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：15 Jan 2020，22：40 摘要 膜蛋白折叠与组装 许多人类疾病，如囊性纤维化，是由膜蛋白的错误折叠引起的 (MPs)在合成和定位过程中。因此，理解这些原则非常重要。 以及MP折叠和组装的机构。问题的一个基本上未被探索的部分是 在细胞环境中理解折叠。为了实现这一目标，我们正在研究 沿着所谓的SecA后膜蛋白的靶向、分泌和插入 活大肠杆菌的翻译途径。我们已经证明SecA马达ATP酶， 作为一个重要的药物靶点，它可以将单跨膜蛋白（S-SMPs）插入E.杆菌 内膜这种简化的体内模型系统消除了许多悬而未决的问题， 关于多跨度MP沿着信号识别颗粒（SRP）折叠的问题 途径，因为我们可以直接进入translocon-双层分配过程。 我们设计了两个不同的嵌合蛋白家族，用于系统地探测S- 使用GGPG-H-GPGG形式的TM片段的SMP稳定性（用于早期研究， 使用无细胞真核系统测定生物疏水性等级）。以确定 为了提高稳定性，我们已经开发了通过天然的方法在体内切割TM片段的方法。 膜内蛋白酶我们已经发现，许多S-SMP在整个 它们的细胞质和周质结构域不能穿过细胞膜。 我们还发现，translocon-膜转移能量不等于 膜到细胞质传递能量，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， 与实验一致，以了解SecYEG易位子的动力学。知之甚少 关于SecA在原子水平上的功能，尽管有数百篇关于这个主题的论文。调用 凭借我们实验室在脂质与蛋白质相互作用方面的专业知识，我们为电子奠定了基础 结合到脂质纳米盘的SecA的结构的低温显微镜（cryo-EM）研究。我们 目标是获得SecA引导的分泌过程的完整结构视图。 SWhite_Abstract_MIRA_2020.docx，2020年1月15日