Bacterial Outer Membrane Biogenesis: The Role of Molecular Chaperones

知识优势：革兰氏阴性菌的包膜由两层膜组成，由含有肽聚糖壁的质周隔室隔开。内膜与胞质溶胶接触，而外膜与细胞外环境接触。OM是革兰氏阴性菌必需的独特结构，由脂多糖（LPS）、磷脂和蛋白质组成。这是一种非常有选择性的渗透性屏障，使细菌能够在恶劣的环境中生存。外膜蛋白（OMPs）是一种完整的膜蛋白，具有β -桶状结构嵌入外膜。在它们的许多功能中，一些omp是介导膜选择性通透性的孔蛋白，而另一些则作为粘附蛋白，负责宿主组织的粘附和定植。omp在细胞质中合成，并通过SEC易位机制在内膜上易位。然而，这些疏水蛋白如何穿过外周质并特异性地插入到OM中并折叠成其典型的b桶结构尚不清楚。许多质周蛋白参与了omp的转运和插入。该项目的重点是了解“17千道尔顿蛋白”（Skp）的作用；一种不依赖atp的周质伴侣，可阻止蛋白质聚集，并被认为可护送omp穿过周质并协助其插入膜。晶体学、核磁共振、电子显微镜和生化方法的结合将用于确定Skp如何结合其货物蛋白并阻止它们聚集。核磁共振技术将用于测试货物蛋白在与伴侣结合时是否发生折叠，或者它们是否保持未折叠状态直到被递送到外膜。由于Skp与atp无关，因此通过生物化学方法探索LPS与Skp结合触发货物释放的假设，可以深入了解货物释放的机制。诱变后的体外和体内试验将用于验证这一假设。这些实验的成功完成将在理解蛋白质如何传递和折叠到细菌外膜方面开辟新的领域。Skp的研究将提供对atp独立伴侣如Prefoldin的一般机制的见解。此外，从革兰氏阴性Skp系统中获得的经验教训可能为蛋白质折叠和插入线粒体外膜的机制提供有价值的线索。更广泛的影响：该教育计划描述了几种策略，以增加代表性不足的群体在科学领域的参与，包括教学、推广和指导组成部分。此外，该计划还整合了一项计划，旨在培养本科生生物化学课程中基于研究的批判性思维。该研究利用了多用户设施，如同步加速器源和机构间核磁共振设施。这为研究生和本科生提供了优秀的尖端技术培训机会。

Intellectual Merit: The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane is in contact with the cytosol while the outer membrane contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide (LPS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments.Outer membrane proteins (OMPs) are integral membrane proteins with beta-barrel structures embedded in the outer membrane. Among their many functions, some OMPs are porins mediating the selective permeability of the membrane while others serve as adhesins responsible for adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the inner membrane by the SEC translocation machinery. However, how these hydrophobic proteins cross the periplasm and insert specifically into the OM and fold into their typical b-barrel structure is poorly understood. A number of periplasmic proteins have been implicated in the transport and insertion of OMPs. This project is focused on understanding the role of the "Seventeen Kilodalton Protein" (Skp); an ATP-independent periplasmic chaperone that prevents protein aggregation and is proposed to escort OMPs across the periplasm and assists in their insertion into membranes. A combination of crystallographic, NMR, electron microscopy and biochemical approaches will be used to determine how Skp binds its cargo proteins and prevents their aggregation. NMR techniques will be used to test whether the cargo proteins undergo folding while bound to the chaperone or if they are kept in an unfolded state until delivered to the outer membrane. Since Skp is ATP-independent, insights into the mechanisms of cargo release will be obtained by biochemically probing the hypothesis that LPS binding to Skp triggers cargo release. Mutagenesis followed by in vitro and in vivo assays will then be used to test this hypothesis. The successful completion of these experiments will break new ground in the understanding of how proteins are delivered and folded into the bacterial outer membrane. The study of Skp will provide insights into the general mechanism of ATP-independent chaperones such as Prefoldin. In addition, the lessons learned from the Gram-negative Skp system may provide valuable clues for the mechanism of protein folding and insertion in the mitochondrial outer membrane. Broader Impacts: The educational program describes several strategies to increase the engagement of underrepresented groups in science including teaching, outreach and mentoring components. In addition the program integrates a plan to foster research-based critical thinking in undergraduate biochemistry classes. The research makes use of multi-user facilities such as synchrotron sources and inter-institutional NMR facilities. This provides outstanding training opportunities in cutting edge technologies for graduate and undergraduate students.