How are Disulfides transported across membranes?

超出提供的空间。二硫键的正确形成对于具有多个二硫键的蛋白质的正确折叠至关重要，包括许多具有药理意义的蛋白质。在过去的几年里，对体内二硫化物氧化机理的了解已经取得了很大的进展，但对于错误形成的二硫化物的还原和异构化机理还不是很清楚。我们最近在体外成功地重建了异构化/还原途径。这种重组为对二硫化物校正过程进行广泛的生物化学和机械分析开辟了道路。DsbC是一种周质蛋白，被认为可以使错误折叠的蛋白异构化。为了保持活性，DsbC必须在非常氧化的周质环境中保持还原。DsbD降低DsbC。DsbD又被细胞质中的硫氧还蛋白还原。我们的目标是解决二硫键如何通过膜运输这一有趣而长期存在的拓扑难题。我们已经成功地确定了DsbD的各个结构域之间以及DsbD、DsbC和硫氧还蛋白之间的电子流动方向。现在我们必须确定二硫化物实际上是如何通过膜运输的。这种传输过程可以被认为是电子向外流动或二硫化物向内流动。我们将测试两个DsbD作用模型，一个DsbD通过简单的二硫键交换反应发挥作用，第二个模型也涉及辅因子依赖的电子传递。为此，我们重点研究通过DsbD的[3]结构域的电子流动，这是跨越膜的结构域。这个结构域似乎包含一个铁辅因子。我们将研究这种铁在DsbD催化的电子传递中的作用。在体内定义DsbC和DsbG底物的努力将给我们提供工具来解决长期存在的问题，即二硫键异构化实际上是如何在体内发生的。总体而言，我们的目标是详细了解DsbC-DsbD二硫键异构酶/还原酶机器的功能，特别是DsbD蛋白如何跨膜运输二硫化物。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The correct formation of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides, including many proteins of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo, but the mechanism of reduction and isomerization of incorrectly formed disulfides is less clear. We have recently succeeded in the reconstitution of the isomerization/reduction pathway in vitro. This reconstitution opens up the way to extensive biochemical and mechanistic analysis of the process of disulfide correction. DsbC is a periplasmic protein that is thought to isomerize misfolded proteins. In order to be active, DsbC must be kept reduced in the very oxidizing periplasmic environment. DsbD reduces DsbC. DsbD in turn is reduced by the cytoplasmic thioredoxin. We aim to solve the interesting and long-standing topological puzzle of how disulfide bonds are transported through membranes. We have succeeded in defining the direction of electron flow between the individual domains of DsbD, and between DsbD, DsbC, and thioredoxin. Now we must determine how disulfides actually are transported through the membrane. This transport process can be thought of as an outward flow of electrons or an inward flow of disulfides. We will test two models for DsbD action, one where DsbD works via simple disulfide exchange reactions, and the second where cofactor dependent electron transport is also involved. To do this we focus on examining electron flow through the [3 domain of DsbD which is the domain that spans the membrane. This domain appears to contain an iron cofactor. We will examine the role of this iron in DsbD catalyzed electron transport. Efforts to define in vivo substrates for DsbC and DsbG will give us tools to cventually address the long-standing question of how disulfide isomerization actually occurs in vivo. Overall, we aim to understand in detail the function of the DsbC-DsbD disulfide isomerase/reductase machine and in particular how disulfides are transported across membranes by the DsbD protein. PERFORMANCE SITE ========================================Section End===========================================