Solution and Solid State NMR studies of Conformational Adaptation and Dynamics in

描述（由申请人提供）：许多rna和dna在与蛋白质或小分子结合时发生显著的构象变化。为了了解运动如何影响RNA和DNA的功能，必须通过分析与功能相关的结构变化相关的运动来增强晶体学和核磁共振获得的静态结构数据。这些动态研究必须确定这种变化发生的速率，以及核酸中存在的任何内在运动的幅度和精确的原子性质。然而，生物动力学是复杂的，并且至少跨越12个数量级的速率，需要多种光谱技术以协调一致的方式应用。如何最好地获取这些信息是本提案的重点。在之前的资助期内，我们已经开发和应用了新的固态核磁共振方法，计算建模技术和超快光谱方法。在两个独立的技术突破中，我们制备了具有足够分辨率的固体核磁共振样品，可以记录RNA的多维固态13C光谱，并记录了分辨率仅为几秒的二维溶液核磁共振光谱，可以实时跟踪核糖开关的构象变化。将多维幻角旋转（MAS）固态核磁共振技术引入核酸；将实时核磁共振方法应用于rna，时间分辨率为几秒；将实验核磁共振结果与长时间尺度分子建模技术相结合，我们将通过以下三种方式提供对三种典型核酸系统分子运动的前所未有的洞察：最终证明（或反驳）从沃森-克里克配对双螺旋中部分预挤出DNA碱基的内在运动是否为HhaI甲基化酶甲基化的胞嘧啶的挤出提供了途径。2. 检查RNA中一个非常常见的蛋白质结合信号，即在K-turn RNA中发现的打断两个螺旋的单链凸起，是否会在高度扭曲的状态下短暂波动，这种状态已经预先为其同源蛋白的结合做了准备。3. 利用实时核磁共振追踪配体诱导的腺嘌呤感应核开关构象变化。在几秒钟的分辨率下，记录化学位移、剩余偶极偶联和顺磁增强，以建立沿着途径和连接两种RNA构象的轨迹的中间产物的结构。通过执行这一技术上雄心勃勃的计划，我们将为RNA和DNA功能的动力学基础提供新的见解，并进一步开发将广泛适用于其他核酸的光谱和计算技术。

DESCRIPTION (provided by applicant): Many RNAs and DNAs function by undergoing significant conformational changes when binding to proteins or small molecules. In order to understand how motions contribute to RNA and DNA function, static structural data obtained by crystallography and NMR must be augmented by analysis of the motions associated with functionally relevant structural changes. These dynamic studies must establish the rates at which such changes occur as well as the amplitudes and precise atomic nature of any intrinsic motion present in the nucleic acids. Yet biological dynamics is complex and spans at least twelve orders of magnitude in rates, requiring multiple spectroscopic techniques to be applied in a concerted fashion. How to best obtain this information is the focus of this proposal. In the previous funding period we have developed and applied new solid state NMR methods, computational modeling techniques and ultrafast spectroscopic methods. In two independent technical breakthroughs, we have prepared solid state NMR samples with sufficient resolution to record multidimensional solid state 13C spectra of RNA and have recorded 2-dimensional solution NMR spectra with resolution of just a few seconds to follow conformational changes in riboswitches in real time. By introducing multidimensional, magic angle spinning (MAS) solid state NMR techniques to nucleic acids; applying real-time NMR methods to RNAs with time resolution of a few seconds; merging experimental NMR results with long time scale molecular modeling techniques, we will provide unprecedented insight into molecular motions in three paradigmatic nucleic acid systems by: 1. Conclusively proving (or disproving) whether intrinsic motions that partially pre-extrude a DNA base from the Watson-Crick paired double helix provide a pathway for the extrusion of the cytosine that is methylated by the HhaI methylase enzyme. 2. Examining whether a very common protein-binding signal in RNA, a single-stranded bulge interrupting two helices as found in K-turn RNAs, fluctuates transiently through a highly kinked state that is already pre-disposed for binding of its cognate protein. 3. Following the ligand-induced conformational change in an adenine-sensing riboswitch by using real time NMR. With resolution of a few seconds, to record chemical shift, residual dipolar couplings and paramagnetic enhancements to establish the structure of intermediates along the pathway and the trajectory linking the two RNA conformations. By executing this technically ambitious program, we will provide new insight into the dynamics underpinning of RNA and DNA function, and further develop spectroscopic and computational techniques that will be widely applicable to other nucleic acids.