Dynamics of human Guanylate Binding Proteins: implications for their structure and oligomerisation

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The ultimate goal of this project is to determine the supertertiary structure ensemble of human Guanylate binding Protein 1 (hGBP1) in solution and to unravel the relevance for its function. hGBP1 is a prototypic large GTP binding protein (GTPase). We are aiming for analysing its intra- and intermolecular protein dynamics in order to understand the molecular mechanism underlying membrane deformation and antimicrobial activity. Sophisticated single-molecule high-precision Förster Resonance Energy Transfer (FRET) experiments and species-specific fluorescence correlation spectroscopy (FCS) as well as kinetic and thermodynamic studies will be employed for defining precisely the architecture of the protein complexes in solution and the time courses of structural changes controlled by the nucleotides.The members of the protein family of large GTPases, like dynamin, Mx, and the numerous hGBP isoforms have many important biological functions in human cells, e.g. endocytosis and the defence of viral or microbrial attack by being part of inflamasomes. Despite these many distinct functions these proteins have many features in common such as the architecture of multiple similar domains, the nucleotide-dependent assembly in tetramers and the association larger oligomers to membranes. We will address many questions common to the members of the superfamily of large GTP binding proteins by pursuing our work on hGBP1 and stepwise including other GBP isoforms such as hGBP2, hGBP4 and hGBP5 (with 50-70% sequence identity but unknown structure) to unravel specific differences and similarities in the mechanism. By generating isoprenylated hGBP1, we also want to study how these structural changes are influenced by the association to membranes.Overall, we want to address the general question how the function of these large and flexible proteins is controlled by intramolecular domain and intermolecular subunit interactions, which modulate their overall supertertiary structure with distinct interface accessibility and dynamics. Especially we want to study how GTP binding and hydrolysis is exploited for function by modulating the molecular energy landscape and hence the dynamic equilibrium between thermodynamically stable conformational states. The detailed picture of a large GTPase, literally 'in action', will help us understanding on a molecular level how the nucleotide-driven protein-protein interactions can lead to concerted biological actions.