Bacterial Outer Membrane Biogenesis: The Role of Molecular Chaperones

细菌外膜生物发生：分子伴侣的作用

• 批准号: 0719225
• 负责人: Marcelo Sousa
• 金额: $ 49.5万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0719225&HistoricalAwards=false
• 关键词: Bacterial; Outer; Membrane; Biogenesis; Role

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.

智力优点：革兰氏阴性细菌的包膜由两层膜组成，两层膜被含有肽聚糖壁的周质隔室分开。内膜与细胞质接触，而外膜与细胞外环境接触。 OM 是革兰氏阴性菌所必需的独特结构，由脂多糖 (LPS)、磷脂和蛋白质组成。它是一种选择性很强的渗透性屏障，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，其 β 桶结构嵌入外膜中。在其众多功能中，一些 OMP 是介导膜选择性通透性的孔蛋白，而另一些则是负责宿主组织粘附和定植的粘附素。 OMP 在细胞质中合成，并通过 SEC 易位机制跨内膜易位。然而，人们对这些疏水蛋白如何穿过周质并特异性插入 OM 并折叠成其典型的 b 桶结构知之甚少。 许多周质蛋白与 OMP 的运输和插入有关。该项目的重点是了解“十七千道尔顿蛋白质”（Skp）的作用；一种不依赖于 ATP 的周质伴侣，可防止蛋白质聚集，并被提议护送 OMP 穿过周质并协助它们插入膜中。将结合晶体学、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并防止其聚集。核磁共振技术将用于测试货物蛋白在与分子伴侣结合时是否经历折叠，或者它们是否保持在未折叠状态直到递送到外膜。由于 Skp 不依赖于 ATP，因此通过生化探索 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。然后将使用诱变和体外和体内测定来检验这一假设。 这些实验的成功完成将为理解蛋白质如何传递并折叠到细菌外膜中开辟新的领域。 Skp 的研究将有助于深入了解 Prefoldin 等不依赖于 ATP 的分子伴侣的一般机制。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：教育计划描述了增加代表性不足的群体参与科学的几种策略，包括教学、推广和指导部分。此外，该计划还整合了一项在本科生物化学课程中培养基于研究的批判性思维的计划。该研究利用了同步加速器源和机构间核磁共振设施等多用户设施。这为研究生和本科生提供了尖端技术方面的绝佳培训机会。