CATALYSIS OF THIOL/DISULFIDE EXCHANGE

二硫键的形成是表达的一个组成部分 大量的胞外蛋白质，包括受体、酶和 荷尔蒙。形成正确的二硫键的机制 在蛋白质折叠和组装过程中不仅对 了解三维蛋白质结构是如何从 初级序列信息在生物信息学中也具有实际意义 含二硫键蛋白和多肽的表达与生产 对治疗很重要。蛋白质二硫键异构酶，一种丰富的 内质网的蛋白质，催化硫醇/二硫键 参与的氧化、还原和重排反应 蛋白质的氧化折叠。拟议中的长期目标 研究是在结构和机制层面上理解如何 蛋白质二硫键异构酶，单独或与 其他蛋白质，促进蛋白质的折叠和组装，这些蛋白质 含有二硫键交联物。定点诱变，动力学 方法，并将应用蛋白质化学来研究 酶的三个二硫醇/二硫键中心的作用机制 单独或集体参与催化。动能 一种或多种特定半胱氨酸突变酶的行为 残基已转换为Ser和Ala将与 野生型蛋白检测相互作用的行为 多个硫醇/二硫中心。共价和非共价相互作用 蛋白质二硫键异构酶及其蛋白质底物之间的相互作用 探索过了。将产生一个半胱氨酸突变体，它可能会捕获 酶与底物形成非产生性共价复合体。这个 酶在分子间和分子间形成中的作用 多链体外组装过程中的分子内二硫键 免疫球蛋白G及其片段(Fab)和 蛋白质二硫键异构酶与其他组装的协同作用 分子伴侣等蛋白质也将被研究。

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.