PROTEIN DISULFIDE BOND ISOMERIZATION IN E COLI

描述：细菌细胞质通常维持在氧化还原状态， 电位，使得不形成二硫键。 内膜外 氧化还原电位通常使得蛋白质中的二硫键形成， 进化很可能利用这些桥梁来增加稳定性， proteins. 使用这种债券会产生两个问题。 首先，半胱氨酸对氧化形成二硫键的速率可 第二，二硫键的交换通常是必要的， 打乱这些键，直到蛋白质中形成正确的键。 E. 大肠杆菌显然具有形成和异构化的途径， 分泌进入和通过周质间隙的蛋白质的二硫键。 所提出的工作集中在两个目标上， 含有多个组装的二硫键，E.大肠杆菌BE 设计来优化这些蛋白质的组装？ 第一个目标 将在哈佛医学院贝克维斯博士的实验室进行研究， 第二个将由乔治博士作为联合体/合同进行 德克萨斯大学奥斯汀分校的Georgiou。 所述目标是表征维持二硫键的途径 键异构酶，DsbC，在还原状态下，分离突变体，影响 这一途径，并寻找其他基因和蛋白质发挥作用， 道路。 为此目的的遗传实验将在 贝克维斯的实验室，以及乔久实验室的生化实验。 以下二硫键蛋白将用于研究，核酸内切酶 牛胰蛋白酶抑制剂和组织纤溶酶原激活剂。 结构-功能研究，即膜拓扑结构，将在 DsbD和DsbC与细胞质和细胞质外的相互作用 将检查组件。 过表达蛋白质产物的基因 将抑制DsbC突变体将寻求。

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.