Time-resolved small angle X-ray scattering

时间分辨小角 X 射线散射

• 批准号: 6760471
• 负责人: LOIS POLLACK
• 金额: $ 23.61万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-06 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760471
• 关键词: Tetrahymena; X ray crystallography; bioimaging /biomedical imaging; conformation; nucleic acid structure; protein folding; ribozymes; structural biology; synchrotrons; time resolved data

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.

RNA在许多生物过程中起着重要的作用，但其构象动力学尚未在最基本的层面上理解：自组装或折叠。该程序项目的长期目标是大RNA的折叠的完整表征：四膜肌核酶。该提案介绍了研究的侵害，旨在探究该核酶通过初步快速崩溃而折叠的全局构象，并随后进行了重大的压实步骤。使用用于同步的小角度X射线散射的流动单元的使用组合，可以按时间尺度访问折叠折叠后的时间尺度到几分钟的结构信息。 先前的工作显示了整个该制度的散射信号的显着变化，对应于与压实和折叠相关的大规模结构重排。 提出了三个具体目标以实现我们的长期目标。为了实现第一个，将使用时间分解的小角度X射线散射来表征折叠开始时发生的快速崩溃。速率常数和瞬态结构信息将在不同的解决方案和序列条件下获得，以研究反族在塌陷中的作用并测试紧凑型状态的结构模型。第二个特定目标是使用小角度X射线散射的后来中间体的结构表征，包括部分折叠状态。还将研究展开的反向过程。最终的特定目的探测沿首选的折叠途径的全局构型，这些折叠途径均在单价和二价阳离子的存在下填充。在此目标下将探讨更与生理的非常相似的解决方案条件。折叠将从离散状态的离散集开始，以确定沿单独途径的瞬态特征。来自这些小角度散射实验的全球结构信息将与从其他项目成员获取的详细的本地信息集成，以实现对该分子折叠的最深理解。