Transport and Insertion of Outer Membrane Proteins

革兰氏阴性菌的包膜由两层膜组成，这两层膜被含有肽聚糖壁的周质隔室隔开。内膜（IM）与细胞溶质接触，而外膜（OM）与细胞外环境接触。OM是革兰氏阴性菌所必需的独特结构，由脂多糖（[PS]）、磷脂和蛋白质组成。它是一种非常有选择性的渗透屏障，允许细菌在恶劣的环境中生存，如肠道，其中OM对胆盐的抗性允许肠道细菌茁壮成长。OM的组分是在感染后首先与宿主接触的组分，并且强烈调节共生菌和致病菌与其宿主的相互作用。OM生物发生过程的清晰把握对于理解宿主-病原体相互作用以及细菌生理学的基本方面至关重要。外膜蛋白（OMPs）是一种完整的膜蛋白，具有13桶结构。许多外膜蛋白具有免疫原性，其中一些外膜蛋白作为粘附素介导宿主组织的粘附和定殖。外膜蛋白在胞浆中合成，并通过SEC转运机制转运到IM中 然而，这些疏水蛋白如何穿过周质并特异性地插入OM中还知之甚少。许多周质蛋白和一种外膜蛋白（0 mp85/YaeT）参与外膜蛋白的转运和插入。在这项提案中，我们将确定的结构和底物特异性的YaeT复杂的组件作为第一步了解的机制OMP运输和组装。结晶学，核磁共振和生物化学方法的组合将结合起来，以表征这一关键的蛋白质复合物。

The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane (IM) is in contact with the cytosol while the outer membrane (OM) contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide ([PS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments such as the gut, where the OM resistance to bile salts allows enteric bacteria to thrive. The components of the OM are the first to come in contact with a host upon infection and strongly modulate the interaction of symbiotic and pathogenic bacteria with their host. A clear grasp of the OM biogenesis process is essential to understand host-pathogen interactions as well as a fundamental aspect of bacterial physiology. Outer membrane proteins (OMPs) are integral membrane proteins, with 13-barrel structures embedded in the OM. Many OMPs are immunogenic and some of them serve as adhesins mediating adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the IM by the SEC translocation machinery However, how these hydrophobic proteins cross the periplasm and insert specifically into the OM is poorly understood. A number of periplasmic proteins and one OMP (0mp85/YaeT) have been implicated in the transport and insertion of OMPs. In this proposal we will determine the structure and substrate specificity of the YaeT complex components as a first step toward understanding the mechanisms of OMP transport and assembly. A combination of crystallographic, NMR and biochemical approaches will be combined to characterize this crucial protein complex.