A MOLECULAR RULER FOR DIRECT MEASUREMENT OF DISTANCE DISTRIBUTIONS IN SOLUTIONS

用于直接测量溶液中距离分布的分子尺

• 批准号: 7370442
• 负责人: PEHR A HARBURY
• 金额: $ 0.43万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7370442
• 关键词: MOLECULAR; RULER; DIRECT; MEASUREMENT; DISTANCE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To gain insight into the molecular events that underlie protein folding, knowledge of the tertiary structure of folding intermediates and molten globules would be extremely informative. However, conventional crystallographic and magnetic resonance techniques cannot characterize the dynamic, three-dimensional conformational ensembles that compose these states. While time-resolved fluorescence resonance energy transfer (FRET) studies can give some information on distance distributions, such analyses rely on assumptions about FRET dye orientations and the shapes of the distributions (generally Gaussian). We propose to use small-angle X-ray scattering in conjunction with heavy-atom replacement (SAXS-HR) to determine directly intramolecular distance distributions in heavy-atom duster labeled biomolecules. The results will constrain molecular-dynamics simulations to provide three-dimensional pictures of protein folding intermediates and molten globules. We propose to perform a proof of principle' test of the SAXS-HR technique: measurements on DNA duplexes and flexible DNA single strands labeled with nanogold clusters will demonstrate that site-specific distance distributions can be recovered by SAXS-HR. These experiments will provide a foundation for studies of the tertiary structure present in the apomyoglobin molten globule folding intermediate.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。为了深入了解蛋白质折叠背后的分子事件，折叠中间体和熔球的三级结构的知识将非常有用。然而，传统的晶体学和磁共振技术无法表征构成这些状态的动态三维构象系综。虽然时间分辨荧光共振能量转移 (FRET) 研究可以提供一些有关距离分布的信息，但此类分析依赖于有关 FRET 染料方向和分布形状（通常是高斯分布）的假设。我们建议将小角度 X 射线散射与重原子置换 (SAXS-HR) 结合使用，直接确定重原子除尘器标记的生物分子中的分子内距离分布。结果将限制分子动力学模拟，以提供蛋白质折叠中间体和熔球的三维图像。我们建议对 SAXS-HR 技术进行原理验证测试：对用纳米金簇标记的 DNA 双链体和柔性 DNA 单链进行测量将证明 SAXS-HR 可以恢复位点特异性距离分布。这些实验将为研究脱辅基肌红蛋白熔球折叠中间体中存在的三级结构提供基础。