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 抽象的 膜蛋白折叠和组装 许多人类疾病，例如囊性纤维化，是由于膜蛋白折叠折叠而引起的 （MP）在其合成和靶向过程中。因此，重要的是要了解原理 MP折叠和组装机制。问题的很大一部分是 了解在蜂窝环境的上下文中折叠。为了实现这一目标，我们正在研究 沿所谓的SECA后膜蛋白的靶向，分泌和插入 生活大肠杆菌的转化途径。我们已经证明了SECA电机ATPase， 一个重要的药物靶标可以在大肠杆菌上插入单跨膜蛋白（S-SMP） 内膜。这种简化的体内模型系统消除了许多未回答的 关于沿信号识别粒子（SRP）折叠多跨MP的问题 途径，因为我们可以直接访问转运双层分区过程。 我们已经设计了两个不同的嵌合蛋白家族，以系统地探测S- 使用GGPG-H-GPGG形式的TM段的SMP稳定性（在早期研究中用于 使用无细胞的真核系统确定生物疏水性量表）。 系统，我们开发了通过本机在体内切割TM段的方法 膜内蛋白酶。我们发现许多S-SMP在 膜仅是因为它们的周质和细胞质结构域不能越过膜。 我们还发现，易位到膜转移能量不等于 膜到胞浆转移能量，稳定性取决于生长 温度。我们工作的一个重要方面是使用分子动力学模拟 与实验的音乐会，以了解Secyeg Clanserocon的动态。鲜为人知 关于在原子级任务中的SECA功能，数百条有关该主题的论文。打电话 根据我们实验室在脂质蛋白质相互作用方面的专业知识，我们为电子产品奠定了基础 与脂质纳米盘结合的SECA结构的冷冻显微镜（冷冻EM）研究。我们的 野心是获得SECA引导分泌过程的完整结构视图。 swhite_abstract_mira_2020.docx，2020年1月15日