FUNCTIONAL ANALYSIS OF A PROTEIN FOLDING CATALYST

蛋白质的不正确折叠至少与 包括老年痴呆症和疯牛病在内的十几种疾病。 二硫键对于蛋白质的折叠和稳定性是非常重要的 这些键的简单还原通常会导致蛋白质 展开 二硫键的形成是一个催化过程。 我们 发现涉及两种催化剂，DsbA作为直接供体， DsbB与新合成的周质蛋白质形成二硫键， 再氧化DsbA。 我们的长期目标是了解这些蛋白质 催化蛋白质折叠。 在这个项目中，我们将寻求答案， 两个基本问题：1）为什么DsbA是如此强大的蛋白质氧化剂？和 2)DsbB的催化特性是什么，使其能够 特异性氧化DsbA？ 已经提出了两种不同的模型， 解释Dsba活性部位二硫化物的极端氧化能力 相对于相关蛋白硫氧还蛋白。 一个模型调用 二硫键菌株，其他静电相互作用影响 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， 区分这些模式。 应变和静电相互作用 在许多蛋白质的折叠和催化功能中发挥重要作用， proteins. 清楚地理解这些力在一个模型中的作用 因此，系统应提供有价值信息， 在其他蛋白质中也有作用。 为了分析DsbB的氧化还原和催化性质，我们建议使用 一个工具箱，与我们成功使用的工具非常相似， DsbA。 这个简单的描述应该告诉我们很多关于 DsbB的作用方式是使DsbA再氧化， 二硫键形成如何与细胞 新陈代谢. 二硫键的形成是为数不多的共价修饰， 发生在蛋白质折叠中。 这使我们能够用短语表达我们的问题， 简单的生物化学术语 我们认为我们现在有潜力 了解DsbA-DsbB二硫化物催化机器的功能。

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.