Time-resolved small angle X-ray scattering

RNA在许多生物过程中扮演着重要的角色，但它的构象动力学在最基本的水平上还没有被了解：自组装或折叠。该计划项目的长期目标是完整地表征一个大的RNA的折叠：四膜虫核酶。这项提议提出了一系列研究，旨在探索这种核酶在经过最初的快速崩溃和随后的重要压实步骤进行折叠时的全球构象。使用专为同步加速器小角X射线散射设计的流动池组合，可以在折叠开始后获取从亚毫秒到几分钟的时间尺度上的结构信息。 以前的工作表明，在整个过程中，散射信号发生了显著的变化，对应于与压实和褶皱相关的大规模结构折返。 为了实现我们的长期目标，我们提出了三个具体目标。为了实现第一个，时间分辨小角x射线散射将被用来表征在折叠开始时发生的快速崩塌。在不同的溶液和序列条件下，将获得速率常数和瞬时结构信息，以研究反离子在坍塌中的作用，并检验致密状态的结构模型。第二个具体目标是利用小角x射线散射对包括部分折叠状态在内的后续中间体的结构进行表征。还将研究展开的反向过程。最终的特定目标是探索沿着首选折叠路径的全局构象，这些折叠路径在单价和二价阳离子存在的情况下被填充。在这一目标下，将探索更接近生理的溶液条件。折叠将从离散的展开状态集合开始，以确定沿不同路径的瞬变的特征。来自这些小角度散射实验的全局结构信息将与从其他项目成员获得的详细的局部信息相结合，以实现对该分子折叠的最深入的了解。

RNA plays an important role in many biological processes, but its conformational dynamics are not yet understood on the most fundamental level: self-assembly or folding. The long term objective of this Program Project is the complete characterization of folding of a large RNA: the Tetrahymena ribozyme. This proposal presents aseries of studies designed to probe the global conformation of this ribozyme as it proceeds towards folding through an initial rapid collapse, and subsequent significant compaction steps. The use of a demonstrated combination of flow cells, designed for synchrotron small angle x-ray scattering, allows access to structural information on time scales ranging from sub-millisecond to minutes after the initiation of folding. Previous work shows significant changes in the scattering signals throughout this regime, corresponding to large scale structural rearragements associated with compaction and folding. Three specific aims are presented to achieve our long term goal. To realize the first, time resolved small angle x-ray scattering will be used to characterize the rapid collapse that occurs at the onset of folding. Rate constants and transient structural information will be obtained under different solution and sequence conditions to investigate the role of counterions in collapse and to test structural models of compact states. The second specific aim is directed at structural characterization of later intermediates including partially folded states, using small angle x-ray scattering. The reverse process of unfolding will also be studied. The final specific aim probes the global conformations along the preferred folding pathways that are populated in the presence of both monovalent and divalent cations. Solution conditions that more closely resemble physiological will be explored under this aim. Folding will be initiated from discrete sets of unfolded states to determine the characteristics of transients along separate pathways. The global structural information from these small angle scattering experiments will be integrated with detailed, local information acquired from other Project members to achieve the deepest possible understanding of the folding of this molecule.