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。 不会形成二硫键的势。内膜外 氧化还原电位通常是这样的，即蛋白质中确实形成了二硫键， 进化论很可能利用这些桥梁来增加稳定性 蛋白质。这种债券的使用产生了两个问题。 首先，半胱氨酸对形成二硫键的氧化速度可以 慢，第二，二硫键的交换通常是必要的，以 打乱这些键，直到蛋白质中形成了正确的键。E. 大肠杆菌显然有一条形成和异构化的途径 蛋白质的二硫键分泌进入和通过周质间隙。 提出的工作集中在两个目标上，即蛋白质是如何 含有多个组装的二硫键，而大肠杆菌如何 是为了优化这种蛋白质的组装而设计的？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， 第二个项目将由乔治博士以联合体/合同的形式执行 乔治欧在德克萨斯大学奥斯汀分校工作。 声明的目标是描述维持二硫化物的途径的特征。 键异构酶，DsbC，处于还原状态，以分离影响 这条途径，并寻找更多的基因和蛋白质发挥作用 这条小路。朝向这些目标的基因实验将在 贝克维斯的实验室，以及乔治欧实验室的生化实验。 下列二硫键蛋白将用于研究，内切酶 I，牛胰酶抑制物和组织型纤溶酶原激活剂。 结构-功能研究，即膜拓扑学，将在 DsbD及其与胞质和胞外的相互作用 将对组件进行检查。蛋白质产物过表达的基因 将抑制DsbC突变体将被寻找。

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.