T-CH3(7)的选择性化学位移及简并性消除，且在侧翼碱基位点C(3)和G(8)处影响较小，表明药物位于十脱氧核糖核苷酸的中央。十脱氧核糖核苷酸上各个化学位移变化的图谱支持了这一结论。同样，亚氨基质子IV和V经历较大位移，II和III经历较小位移，符合这一结论，而药物络合使得能够检测到亚氨基质子I。吡咯H5与AH2(5)之间有强核Overhauser效应（NOE），与AH1'(5)、TH3'(6)和AH2'(5)之间有较弱NOE，这确定了配体位于小沟中。相邻杂环部分之间的配体内NOE表明，左旋肌动蛋白在结合态和游离态均围绕N6-C9键发生螺旋扭转。核Overhauser效应光谱（NOESY）和相关光谱（COSY）实验还表明，络合后C16亚甲基质子简并性的消除可能源于围绕C15-N9、C15-C16和C16-C17键的旋转受限。配体凹面上的酰胺NH基团（N4H、N6H、N9H）与小沟底部的腺嘌呤N3或胸腺嘧啶O2之间的特定氢键与药物取代水合壳层一致。对1:1复合物中十脱氧核糖核苷酸的NOE测量证实其以右手螺旋形式存在，属于B家族。交换NMR效应使得能够估计在294 K时，左旋肌动蛋白在十聚体上两个等效位点之间的双位点交换速率约为44 s-1，ΔG++约为70±5 kJ mol-1。考虑了该交换过程的其他机制。
