How are Disulfides transported across membranes?

二硫化物如何跨膜运输？

• 批准号: 6837677
• 负责人: JAMES BARDWELL
• 金额: $ 19.98万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6837677
• 关键词: How; Disulfides; transported; across; membranes

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===========================================

超出所提供的空间。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，包括许多具有药理学重要性的蛋白质。在过去的几年里，在理解体内二硫化物氧化的机制方面取得了很大的进展，但是不正确形成的二硫化物的还原和异构化的机制还不太清楚。我们最近成功地在体外重建的异构化/还原途径。这种重建开辟了广泛的生化和机械分析的过程中的二硫键校正的方式。DsbC是一种周质蛋白，被认为是异构化错误折叠的蛋白质。为了保持活性，DsbC必须在氧化性很强的周质环境中保持还原状态。DsbD降低DsbC。DsbD又被细胞质硫氧还蛋白还原。我们的目标是解决有趣的和长期存在的拓扑难题二硫键如何通过膜运输。我们已经成功地确定了DsbD的各个结构域之间，DsbD，DsbC和硫氧还蛋白之间的电子流方向。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的向外流动或二硫化物的向内流动。我们将测试两个模型DsbD行动，一个DsbD工程通过简单的二硫键交换反应，第二个辅因子依赖的电子传递也参与。为了做到这一点，我们专注于检查通过DsbD的[3]结构域的电子流，该结构域跨越膜。该结构域似乎含有铁辅因子。我们将研究这种铁在DsbD催化的电子传递中的作用。确定DsbC和DsbG的体内底物的努力将为我们提供工具，最终解决长期存在的问题，即二硫键异构化在体内实际上是如何发生的。总的来说，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，特别是二硫键是如何通过DsbD蛋白跨膜转运的。性能现场=