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.

研究项目的一般目标是投资如何受到蛋白质的结构变化来调节人类鸟根结合蛋白1（HGBP1）的酶促活性，这取决于存在不同核苷酸的存在。此外，将探索翻译后修饰（Farnesylation）对蛋白质结构变化及其在聚集和蛋白质解离后的作用的影响。我们将使用X射线和中子（SAX和SANS），动态光散射（DLS）和中子自旋回波光谱（NSE）结合单分子荧光共振能量（FRET），以能够研究多组合蛋白的分子功能。溶液中单体，二聚体和四聚体状态中HGBP1的结构。为此，我们将将SAN和SAXS实验与FRET测量相结合，以允许对粗粒和完全原子的计算机模拟进行精致的解释。还将探索HGBP1单体和二聚体的结构的温度依赖性。第二个目标是使用NSE在某些NS的时间尺度上测量一些NS的时间尺度的集体域运动。目的是了解不同构象中蛋白质的潜在动力学。 NSE已完成荧光相关光谱（FCS），该光谱法提供了有关从一些µs到MS的时间尺度上较慢的集体运动的信息。 FCS实验将在Seidel教授（Heinrich HeineUniversität，Düsseldorf）的小组中进行。通过结合NSE和FCS结果，我们将从强大的协同效应中获利。该项目的第三个方面是投资Farnesylation对HGBP1月结构的影响以及聚集物种结构的表征。聚集过程中的结构变化将使用DLS和SAXS随时间的函数进行研究。

项目成果

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

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