1H NMR study of the base-pairing reactions of d(GGAATTCC): salt and polyamine effects on the imino proton exchange.
1H NMR study of the base-pairing reactions of d(GGAATTCC): salt and polyamine effects on the imino proton exchange.
复制标题
d(GGAATTCC) 碱基配对反应的 1H NMR 研究：盐和多胺对亚氨基质子交换的影响。
DOI：
10.1021/bi00404a018
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Bloomfield,VA
中科院分区：
文献类型：
--
作者：
Braunlin,WH;Bloomfield,VA
明尼苏达大学生物化学系，圣保罗，明尼苏达州55108接收1987年7月6日;修订的Mandarin pt接收1987年10月21日摘要：盐和多胺对DNA的物理性质有多种影响，包括对热熔融的稳定性。我们希望通过确定它对碱基打开和关闭反应动力学的影响，如NMR所测量的，来更深入地了解这种稳定化的机制。由于亚精胺（3+）的结合受盐的影响，并且亚精胺在质子交换反应中可能起碱催化剂的作用，我们研究了盐和碱催化剂对模型寡聚DNA亚氨基质子交换动力学的影响。选择性纵向NMR弛豫速率的氢键亚胺质子的自我互补的八脱氧核糖核苷酸d（GGAATTCC）监测therate的碱催化的化学交换这些质子与溶剂水。由此获得的交换速率提供了DNA双链体的碱基对开放反应的灵敏量度。在低pH和不添加碱催化剂的条件下，NMR弛豫速率允许测定kd，八聚体双链体解离成单链的速率常数。滴定与碱催化剂三（羟甲基）氨基甲烷允许确定kop，单个碱基对的局部开放的速率常数，解离之前。一个显着的Na+浓度依赖性被发现kd。通过对这种依赖关系的分析，确定在解离过程中释放出0.6±0.1个钠离子。螺旋解离的活化能（200±5 kJ/mol）不依赖于钠离子浓度，表明解离是由结合钠离子的释放熵驱动的。与以前的结果一致，没有发现KOP的可测量的盐依赖性，其在25 ℃下等于约100 s-1。在低盐条件下，三价阳离子亚精胺降低了螺旋解离速率，同样不影响该过程的活化能。二质子化亚精胺（2+）是一种非常有效的亚氨基质子交换催化剂。^）我们目前对DNA构象、动力学以及与蛋白质和小分子相互作用的细节的理解，随着生产毫克量的高纯度、确定序列的寡脱氧核苷酸的有效技术的发展而发展。因为可以研究单个质子共振，并且因为交叉弛豫效应比聚合物DNA中的显著得多，所以寡核苷酸的NMR研究提供了许多以前无法获得的关于双螺旋DNA的溶液结构和动力学的信息（Patel等人，1982 c; Kearns，1984; Wemmer & Reid，1985）。离子对核酸的行为有许多重要的影响。简单的盐提高双螺旋DNA的热熔融温度并影响配体的结合（Record等人，1981年）。多胺如亚精胺和精胺具有这些一般作用（布卢姆菲尔德和威尔逊，1981），并且还影响核酸的构象，诱导tRNA的双螺旋区域的弯曲（奎格利等人，1978），向DNA的左手Z形式的转变（Rich等人，1984; Behe & Felsenfeld，1981），以及DNA凝聚成紧凑的环形结构（Goelman & Schellman，1976; Wilson &布卢姆菲尔德，1979）。为了从分子水平上了解这些效应是如何产生的，我们用核磁共振研究了盐和亚精胺对碱基配对的影响。
Department of Biochemistry, The University of Minnesota, St. Paul, Minnesota 55108 Received July 6, 1987; Revised Manuscript Received October 21, 1987 abstract: Salts and polyamines have a variety of effects on the physical properties of DNA, including stabilization against thermal melting. Wewished to gain greater insightinto themechanism of this stabilization by ascertaining its effect on the dynamics of base opening and closing reactions, as measured by NMR. Since the binding of spermidine (3+) is influenced by salt, andsince spermidine may act as a base catalyst in proton exchange reactions, we have undertaken a study of salt and base catalyst effects on the imino proton exchange kinetics of a model oligomeric DNA. The selective longitudinal NMR relaxation rates of the hydrogen-bonded imino protons of the self-complementary octadeoxyribonucleotide d (GGAATTCC) monitor therate of the base-catalyzed chemical exchange of these protons with solvent water. The exchange rates thus obtained provide a sensitive measure of the base-pair opening reactions of the DNA duplex. Under conditions of low pH and no added base catalyst, the NMR relaxation rates allow the determination of kd, the rate constant for the dissociation of the octameric duplex into single strands. Titration with the base catalyst tris (hydroxymethyl) aminomethane allows the determination of kop, the rate constant for the localized opening of individual base pairs, prior to dissociation. A significant Na+ con-centration dependence is found for kd. From an analysis of this dependence, it is determined that 0.6±0.1 sodium ion is released during the dissociationevent. The activation energy for helix dissociation (200±5 kJ/mol) is not dependent on the sodium ion concentration, indicating that the dissociation is entropically driven by the release of bound sodium ions. In agreement with previous results, no measurable salt dependence is found for kop, which is equal to about 100 s-1 at 25 C. Under low-salt conditions, the trivalent cation spermidine decreases the rate of helix dissociation, again without affecting the activation energy for this process. Diprotonated spermidine (2+) acts as an extremely effective catalyst of iminoproton exchange.^) ur current understanding of the details of DNA conformation, dynamics, and interactions with proteins and small molecules has grown in concert with the development of an efficient technology for producing milligram quantities of highly pure, defined-sequence oligodeoxynucleotides. Because individual proton resonances may be studied, and since cross-relaxation effects are much less pronounced than in polymeric DNA, NMR studies of oligonucleotides have provided much previously inaccessible information concerningthe solution structure and dynamics of double-helical DNA (Patel et al., 1982c; Kearns, 1984; Wemmer & Reid, 1985). Ions have many important effects on the behavior of nucleic acids. Simple salts raise the thermal melting temperature of double-helical DNA and affect the binding of ligands (Record et al., 1981). Polyamines such as spermidine and spermine have these general effects (Bloomfield & Wilson, 1981) and also influence the conformation of nucleic acids, inducing bending of double-helical regions of tRNA (Quigley et al., 1978), transition to the left-handed Z form of DNA (Rich et al., 1984; Behe & Felsenfeld, 1981), and condensation of DNA into compact toroidal structures (Gosule & Schellman, 1976; Wilson & Bloomfield, 1979). In an attempt to understand how these effects may be produced at the molecular level, we have undertaken an investigation by NMRof salt and sper-midine effects on the base-pairing …