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鸟苷酸结合蛋白 1 (hGBP1) 的酶活性如何受到蛋白质结构变化的调节，而结构变化取决于不同核苷酸的存在。此外，还将探讨翻译后修饰（法尼基化）对蛋白质结构变化的影响及其对蛋白质聚集和解离的作用。我们将使用X射线和中子小角散射（SAXS和SANS）、动态光散射（DLS）和中子自旋回波光谱（NSE）的方法与单分子荧光共振能量转移（FRET）相结合，能够研究多结构域蛋白质中生化过程的分子功能。该项目的第一部分是确定hGBP1在溶液中的单体、二聚和四聚状态的溶液结构。为此，我们将 SANS 和 SAXS 实验与 FRET 测量相结合，以便对粗粒度和完全原子计算机模拟进行精细解释。还将探讨 hGBP1 单体和二聚体结构的温度依赖性。第二个目标是使用 NSE 测量 hGBP1 单体和二聚体在几纳秒到几百纳秒的时间尺度内的集体域运动。 The aim is to get an understanding of the underlying dynamics of the protein in the different conformations. NSE 是荧光相关光谱 (FCS) 的补充，后者提供了从几微秒到毫秒的时间尺度上较慢的集体运动的信息。 FCS 实验将由 Seidel 教授（杜塞尔多夫 Heinrich Heine Universität）小组进行。通过结合 NSE 和 FCS 结果，我们将从强大的协同效应中受益。该项目的第三个方面将是研究法尼基化对 hGBP1 单体结构的影响以及聚集物种结构的表征。将使用 DLS 和 SAXS 作为时间函数来研究聚合过程中的结构变化。

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.