Probing TonB-dependent transporters reconstituted in planar lipid bilayers
Udho, Eshwar Bansraj
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The siderophore-bound iron and vitamin B12 transporters, called TonB-dependent transporters (TBDTs), are gram negative outer membrane proteins consisting of a 22-stranded beta-barrel with an amino terminal plug domain that occludes this beta-barrel by folding into it. These proteins interact with the TonB protein of the inner membrane TonB complex to harness the inner membrane's proton motive force to form a pathway through the lumen of the beta-barrel to allow siderophore or vitamin B12 permeation.;This thesis is concerned with the reconstitution and properties of three TBDTs---FhuA, Cir, and BtuB---in planar phospholipid bilayer membranes. I show that in order for them to be inserted into the membrane, an osmotic gradient (provided by either 3 M glycerol or 4 M urea) must be present across the membrane (insertion is bidirectional into the membrane), and that once inserted into the membrane, they are opened by the denaturing property of 4 M urea. The opening is partly reversible with the removal of urea in the case of FhuA and Cir. Physiological concentrations of ligands (ferrichrome for FhuA and colicin Ia for Cir) were found able to influence transporter behavior in the presence of 4 M urea, suggesting that the transporters retain physiological structure and function on the membrane.;I exposed FhuA to trypsin in the presence of 4 M urea on the bilayer and found that trypsin exposure prevented the loss of conductance upon urea removal, indicating that trypsin may be cleaving the plug as it comes out of the barrel. The first 67 residues of FhuA were found to be trypsin accessible in the presence of 4 M urea as assessed by gel-shift and mass spectrometry assays. I prepared plugless FhuA barrels and found that they could induce conductance across the membrane in the presence of either cis 3 M glycerol or cis 4 M urea, a conductance that is partly lost with the addition of either cis or trans FhuA plug, suggesting that the exogenous plug can enter an empty barrel. This FhuA plug was found by NMR and circular dichroism to be unfolded in solution.
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