A MOLECULAR RULER FOR DIRECT MEASUREMENT OF DISTANCE DISTRIBUTIONS IN SOLUTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 为了深入了解蛋白质折叠背后的分子事件，了解折叠中间体和熔融球体的三级结构将是非常有用的。然而，传统的结晶学和磁共振技术不能表征组成这些状态的动态、三维构象系综。虽然时间分辨荧光共振能量转移(FRET)研究可以提供一些关于距离分布的信息，但这种分析依赖于关于FRET染料取向和分布形状(通常是高斯分布)的假设。我们建议使用小角X射线散射结合重原子置换(SAXS-HR)来直接确定重原子灰尘标记的生物分子中的分子内距离分布。这些结果将限制分子动力学模拟提供蛋白质折叠中间体和熔融球体的三维图像。我们建议对SAXS-HR技术进行原理证明测试：对用纳米金簇标记的DNA双链和柔性DNA单链的测量将证明SAXS-HR可以恢复特定位置的距离分布。这些实验将为研究去肌红蛋白熔化球体折叠中间体中存在的三级结构提供基础。

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.