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

本项目的最终目标是确定人鸟苷酸结合蛋白1 （hGBP1）在溶液中的超三级结构集合，并揭示其功能的相关性。hGBP1是一种典型的大型GTP结合蛋白（GTP酶）。我们的目标是分析其分子内和分子间的蛋白质动力学，以了解膜变形和抗菌活性的分子机制。复杂的单分子高精度Förster共振能量转移（FRET）实验和物种特异性荧光相关光谱（FCS）以及动力学和热力学研究将用于精确定义溶液中蛋白质复合物的结构和由核苷酸控制的结构变化的时间过程。大型gtp酶蛋白家族的成员，如dynamin， Mx和众多hGBP亚型，在人类细胞中具有许多重要的生物学功能，例如作为炎症小体的一部分，内吞作用和防御病毒或微生物的攻击。尽管这些蛋白质有许多不同的功能，但它们有许多共同的特征，如多个相似结构域的结构，四聚体中核苷酸依赖的组装以及大的低聚物与膜的结合。我们将通过对hGBP1的研究，并逐步包括其他GBP亚型，如hGBP2、hGBP4和hGBP5（具有50-70%的序列同一性，但结构未知），解决大型GTP结合蛋白超家族成员共同的许多问题，以揭示其机制中的具体差异和相似之处。通过生成异丙烯化的hGBP1，我们还想研究这些结构变化是如何受到与膜结合的影响的。总的来说，我们希望解决这些大而灵活的蛋白质的功能如何受到分子内结构域和分子间亚基相互作用的控制的一般问题，这些相互作用调节了它们具有不同界面可及性和动力学的整体超三级结构。我们特别想研究GTP的结合和水解是如何通过调节分子能量景观来发挥作用的，从而在热力学稳定的构象状态之间实现动态平衡。一个大的GTPase的详细图片，字面上“在行动”，将帮助我们在分子水平上理解核苷酸驱动的蛋白质-蛋白质相互作用如何导致协调一致的生物行为。

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.