Bacterial Outer Membrane Biogenesis: The Role of Molecular Chaperones

智力价值：革兰氏阴性细菌的包膜由两层膜组成，由含有肽多聚糖壁的周质隔间隔开。内膜与胞浆接触，外膜与细胞外环境接触。OM是一种独特的结构，对革兰氏阴性菌是必不可少的，由脂多糖(LPS)、磷脂和蛋白质组成。外膜蛋白是一种选择性很强的通透性屏障，使细菌能够在恶劣的环境中生存。外膜蛋白(OMP)是一种完整的膜蛋白，外膜内嵌有β-桶状结构。在它们的众多功能中，一些是介导膜选择性通透性的孔蛋白，另一些是粘附素，负责宿主组织的黏附和定植。OMP是在胞浆中合成的，并通过SEC转位机制跨内膜转运。然而，这些疏水蛋白是如何穿过周质并特异性地插入OM并折叠到其典型的b-Barrel结构中的，人们对此知之甚少。许多周质蛋白参与了OMPS的运输和插入。这个项目的重点是了解“17kodalton蛋白”(Skp)的作用；Skp是一种不依赖于ATP的周质伴侣蛋白，可以防止蛋白质聚集，并护送OMP穿过周质并帮助它们插入细胞膜。将结合结晶学、核磁共振、电子显微镜和生化方法来确定Skp如何结合其货物蛋白质并防止它们聚集。核磁共振技术将被用来测试货物蛋白质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，直到被运送到外膜。由于Skp是不依赖于ATP的，因此，通过生化方法探讨与Skp结合的内毒素可触发货物释放的假说，可以更深入地了解货物释放的机制。突变之后的体外和体内试验将被用来检验这一假说。这些实验的成功完成将在理解蛋白质如何输送和折叠到细菌外膜方面开辟新的天地。对Skp的研究将为深入了解前折叠蛋白等ATP非依赖性伴侣的一般机制提供依据。此外，从革兰氏阴性Skp系统中吸取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：教育计划描述了几种战略，以增加未被充分代表的群体对科学的参与，包括教学、外联和指导部分。此外，该计划还整合了一项计划，在本科生物化学课程中培养基于研究的批判性思维。这项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.