TonB Dependent Transport

Shultis, David Donahue, Department of Molecular Physiology and Biological Physics, University of Virginia
Wiener, Michael, Department of Molecular Physiology and Biological Physics, University of Virginia

TonB dependent transport is responsible for the uptake of large and/or scarce micronutrients, such as vitamin B 12 (cyanocobalamin CN-Cbl), heme, and iron siderophores, across the outer membrane of Gram-negative bacteria in an energy dependent manner. Transport requires the following cytoplasmic membrane proteins: ExbB, ExbD, TonB, and an outer membrane protein from a class of proteins called TonB Dependent Transporters (TBDT). This dissertation structural and biochemical experiments focused on providing novel information on TonB dependent transport function, and is subdivided into three parts. The first part focuses on molecular recognition between the C-terminal domain of TonB 147-239 and the outer membrane protein BtuB, a TBDT, by X-ray crystallography. Analysis of the BtuB TonB crystal structure supports a published active transport hypothesis involving mechanical force transduction and molecular rearrangement of the lumen of BtuB. The second part of the dissertation describes the structural determination of the soluble periplasmic C-terminal domain of the cytoplasmic membrane protein ExbD in the absence and presence a TonB peptide, residues 43-54: coined the "D-strand," by NMR spectroscopy and X-ray crystallography respectively. Analysis of the solution NMR structure, in light of a previously known loss of function mutation in ExbD, L132Q, was used to form the D- strand hypothesis: TonB dependent transport requires -strand recruitment between the C-terminal domain of ExbD 59-141 and TonB 43-54 . This hypothesis was confirmed by the crystallographic complex of ExbD and the TonB D-strand at 1.25 Å resolution as well as by ExbD NMR chemical shift perturbation and isothermal calorimetry titration experiments with the TonB D-strand peptide. These in vitro experimental results need to ii be confirmed by in vivo experiments that are outside the scope of this dissertation. The third part of the dissertation describes (unsuccessful) experimental efforts to determine the stoichiometry of the proteins required in TonB dependent transport by immunoprecipitation and tandem mass spectrometry from Escherichia coli (E. coli) and Neisseria gonorrhoeae (N. gonorrhoeae). This section is covered as an appendix.

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PHD (Doctor of Philosophy)
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