Time-resolved hydroxyl radical footprinting

时间分辨羟基自由基足迹

• 批准号: 6760481
• 负责人: Michael D. Brenowitz
• 金额: $ 25.26万
• 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/6760481
• 关键词: X ray crystallography; chemical kinetics; conformation; hydroxyl radical; intermolecular interaction; nucleic acid structure; protein folding; ribozymes; structural biology; synchrotrons; time resolved data

Time-resolved synchrotron x-ray hydroxyl radical footprinting can simultaneously follow the formation of individual tertiary contacts within RNA on time scales ranging from millisecond to minutes. This breadth of accessible timescales allows direct visualization of a large proportion of the tertiary transitions of Tetrahymena ribozyme folding. Synchrotron x-ray footprinting will be used in Overall Specific Aim 1 in conjunction with small angle x-ray scattering to determine if the ribozyme "collapses" nonspecifically prior to proceeding down a specific folding pathway or whether the specific tertiary contacts guide the initial electrostatic collapse. In Overall Specific Aim 2, hydroxyl radical footprinting will be used to explore the structure of the structures present in the unfolded ribozyme. Time-resolved synchrotron x-ray footprinting studies will characterize the folding pathways that predominate at physiological conditions and the native and non-native intermediates of these pathways. Selected ribozyme mutants that perturb the stability of the P4-P6 domain, the peripheral helices and the catalytic core will be analyzed by synchrotron x-ray footprinting in order to explore the structures and lifetimes of the intermediate species. Overall Specific Aim 3 seeks to map the folding 'landscape' for the Tetrahymena ribozyme and the preferred pathways followed by the RNA within it. Solution variables including monovalent and divalent ion concentration will be probed individually and in combination in order to comprehensively map the preferred pathways within the RNA folding landscape. Lastly, the folding of ribozymes in which individual tertiary contacts have been perturbed by mutation will be analyzed in order to distinguish the contributions of inter and intradomain interactions to ribozyme folding. These studies will identify the tertiary contacts that constitute kinetically trapped intermediates, identify preferred pathways through the folding landscape and generate predictions of tertiary contacts that constitute kinetically trapped intermediates for analysis by single molecule FRET.

时间分辨同步辐射X射线羟基自由基足迹可以同时遵循的时间尺度从毫秒到分钟的RNA内的个人三级接触的形成。这种广度的可访问的时间尺度允许直接可视化的大比例的四膜虫核酶折叠的三级转换。同步加速器X射线足迹法将在总体特异性目标1中与小角度X射线散射结合使用，以确定核酶是否在沿着特定折叠途径进行之前非特异性地“塌陷”，或者特定的三级接触是否引导初始静电塌陷。在总体特定目标2中，羟基自由基足迹法将用于探索未折叠核酶中存在的结构的结构。时间分辨同步辐射X射线足迹研究将表征在生理条件下占主导地位的折叠途径以及这些途径的天然和非天然中间体。选择的干扰P4-P6结构域的稳定性的核酶突变体，外周螺旋和催化核心将通过同步加速器X射线足迹法进行分析，以探索中间物种的结构和寿命。总体具体目标3旨在绘制四膜虫核酶的折叠“景观”和其中RNA遵循的优选途径。将单独和组合探测包括单价和二价离子浓度在内的溶液变量，以全面绘制RNA折叠景观中的优选途径。最后，将分析其中个别三级接触已被突变扰动的核酶的折叠，以区分结构域间和结构域内相互作用对核酶折叠的贡献。 核酶折叠这些研究将确定三级接触，构成动力学捕获的中间体，确定通过折叠景观的首选途径，并产生预测的三级接触，构成动力学捕获的中间体，通过单分子FRET分析。