MEMBRANE PROTEIN STABILITY, SOLUBILIZATION, AND REFOLDING

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在酵母和大肠杆菌中表达完整的膜蛋白的努力在很大程度上是不成功的，这些蛋白通常用于生产可溶性蛋白。因此，我们寻求开发利用大肠杆菌生产膜蛋白的方法，而不需要专门的设备或大量的资本支出。从大肠杆菌中回收活性膜蛋白的效率低下是由于缺乏对链内相互作用、折叠途径、与脂类和洗涤剂的相互作用以及稳定性决定因素的了解，特别是与可溶性蛋白相比。尽管迫切需要更好的方法来表达和研究这些蛋白质，但几乎没有系统的研究来确定复性尝试失败的原因。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颖的复性策略，从而能够从大肠杆菌中大规模生产和回收活性膜蛋白。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的“黄金标准”。我们还将建立一种策略，以选择最适合纯化、增溶和稳定天然结构的洗涤剂和脂类。除了指导重新折叠战略外，这些信息本身也将是有用的。同时，我们将研究在大肠杆菌中产生的以非活性形式作为包涵体的膜蛋白的构象和性质。我们发现，这些蛋白质经常折叠成与活性状态非常相似的构象。这种构象的性质，再加上我们对天然状态的折叠和稳定性的了解，将指导我们开发有效地复性失活的膜蛋白的方法。我们的结果将增加对膜蛋白插入、折叠以及与洗涤剂和脂类相互作用的机制的了解，并将促进在大肠杆菌中生产大量具有活性的完整膜蛋白。

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.