TARGETING AND ASSEMBLY OF E COLI OUTER MEMBRANE PROTEINS

大肠杆菌外膜蛋白的靶向和组装

• 批准号: 2185645
• 负责人: RAJEEV MISRA
• 金额: $ 10.47万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185645
• 关键词: Escherichia coli; bacterial capsules; bacterial polysaccharides; bacterial proteins; gene expression; genetic mapping; gram negative bacteria; host organism interaction; intermolecular interaction; lipopolysaccharides; membrane permeability; membrane proteins; nucleic acid sequence; pore forming protein; protein biosynthesis; protein folding; protein signal sequence; site directed mutagenesis; suppressor mutations; temperature sensitive mutant

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.

细菌细胞表面与宿主相互作用，形成致病性或 共生协会。 这些互动直接受到 革兰氏阴性细菌的外膜（例如大肠杆菌） 充当环境和内部的界面 细胞。 越来越清楚的是外膜是 对于使细菌抗宿主防御因素极为重要 通过对胆汁洗涤剂作用的有效障碍 盐，消化酶降解，并被疏水杀死 抗生素，例如Novobiocin和红霉素。 小水溶性 养分和抗生素，例如青霉素和头孢霉素渗透 通过通过一组独特的一组孔或通道扩散来扩散该单元 蛋白质，称为porins。 OMPF是形成的孔蛋白的一个例子 非特异性毛孔，而羔羊形成孔特异性毛孔 麦芽糊精的扩散（麦芽糖量多达7个葡萄糖单元）。 这些 孔蛋白蛋白还提供细菌病毒的受体位点。 波林 蛋白质与其他膜成分紧密相关 作为脂多糖（LPS）和肽聚糖。 这项研究的长期目标是了解 各种的外膜和分子相互作用 膜组件。 该提议的具体目的是检查如何 蛋白质（OMPF和LAMB）针对外膜，什么 LP在此过程中扮演的角色（如果有的话）。 这些目标将是 通过利用遗传和生化的结合来实现 方法。 我们已经设计了一个遗传选择方案，以分离 温度敏感的折叠突变，有条件地影响 OMPF和羔羊的组装。 组装的条件积累 中级将使我们能够确定其结构和细胞 地点。 描述的实验方法是研究的新颖 膜蛋白，并将揭示涉及的关键残基 外膜蛋白的靶向和组装。 这些研究将是 扩展以通过抑制器检查其他组件的作用 分析。