ANALYSIS OF PROTEIN DISULFIDE BOND FORMATION IN E. COLI

这一提议旨在对蛋白质折叠的一个重要特征--二硫键的形成过程产生新的见解，并为提高多二硫蛋白的生产提供生物技术工具。我们将通过以下方式研究大肠杆菌周质中二硫键异构化的过程：a)我们将确定确保二硫键异构酶保持还原状态的电子从细胞质到周质的途径；b)我们将确定二硫键异构酶DsbC和DsbG以及它们还原所必需的蛋白质DsbD的专一性；c)我们将通过突变和基因打乱来改变这些蛋白的特异性和活性，以了解它们的作用机制，并促进异源二硫键蛋白的生产；D)我们将确定新基因的特征，这些基因在多拷贝或被突变改变时，增强周质中多二硫键蛋白的产生。我们还将研究改变了硫醇：二硫键氧化还原环境的大肠杆菌菌株细胞质中二硫键的形成和异构化过程。从缺少两个主要的硫醇：二硫化物氧化还原途径的菌株开始，我们分离出了抑制突变，这些突变可以恢复细胞质的足够还原能力以供生长，但仍然允许细胞质形成蛋白质二硫键。对这些抑制因子的研究已经揭示了大量细胞质氧化还原蛋白之间的新的相互作用。我们将确定改变的基因和抑制突变产生矛盾的减少但氧化细胞质的机制。我们将分析抑制二硫键形成和异构化的动力学，以及生产异源二硫键蛋白的效率。因此，这项建议统一了对硫醇中电子转移过程的广泛基础研究：氧化还原反应，可能有利于生物技术生产医学上有用的产品。

This proposal is designed to yield new insights into an important feature of protein folding, the process of disulfide bond formation, and to provide biotechnological tools for enhanced production of multidisulfide proteins. I. We will study the process of disulfide bond isomerization in the Escherichia coli periplasm in the following ways: a) We will determine the pathways of electrons from cytoplasm to periplasm that insure the maintenance of disulfide bond isomerases in the reduced state; b)We will determine the specificity of the disulfide bond isomerases, DsbC and DsbG, and of the protein necessary for their reduction, DsbD; c)We will alter the specificity and activity of these proteins using mutagenesis and gene scrambling in order both to understand the mechanism of their action and to enhance the production of heterologous disulfide-bonded proteins; d)We will characterize new genes, which in multi-copy or when altered by mutation, enhance the production of multi-disulfide proteins in the periplasm. II. We will also study the process of disulfide bond formation and isomerization in the cytoplasm of strains of E. coli with altered thiol:disulfide redox environments. Beginning with strains missing the two major thiol: disulfide redox pathways, we have isolated suppressor mutations that restore sufficient reductive power to the cytoplasm for growth, but still allow cytoplasmic formation of protein disulfide bonds. Studies of these suppressors are already revealing novel interactions among the large set of cytoplasmic redox proteins. We will determine the altered genes and the mechanisms whereby suppressor mutations generate the paradoxical reducing but oxidizing cytoplasms. We will analyze suppressor for the kinetics of disulfide bond formation and isomerization and for the efficiency in the production of heterologous disulfide bonded proteins. This proposal thus unites extensive basic studies on processes of electron transfer in thiol: redox reactions with likely benefits for the biotechnological production of medically useful products.