TARGETING AND ASSEMBLY OF E COLI OUTER MEMBRANE PROTEINS

细菌细胞表面与宿主相互作用，形成致病或 共生关系 这些相互作用直接受到 革兰氏阴性菌（如大肠杆菌）的外膜， 作为环境和内部之间的界面， cell. 越来越清楚的是，外膜是 在使细菌对宿主防御因子产生抗性方面极其重要 通过提供对抗胆汁的洗涤作用的有效屏障 盐、消化酶降解和疏水性 抗生素如新生霉素和红霉素。 水溶性小 营养素和抗生素，如青霉素和头孢菌素， 细胞通过扩散通过由一组独特的 蛋白质，称为孔蛋白。 OmpF是孔蛋白的一个例子， 非特异性孔，而LamB形成特异性促进 麦芽糖糊精的扩散（麦芽低聚物高达7个葡萄糖单位）。 这些 孔蛋白还为细菌病毒提供受体位点。 孔蛋白 蛋白质与其他膜成分紧密结合， 如脂多糖（LPS）和肽聚糖。 这项研究的长期目标是了解 外膜和分子之间的相互作用 膜组件 这项建议的具体目的是研究如何 蛋白质（OmpF和LamB）靶向外膜， 如果有的话，LPS在这个过程中起作用。 这些目标将 通过利用遗传和生物化学的结合， 方法。 我们设计了一个遗传选择方案， 温度敏感性折叠突变，条件性影响 OmpF和LamB的组装。 汇编的条件积累 中间体将使我们能够确定它们的结构和细胞 位置. 所描述的实验方法是新颖的研究 膜蛋白，并将揭示参与的关键残基， 外膜蛋白的靶向和组装。 这些研究报告将 通过抑制剂扩展到检查其他成分的作用 分析.

Bacterial cell surfaces interact with the host to form a pathogenic or symbiotic association. These interactions are directly influenced by the outer membrane of Gram negative bacteria (such as Escherichia coli) which serves as the interface between the environment and the interior of the cell. It is becoming increasingly clear that the outer membrane is extremely important in making bacteria resistant to host defence factors by providing an effective barrier against the detergent action of bile salts, degradation by digestive enzymes, and killing by hydrophobic antibiotics such as novobiocin and erythromycin. Small water-soluble nutrients and antibiotics such as penicillin and cephalothin penetrate the cell by diffusing through pores or channels formed by a unique set of proteins, called porins. OmpF is an example of a porin that forms nonspecific pores, whereas LamB forms pores that specifically facilitate diffusion of maltodextrins (maltooligomers up to 7 glucose units). These porin proteins also provide receptor sites for bacterial viruses. Porin proteins exist in tight association with other membrane components such as lipopolysaccharide (LPS) and peptidoglycan. The long term goal of this research is to understand the biogenesis of the outer membrane and molecular interactions between the various membrane components. The specific aim of this proposal is to examine how proteins (OmpF and LamB) are targeted to the outer membrane and what role, if any, LPS plays in this process. These objectives will be achieved by utilizing a combination of genetic and biochemical approaches. We have devised a genetic selection scheme to isolate temperature sensitive folding mutations that conditionally affect assembly of OmpF and LamB. The conditional accumulation of assembly intermediates will allow us to determine their structure and cellular location. The experimental approach described is novel for studying membrane proteins and will reveal the key residues involved in the targeting and assembly of outer membrane proteins. These studies will be extended to examine the role of other components through suppressor analysis.