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资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。为了深入了解蛋白质折叠的分子事件，对折叠中间体和熔融球的三级结构的了解将是非常有用的。但是，常规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合奏。尽管时间分辨荧光共振能量传递（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染料方向的假设以及分布的形状（通常是高斯）。我们建议将小角度的X射线散射与重原子替换（SAXS-HR）结合使用，以确定在重原子duster标记的生物分子的重原子内分子内距离分布。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融球的三维图片。我们建议对SAXS-HR技术进行原理测试证明：对DNA双链体的测量以及用纳米质量簇标记的柔性DNA单链将证明可以通过SAXS-HR恢复特定于位点特异性的距离分布。这些实验将为对Apomyoglobin熔融球折叠中间的三级结构的研究提供基础。