MEMBRANE PROTEIN STABILITY, SOLUBILIZATION, AND REFOLDING

膜蛋白稳定性、溶解和重折叠

• 批准号: 7381190
• 负责人: Clifford Robinson
• 金额: $ 29.5万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381190
• 关键词: MEMBRANE; PROTEIN; STABILITY; SOLUBILIZATION; REFOLDING

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Efforts to express integral membrane proteins in yeast and E. coli, commonly used to produce soluble proteins, have been largely unsuccessful. Thus, we seek to develop approaches that use E. coli for membrane protein production that do not require specialized equipment or large capital outlays. Inefficiencies of active membrane protein recovery from E. coli arise from a lack of knowledge of intrachain interactions, folding pathways, interactions with lipids and detergents, and determinants of stability, especially as compared with soluble proteins. Despite the clear and urgent need for better methods to express and study these proteins, there have been few if any systematic studies to identify why refolding attempts fail. We believe that understanding the conformations and stabilities of native and inactive states will allow us to develop improved and novel refolding strategies that will enable large-scale production and recovery of active membrane proteins from E. coli. Our approach starts with an analysis of the stability and properties of native, active, correctly folded membrane proteins, which will be used as the "gold standard" in our analysis. We will also establish a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure. In addition to guiding refolding strategies, this information will be useful in its own right. In parallel, we will study the conformation and properties of membrane proteins produced in E. coli in inactive forms as inclusion bodies. We have found that these proteins often are folded into a conformation that closely resembles the active state. The properties of this conformation, together with our knowledge of the folding and stability of the native state, will guide our development of methods to refold inactive membrane proteins efficiently. Our results will provide increased knowledge of the mechanism of membrane protein insertion, folding, and interactions with detergents and lipids, and will facilitate production of significant quantities of active integral membrane proteins in E. coli.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在酵母和E.通常用于生产可溶性蛋白质的大肠杆菌基本上不成功。因此，我们寻求开发使用E.大肠杆菌的膜蛋白生产，不需要专门的设备或大量的资本支出。从E.大肠杆菌中的蛋白质是由于缺乏对链内相互作用、折叠途径、与脂质和去污剂的相互作用以及稳定性决定因素（特别是与可溶性蛋白质相比）的了解而产生的。尽管明确和迫切需要更好的方法来表达和研究这些蛋白质，但很少有系统的研究来确定为什么重折叠尝试失败。我们相信，了解天然和非活性状态的构象和稳定性将使我们能够开发改进的和新的重折叠策略，这将使大规模生产和回收的活性膜蛋白从大肠杆菌。杆菌我们的方法从分析天然的、活性的、正确折叠的膜蛋白的稳定性和性质开始，这将被用作我们分析中的“金标准”。我们还将建立一个策略，选择洗涤剂和脂质的纯化，溶解和稳定的天然结构的最佳。除了指导重折叠策略外，这些信息本身也很有用。同时，我们将研究E.大肠杆菌中以包涵体形式存在。我们发现，这些蛋白质通常折叠成与活性状态非常相似的构象。这种构象的性质，连同我们的知识的折叠和稳定性的天然状态，将指导我们的方法的发展，以重新折叠非活性膜蛋白有效。我们的研究结果将为膜蛋白插入、折叠以及与去污剂和脂质相互作用的机制提供更多的知识，并将促进E.杆菌