PROTEIN DISULFIDE BOND ISOMERIZATION IN E COLI

大肠杆菌中蛋白质二硫键异构化

• 批准号: 2872723
• 负责人: JONATHAN BECKWITH
• 金额: $ 23.64万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872723
• 关键词: Escherichia coli; bacterial proteins; disulfide bond; endonuclease; genetic manipulation; intermolecular interaction; membrane proteins; microorganism metabolism; plasminogen activator; protein biosynthesis; protein disulfide isomerase; protein structure function; trypsin inhibitors

DESCRIPTION: The bacterial cytoplasm is normally maintained at a redox potential such that disulfide bonds do not form. Outside the inner membrane the redox potential often is such that disulfide bonds in proteins do form, and evolution likely has utilized these bridges to add stability to proteins. Two problems are generated by the utilization of such bonds. First, the rate of oxidation of cysteine pairs to form a disulfide bond can be slow, and second, interchange of disulfide bonds is often necessary to shuffle these bonds until the correct set has been made in a protein. E. coli apparently possesses a pathway for the formation and isomerization of disulfide bonds of proteins secreted into and through the periplasmic space. The work proposed is focussed on two objectives, how are proteins that contain multiple disulfide bonds assembled, and how can E. coli be engineered to optimize the assembly of such proteins? The first objective will be studied in Dr. Beckwith's laboratory in the Harvard Medical School, and the second will be carried out as a consortium/contract by Dr. George Georgiou at the University of Texas, Austin. The stated goals are to characterize the pathway that maintains disulfide bond isomerase, DsbC, in a reduced state, to isolate mutants that affect this pathway, and to seek additional genes and proteins that play a role in the pathway. Genetic experiments toward these ends will be performed in Beckwith's laboratory, and biochemical experiments in Georgiou's laboratory. The following disulfide proteins will be used in the studies, endonuclease I, bovine pancreatic trypsin inhibitor, and tissue plasminogen activator. Structure-function studies, meaning membrane topology, will be performed on DsbD and the interactions of DsbC with cytoplasmic and extracytoplasmic components will be examined. Genes whose overexpressed protein products will suppress DsbC mutants will be sought.

描述：细菌细胞质通常保持在氧化还原 潜力使二硫键不会形成。 内膜外 氧化还原电位通常是使蛋白质中的二硫键确实形成， 进化可能已经利用了这些桥来增加 蛋白质。 这种债券的利用产生了两个问题。 首先，半胱氨酸对形成二硫键键的氧化速率 要慢，其次，二硫键的互换通常是必需的 洗牌这些键，直到在蛋白质中制成正确的集合为止。 E. 大属显然拥有形成和异构化的途径 蛋白质的二硫键分泌到周质空间中。 提出的工作集中在两个目标上，蛋白质如何 包含组装的多个二硫键，以及大肠杆菌如何成为 设计以优化这种蛋白质的组装？ 第一个目标 将在哈佛医学院的贝克威斯博士的实验室中进行研究， 第二个将由乔治博士作为财团/合同进行。 奥斯丁德克萨斯大学的乔治。 既定的目标是表征维持二硫化的途径 键异构酶DSBC以降低的状态分离出影响的突变体 这条途径，并寻求其他基因和蛋白质 路径。 对这些目的的基因实验将在 贝克维斯的实验室和乔治实验室的生化实验。 以下二硫键蛋白将用于内切酶 I，牛胰腺胰蛋白酶抑制剂和组织纤溶酶原激活剂。 结构功能研究（意味着膜拓扑）将在 DSBD以及DSBC与细胞质和外质的相互作用 将检查组件。 过表达蛋白质产物的基因 将寻求抑制DSBC突变体。