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

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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.