A MOLECULAR RULER FOR DIRECT MEASUREMENT OF DISTANCE DISTRIBUTIONS IN SOLUTIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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，了解折叠中间体和熔融小球的三级结构将非常有用。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。虽然时间分辨荧光共振能量转移（FRET）研究可以提供距离分布的一些信息，但这种分析依赖于对FRET染料取向和分布形状（通常为高斯）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）来直接确定重原子粉尘标记生物分子的分子内距离分布。该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体的三维图像。我们建议对SAXS-HR技术进行原理验证测试：对DNA双链和用纳米金簇标记的柔性DNA单链的测量将证明SAXS-HR可以恢复特定位点的距离分布。这些实验将为研究无肌红蛋白熔融球状折叠中间体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.