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通过经历显著的构象变化来发挥功能。为了了解运动如何影响RNA和DNA的功能，通过晶体学和NMR获得的静态结构数据必须通过分析与功能相关的结构变化相关的运动来增强。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，跨越至少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。如何最好地获得这些信息是本提案的重点。 在上一个资助期间，我们开发并应用了新的固态NMR方法，计算建模技术和超快光谱方法。在两项独立的技术突破中，我们已经制备了具有足够分辨率的固态NMR样品，以记录RNA的多维固态13 C光谱，并记录了具有仅几秒分辨率的二维溶液NMR光谱，以真实的时间跟踪核糖开关的构象变化。通过引入多维，魔角旋转（MAS）固态核磁共振技术的核酸;应用实时核磁共振方法的RNA与几秒钟的时间分辨率;合并实验核磁共振结果与长时间尺度的分子模拟技术，我们将提供前所未有的洞察分子运动在三个范式的核酸系统：1。结论性地证明（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中非常常见的蛋白质结合信号，即在K-转角RNA中发现的中断两个螺旋的单链凸起，是否通过高度扭结状态瞬时波动，该高度扭结状态已经预先设置用于结合其同源蛋白质。3.利用真实的时间核磁共振研究配体诱导的腺嘌呤敏感核糖开关的构象变化。以几秒的分辨率记录化学位移、残留偶极耦合和顺磁增强，以建立中间体的结构，沿着连接两种RNA构象的路径和轨迹。通过执行这个技术上雄心勃勃的计划，我们将提供新的见解，以支持RNA和DNA功能的动力学，并进一步开发光谱和计算技术，将广泛适用于其他核酸。

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.