Characterization of Outer Membrane Interactions, Dimerization Behavior and Structure of the Escherichia coli Inner Membrane Protein TonB
Staley, Jacob, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Cafiso, David, AS-Chemistry, University of Virginia
In order to survive and proliferate, Gram-negative bacteria must import large, rare nutrients such as siderophores and vitamin B12 across the protective barrier of their outer membrane. They have evolved a transport system called the TonB-dependent transport system involving the inner membrane protein TonB to achieve this. It is called this because TonB-dependent transporters in the outer membrane require energy from the inner membrane proton motive force to function, which is transduced through TonB. In this study, the behavior of the E. coli TonB protein in the presence of purified E. coli outer membranes was monitored through site-directed spin labeling and continuous wave/ pulsed EPR techniques. First, distance measurements were performed at multiple sites within the protein to monitor changes in distance distributions of the TonB homodimer in the presence of purified outer membranes. Sedimentation assays were also performed to monitor binding interactions between TonB and non-specific targets in the outer membrane. Lastly, the rigid polyproline region of TonB has been shown to form a trans polyproline II helix in vitro. Pulsed EPR was used to assess whether this helix is able to undergo isomerization in vitro to a cis polyproline I helix in the presence of aliphatic alcohol and reduce in total helix length.
The work in this study shows that in the presence of TonB the homodimer is able to undergo different conformations that are not explained by previously published crystal structures. TonB also appears to show non-specific binding interactions with purified E. coli outer membrane. Most significantly, the polyproline II helix that spans the periplasm in vivo possesses the ability to isomerize to a polyproline I helix, which is a mechanism of action that supports the pulling model of energy transduction.
MS (Master of Science)
TonB, Outer Membrane Transport, TBDT, Polyproline, EPR, DEER