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

溶液中人鸟苷酸结合蛋白 1 的结构和动力学 - 将 X 射线和中子散射实验与单分子 FRET 测量相关联

• 批准号: 280164000
• 负责人: Privatdozent Dr. Andreas Stadler
• 金额: --
• 依托单位: Neutronenstreuung und biologische Materie (JCNS-1/IBI-8)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/280164000?language=en
• 关键词: Structure; Dynamics; Human; Guanylate; Binding

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.

该研究项目的总体目标是研究人类鸟苷酸结合蛋白1（hGBP1）的酶活性如何通过蛋白质的结构变化进行调节，这取决于不同核苷酸的存在。此外，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将被探讨。我们将使用X射线和中子的小角散射方法（SAXS和SANS），动态光散射（DLS），和中子自旋回波光谱（NSE）与单分子荧光共振能量转移（FRET）相结合，能够研究多个生物化学过程中的分子功能。该项目的第一部分是确定hGBP1在溶液中的单体、二聚体和四聚体状态的溶液结构。为此，我们将结合联合收割机SANS和SAXS实验与FRET测量，允许粗粒度和完全原子计算机模拟的精细解释。hGBP1单体和二聚体的结构的温度依赖性也将被探索。第二个目标将是测量集体域运动的时间尺度上的一些ns到几个100 ns的单体和二聚体的hGBP1使用NSE。目的是了解不同构象中蛋白质的潜在动力学。NSE是荧光相关光谱（FCS）的补充，它提供了从几微秒到毫秒的时间尺度上的较慢的集体运动的信息。FCS实验将在Seidel教授（Heinrich海涅大学，杜塞尔多夫）的小组中进行。通过将NSE和FCS的结果结合起来，我们将受益于强大的协同效应。本项目的第三个方面将是调查法尼基化对hGBP1单体结构的影响，并对聚集物种的结构进行表征。聚集过程中的结构变化将使用DLS和SAXS作为时间的函数进行研究。

项目成果

Assembly Mechanism of Farnesylated hGBP1 Studied by Time-Resolved Saxs and Electron Microscopy

时间分辨萨克斯和电子显微镜研究法尼基化 hGBP1 的组装机制

• DOI: 10.1016/j.bpj.2018.11.880
• 发表时间: 2019
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Charlotte Lorenz;Andreas M Stadler
• 通讯作者: Andreas M Stadler

Farnesylation of human guanylate‐binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization

人鸟苷酸结合蛋白 1 的法呢基化作为防止结构重排和非诱导二聚化的安全机制

• DOI: 10.1111/febs.15015
• 发表时间: 2020
• 期刊: The FEBS Journal
• 作者: Charlotte Lorenz;Semra Ince;Tao Zhang;Anneliese Cousin;Renu Batra-Safferling;Luitgard Nagel-Steger;Christian Herrmann;Andreas M. Stadler
• 通讯作者: Andreas M. Stadler