Biophysical characterization of the energy and TonB-dependence of the ferric enterobactin transport protein FepA

K-REx Repository

Show simple item record

dc.contributor.author Jordan, Lorne Donnell
dc.date.accessioned 2015-08-17T13:15:39Z
dc.date.available 2015-08-17T13:15:39Z
dc.date.issued 2015-08-01 en_US
dc.identifier.uri http://hdl.handle.net/2097/20407
dc.description.abstract The goal of the research included in this dissertation is to provide a more complete model of the role of TonB, an energy transducing protein that resides in the inner membrane and is an essential component of the iron transport of Escherichia coli under iron-starved conditions. Using fluorescent hybrid proteins, the anisotropy of TonB in the cytoplasmic membrane (CM) of Escherichia coli was determined. With the aim of understanding the bioenergetics of outer membrane (OM) iron transport, the dependence of TonB motion on the electrochemical gradient and the effect of CM proteins ExbB and ExbD on this phenomenon was monitored and analyzed. The native E. coli siderophore, enterobactin chelates Fe⁺³ in the environment and ferric enterobactin (FeEnt) enters the cell by energy- and TonB-dependent uptake through FepA, its OM transporter. The TonB-ExbBD complex in the CM is hypothesized to transfer energy to OM transporters such as FepA. We observed the polarization of GFPTonB hybrid proteins and used metabolic inhibitors (CCCP, azide and dinitrophenol) and chromosomal deletions of exbBD to study these questions. The results showed higher anisotropy (R) values for GFP-TonB in energy-depleted cells, and lower R-values in bacteria lacking ExbBD. Metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings suggest that TonB undergoes constant, energized motion in the bacterial CM, and that ExbBD mediates its coupling to the electrochemical gradient. By spectroscopic analyses of extrinsic fluorophore labeled site-directed Cys residues in 7 surface loops of Escherichia coli FepA, binding and transport of ferric enterobactin (FeEnt) was characterized. Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m-chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB-dependent metal transport. en_US
dc.description.sponsorship National Science Foundation Bridge-to-Doctorate Fellowship en_US
dc.language.iso en_US en_US
dc.publisher Kansas State University en
dc.subject Iron transport en_US
dc.subject Escherichia coli en_US
dc.subject TonB-dependent en_US
dc.subject Fluorescence anisotropy en_US
dc.subject FepA en_US
dc.subject Enterobactin en_US
dc.title Biophysical characterization of the energy and TonB-dependence of the ferric enterobactin transport protein FepA en_US
dc.type Dissertation en_US
dc.description.degree Doctor of Philosophy en_US
dc.description.level Doctoral en_US
dc.description.department Biochemistry and Molecular Biophysics en_US
dc.description.advisor Phillip E. Klebba en_US
dc.subject.umi Biochemistry (0487) en_US
dc.subject.umi Biophysics (0786) en_US
dc.date.published 2015 en_US
dc.date.graduationmonth August en_US


Files in this item

This item appears in the following Collection(s)

Show simple item record

Search K-REx


Advanced Search

Browse

My Account

Statistics








Center for the

Advancement of Digital

Scholarship

cads@k-state.edu