UBC Theses and Dissertations
Development of methods for study of membrane proteins in the absence of detergents. Carlson, Michael
Membrane proteins are sequestered in a hydrophobic lipid environment, and are therefore resistant to characterization by traditional biochemical techniques which occur in aqueous solution. Traditionally, detergents have been used to solubilize membrane proteins, but these surfactants have detrimental effects on protein form and function. In this thesis, the form, function, and potential mechanism of membrane protein complexes are investigated in detergent-free buffer. Our first study is motivated by the lack of flexible reconstitution scaffolds that maintain a lipid-protein environment for membrane protein stabilization. To this end, we design the peptidisc, a simple method for the universal stabilization of membrane protein complexes in detergent-free solution. Analysis of 5 different membrane protein complexes reconstituted in peptidiscs demonstrate that the method maintains function and increases stability of incorporated membrane proteins. We extend the method to reconstitute the entire membrane proteome of the bacterium Escherichia coli, demonstrating the peptidisc as a “one-size fits all” scaffold. We measure the co-fractionation of reconstituted proteins to identify proteins complexes captured in the peptidisc. The method provides a high-throughput, detergent free approach for identifying the protein-protein interactions of a biological membrane. In the final study, we discover that conformational flexibility of the maltose importer MalFGK2 chloride channel activity in the maltose importer MalFGK2 is linked directly to the transient chloride channel activity. This finding provides support for the expanded alternating access model, which include discrete substeps in the transport model. I discuss the ramifications of the expanded alternating access model on membrane transporter and channel evolution and function. In addition, the development of the peptidisc method is discussed in the broader context of current, detergent free methods for analysis of membrane protein structure, function and interactions.
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