FUNCTIONAL ANALYSIS OF A PROTEIN FOLDING CATALYST

蛋白质折叠催化剂的功能分析

• 批准号: 6351237
• 负责人: JAMES BARDWELL
• 金额: $ 19.27万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351237
• 关键词: Escherichia coli; active sites; catalyst; crosslink; cysteine; disulfide bond; enzyme mechanism; ionic bond; molecular chaperones; oxidation reduction reaction; oxidizing agents; protein folding; protein structure function

Improper folding of proteins has been directly implicated in at least a dozen disease states including Alzheimer's and mad cow disease. Disulfide bonds are important enough for protein folding and stability that simple reduction of these bonds will often cause proteins to unfold. The formation of disulfide bridges is a catalyzed process. We found two catalysts to be involved, DsbA acts as the direct donor of DiSulfide Bonds to newly synthesized periplasmic proteins, DsbB acts to reoxidize DsbA. Our long term goal is to understand how these proteins act to catalyze protein folding. In this project we will seek to answer two basic questions: 1) Why is DsbA so powerful a protein oxidant? and 2) What are the catalytic properties of DsbB that allow it to specifically oxidize DsbA? Two distinct models have been proposed to explain the extreme oxidizing power of Dsba's active site disulfide relative to the related protein, thioredoxin. One model invokes disulfide strain, the other electrostatic interactions that affect the pKa of an active site cysteine. We have designed a multifaceted genetic, biophysical and structural approach that should clearly distinguish between these models. Strain and electrostatic interactions play important roles in the folding and catalytic function of many proteins. Clearly understanding the role of these forces in one model system should thus provide valuable information for understanding their role in other proteins as well. To analyze the redox and catalytic properties of DsbB we propose to use a workbox of tools very similar to those we have successfully used with DsbA. This straightforward characterization should tell us much about the way DsbB functions to reoxidize DsbA and may open the door to investigation of how disulfide bond formation is linked to cellular metabolism. Disulfide bond formation is one of the few covalent modifications that occurs in protein folding. This allows us to phrase our questions in simple biochemical terms. We feel that we now have the potential to understand the function of the DsbA-DsbB disulfide catalytic machine.

蛋白质的不正确折叠至少直接涉及 包括阿尔茨海默病和疯牛病在内的十几种疾病。 二硫键对于蛋白质折叠和稳定性非常重要 这些键的简单还原通常会导致蛋白质 展开。 二硫桥的形成是一个催化过程。 我们 发现涉及两种催化剂，DsbA 充当直接供体 DsbB 与新合成的周质蛋白形成二硫键 再氧化 DsbA。 我们的长期目标是了解这些蛋白质如何 起到催化蛋白质折叠的作用。 在这个项目中我们将寻求答案 两个基本问题： 1) 为什么 DsbA 是一种如此强大的蛋白质氧化剂？和 2) DsbB 的催化特性是什么？ 特异性氧化DsbA？ 提出了两种不同的模型 解释 Dsba 活性位点二硫化物的极端氧化能力 相对于相关蛋白质硫氧还蛋白。 一种模型调用 二硫键应变，其他影响的静电相互作用 活性位点半胱氨酸的 pKa。 我们设计了一个多方面的 遗传、生物物理和结构方法应明确 区分这些模型。 应变和静电相互作用 在许多物质的折叠和催化功能中发挥重要作用 蛋白质。 清楚地理解这些力量在一个模型中的作用 因此，系统应该提供有价值的信息来了解他们的情况 在其他蛋白质中也有作用。 为了分析 DsbB 的氧化还原和催化特性，我们建议使用 与我们成功使用的工具非常相似的工具箱 DsbA。 这个简单的表征应该告诉我们很多关于 DsbB 重新氧化 DsbA 的方式可能会打开大门 研究二硫键形成如何与细胞相关 代谢。 二硫键形成是少数能够形成二硫键的共价修饰之一 发生在蛋白质折叠中。 这使我们能够用以下方式表达我们的问题 简单的生化术语。 我们认为我们现在有潜力 了解DsbA-DsbB二硫化物催化机的功能。