"Membrane proteins count for 2530%0303 of all proteins and carry out a variety of critical biological processes, such as nutrient transport, signal transduction, catalysis and generation of metabolic energy. Despite the importance, understandings of membrane protein folding lag far behind those of water-soluble proteins. The knowledge gap stems from inherent difficulties in controlling the reversible folding of membrane proteins in lipid bilayers, which is necessary for thermodynamic analysis of driving forces and mechanisms of folding. Steric trapping is a promising tool to reversibly control membrane protein folding. It utilizes the strong binding affinity between the biotin affinity tag and the bulky tag-binding protein streptavidin. In my Ph.D. research, I developed an array of novel methods by synthesizing a set of novel biotinylated protein probes, advancing the steric trapping method for a general application. Applying those methods to studying the folding of a helical-bundle membrane protein, rhomboid protease GlpG in detergent micelles, I mapped its folding energy landscape by revealing subglobal unfolding of the region encompassing the active sites, and quantifying a network of cooperative and localized interactions to maintain the stability. Combining computational methods, I elucidated the role of packing interactions in the stability and function of GlpG, showing that the advanced steric trap method can be used for studying driving forces in membrane protein folding. By using the novel biotinylated spin label, I was able to determine the inter-spin distance between the two biotinylated sites in the sterically trapped denatured state by double electron-electron resonance spectroscopy in the native lipid bilayer environments. These novel steric trapping methods can be applied to investigate a variety of problems in the folding and stability of membrane proteins directly under native lipid and solvent conditions."--Pages ii-iii.
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