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.
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