Structure and Dynamics of Human Guanylate Binding Protein 1 in Solution - Correlating X-ray and Neutron Scattering Experiments with Single-Molecule FRET Measurements

该研究项目的总体目标是研究人类鸟苷结合蛋白1(HGBP1)的酶活性是如何受到蛋白质结构变化的调节的，这种变化依赖于不同核苷酸的存在。此外，还将探讨翻译后修饰(法尼化)对蛋白质结构变化的影响，以及它对蛋白质聚集和随后解离的作用。我们将使用X射线和中子的小角散射(SAXS和SANS)、动态光散射(DLS)和中子自旋回波谱(NSE)结合单分子荧光共振能量转移(FRET)来研究多结构域蛋白质中生化过程的分子功能。本项目的第一部分是确定hGBP1在溶液中以单体、二聚体和四聚体状态的溶液结构。为此，我们将结合SANS和SAXS实验与FRET测量，以允许对粗粒度和完全原子化的计算机模拟进行精细解释。第二个目标是使用NSE测量单体和二聚体hGBP1在一些ns到几百ns的时间尺度上的集体磁区运动。其目的是了解蛋白质在不同构象中的潜在动力学。NSE是对荧光相关光谱的补充，荧光相关光谱提供了从S到毫秒的时间尺度上较慢的集体运动的信息。FCS实验将在塞德尔教授(海因里希·海涅大学，杜塞尔多夫)的团队中进行。通过结合NSE和FCS的结果，我们将受益于强大的协同效应。该项目的第三个方面将是研究法尼化对hGBP1单体结构的影响，并对聚集物种的结构进行表征。将使用DLS和SAXS作为时间函数来研究聚集过程中的结构变化。

The general objective of the research project is the investigation how the enzymatic activity of the human guanylate-binding protein 1 (hGBP1) is regulated by structural changes of the protein, which depend on the presence of different nucleotides. Furthermore, the impact of a posttranslational modification (farnesylation) on structural changes of the protein and its role for aggregation and following dissociation of the protein will be explored. We will use the methods of small-angle scattering by X-rays and neutrons (SAXS and SANS), dynamic light scattering (DLS), and neutron spin-echo spectroscopy (NSE) in combination with single-molecule fluorescence resonance energy transfer (FRET) to be able to investigate the molecular functionality of biochemical processes in multi-domain proteins.The first part of the project is to determine the solution structures of hGBP1 in the monomeric, dimeric and tetrameric state in solution. For that purpose we will combine SANS and SAXS experiments with FRET measurements to allow a refined interpretation of coarse-grained and fully atomistic computer simulations. The temperature dependence of the structures of hGBP1 monomer and dimer will also be explored.The second objective will be to measure collective domain motions in the time scale of some ns up to several 100 ns of the monomeric and dimeric hGBP1 using NSE. The aim is to get an understanding of the underlying dynamics of the protein in the different conformations. NSE is complementary to fluorescence correlation spectroscopy (FCS), which provides information on slower collective motions on a time scale from some µs to ms. The FCS experiments will be performed in the group of Prof. Seidel (Heinrich Heine Universität, Düsseldorf). We will profit from strong synergic effects by combining the NSE and the FCS results.The third aspect of the project will be the investigation of the impact of farnesylation on the structure of the hGBP1 monomer and the characterization of the structure of the aggregated species. Structural changes during the aggregation process will be investigated using DLS and SAXS as a function of time.