CATALYSIS OF THIOL/DISULFIDE EXCHANGE

二硫键的形成是表达 许多细胞外蛋白质，包括受体、酶和 荷尔蒙 正确二硫键形成的机制 在蛋白质折叠和组装过程中， 了解三维蛋白质结构是如何从 主要序列信息，而且具有实际意义 含二硫键的蛋白质和肽的表达和生产 有治疗意义 蛋白质二硫键异构酶，一种丰富的 内质网蛋白，催化巯基/二硫化物 氧化、还原和重排反应， 蛋白质的氧化折叠。 拟议的长期目标 研究的目的是在结构和机制层面上了解 蛋白质二硫键异构酶，无论是本身或与 其他蛋白质，促进蛋白质的折叠和组装， 含有二硫键交联。 定点突变，动力学 方法和蛋白质化学将被应用于研究 酶的三个二硫醇/二硫键中心 单独或集体参与催化。 动力学 突变酶的行为，其中一个或多个特定的半胱氨酸 将已转化为Ser和Ala的残基与已转化为Ser和Ala的残基进行比较。 野生型蛋白的行为，以检测 多个硫醇/二硫化物中心。 共价和非共价相互作用 蛋白质二硫键异构酶和它的蛋白质底物之间的相互作用， 探讨了 将产生单个半胱氨酸突变体，其可以捕获 酶与底物形成非生产性共价复合物。 的 酶参与分子间和 多链体外组装过程中的分子内二硫键 蛋白质如免疫球蛋白G及其片段（Fab），以及 蛋白质二硫键异构酶与其他组装体的协同作用 还将研究蛋白质如分子伴侣蛋白。

Disulfide bond formation is an integral part of the expression of numerous extracellular proteins including receptors, enzymes, and hormones. The mechanisms by which correct disulfide bonds are formed during protein folding and assembly is not only important to understanding how three-dimensional protein structure is generated from primary sequence information but also has practical significance in the expression and production of disulfide-containing proteins and peptides of therapeutic importance. Protein disulfide isomerase, an abundant protein of the endoplasmic reticulum, catalyzes thiol/disulfide oxidation, reduction, and rearrangement reactions involved in the oxidative folding of proteins. The long-range goal of the proposed research is to understand at a structural and mechanistic level how protein disulfide isomerase, either by itself or in conjunction with other proteins, facilitates the folding and assembly of proteins that contain disulfide crosslinks. Site-directed mutagenesis, kinetic methods, and protein chemistry will be applied to investigate the mechanism by which the three dithiol/disulfide centers of the enzyme participate individually or collectively in catalysis. The kinetic behavior of mutant enzymes in which one or more specific cysteine residues have been converted to Ser and Ala will be compared to the behavior of the wildtype protein to detect interactions between the multiple thiol/disulfide centers. Covalent and noncovalent interactions between protein disulfide isomerase and its protein substrates will be explored. Single cysteine mutants will be produced that may trap the enzyme in non-productive covalent complexes with the substrates. The participation of the enzyme in the formation of intermolecular and intramolecular disulfide bonds during the in vitro assembly of multichain proteins such as immunoglobulin G and its fragments (Fab) and the cooperation between protein disulfide isomerase and other assembly proteins such as the molecular chaperonins will -also be investigated.