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Development and Application of Whole Cell and Intact Outer-Membranes for Double Electron-Electron Resonance on Membrane Proteins

Sikora, Arthur
Thesis/Dissertation; Online
Sikora, Arthur
Cafiso, David
In order to fully understand the function of a protein its native environment must be taken into account. Native membranes contain many unique features inaccessible in reconstituted systems that may greatly influence membrane protein function. Despite its importance, an unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. BtuB, the Escherichia coli outer membrane TonB-dependent cyanocobalamin transporter has been widely studied but its transport mechanism is not fully understood. TonB dependent transporters are essential for the success of many pathogenic bacteria, making these interesting targets for new antibiotics. These proteins are a family of outer membrane β-barrels that Gram-negative bacteria use to transport essential nutrients such as vitamin B12 or iron. Although high-resolution crystal structures have been obtained for many of these proteins, the mechanism of substrate transport is still unclear. The outer membrane environment is low in free reactive cysteines and thus provides low background signal for Double Electron-Electron Resonance (DEER) measurements DEER is a well-established technique to follow conformational changes in purified membrane protein complexes. This work details the development of DEER in whole living cells and intact outer membranes. Our approach avoids detergent extraction, purification and reconstitution usually required for these systems. With this approach structure, function, conformational changes and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment. We then observe and characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the DEER data with high-resolution crystal structures. These comparisons reveal that ligand binding in whole cells is reflective of previous studies done in reconstituted systems. Finally using the native-system DEER technique we show that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the second extracellular loop to create a more open loop configuration, and it diminishes the affinity of BtuB for substrate. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, a feature that appears to be critical to the TonB-dependent transport mechanism.
University of Virginia, Department of Chemistry, PHD (Doctor of Philosophy), 2016
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PHD (Doctor of Philosophy)
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