Membrane Protein Folding and Assembly

膜蛋白折叠和组装

• 批准号: 10411888
• 负责人: STEPHEN H. WHITE
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10411888
• 关键词: ATP phosphohydrolase; Biological; Biological Models; Cells; Chimeric Proteins; Cryoelectron Microscopy; Cystic Fibrosis; Cytoplasm; Cytoplasmic Tail; Disease; Drug Targeting; Electrons; Engineering; Escherichia coli; Foundations; Goals; Growth; Hydrophobicity; Lipids; Membrane; Membrane Proteins; Methods; Motor; Paper; Pathway interactions; Peptide Hydrolases; Process; Protein Family; Proteins; Signal Recognition Particle; Structure; System; Temperature; Work; experimental study; human disease; in vivo; in vivo Model; insight; molecular dynamics; nanodisk; periplasm; protein folding; protein misfolding; secretion process

Save date: 15 Jan 2020, 22:40 ABSTRACT Membrane Protein Folding and Assembly Many human diseases, such as cystic fibrosis, result from misfolding of membrane proteins (MPs) during their synthesis and targeting. It is therefore important to understand the principles and mechanism of MP folding and assembly. A largely unexplored part of the problem is to understand folding in the context of the cellular milieu. Toward that goal, we are studying the targeting, secretion, and insertion of membrane proteins along the so-called SecA post- translational pathway of living Escherichia coli. We have shown that the SecA motor ATPase, a significant drug target, can insert single-span membrane proteins (S-SMPs) across the E. coli inner membrane. This simplified in vivo model system eliminates the many unanswered questions about the folding of multi-span MPs along the signal recognition particle (SRP) pathway, because we gain direct access to the translocon-bilayer partitioning process. We have engineered two different chimeric protein families for probing systematically S- SMP stability using TM segments of the form GGPG-H-GPGG (used in an earlier study to determine a biological hydrophobicity scale using a cell-free eukaryotic system). To determine stabilities, we have developed methods for cleaving TM segments in vivo via native intramembrane proteases. We have discovered that many S-SMPs are stable across the membrane only because their periplasmic & cytoplasmic domains cannot cross the membrane. We have also discovered that translocon-to-membrane transfer energetics are not equal to membrane-to-cytoplasm transfer energetics and that stability depends upon growth temperature. An important aspect of our work is the use of Molecular Dynamics simulations in concert with experiments to understand the dynamics of the SecYEG translocon. Little is known about SecA function at the atomic level despite hundreds of papers on the subject. Calling upon our lab’s expertise in lipid-protein interactions, we have laid the foundation for electron cryomicroscopic (cryo-EM) studies of the structure of SecA bound to lipid nanodiscs. Our ambition is to obtain a complete structural view of the SecA-guided secretion process. SWhite_Abstract_MIRA_2020.docx, 15 January 2020

保存日期：2020年1月15日22：40 摘要 膜蛋白的折叠和组装 许多人类疾病，如囊性纤维化，都是由膜蛋白的错误折叠引起的。 (MPS)在合成和靶向过程中。因此，理解这些原则是很重要的 以及MP折叠和组装的机理。这个问题的一个很大程度上未被探索的部分是 在细胞环境的背景下理解折叠。为了实现这个目标，我们正在研究 膜蛋白的靶向、分泌和插入沿所谓的SECA后- 活体大肠杆菌的翻译途径。我们已经证明了赛卡马达ATPase， 一个重要的药物靶点，可以在大肠杆菌中插入单跨膜蛋白(S-SMPS) 内膜。这一简化的体内模型系统消除了许多未回答的问题 关于多跨MPS沿信号识别粒子(SRP)折叠的问题 途径，因为我们获得了直接进入转位-双层分割过程的途径。 我们已经设计了两个不同的嵌合蛋白家族来系统地探测S- 使用GGPG-H-GPGG形式的TM片段的SMP稳定性(在早期的研究中使用 使用无细胞真核系统确定生物疏水性等级)。要确定 稳定性，我们已经开发出在体内通过天然切割TM片段的方法 膜内蛋白酶。我们发现，许多S-SMP在全国范围内是稳定的 仅因为它们的周质和细胞质区域不能穿过膜。 我们还发现，转位子到膜的转移能不等于 膜到细胞质转移的能量学及其稳定性依赖于生长 温度。我们工作的一个重要方面是使用分子动力学模拟 结合实验了解SecYEG易位子的动力学。鲜为人知 关于Seca在原子水平上的作用，尽管有数百篇关于这个主题的论文。叫唤 基于我们实验室在脂质-蛋白质相互作用方面的专业知识，我们已经为电子 冷冻显微镜(Cryo-EM)研究与脂质纳米盘结合的SecA的结构。我们的 野心是对SecA引导的分泌过程获得一个完整的结构视图。 SWhite_Abstract_Mira_2020.docx，2020年1月15日