Investigating the Signal Transduction Pathway for the Outer Membrane Transporter FecA

Yen, Cheng Huang, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Columbus, Linda, Department of Chemistry, University of Virginia
Landers, James, Department of Chemistry, University of Virginia
Venton, Jill, Department of Chemistry, University of Virginia

In Gram-negative bacteria, the outer membrane serves as a protective barrier that is permeable only to solutes smaller than 600Da. In order to import large trace nutrients into the cytoplasm, Gram-negative bacteria utilize TonB-dependent transporters. These transporters couple to the protein TonB, which presumably provides the energy for transport through the proton motive force and the inner membrane proteins ExbB and ExbD. Although the transport mechanism is not well understood, one of the common characteristics displayed by TonB-dependent transporters is the substrate-induced unfolding of an N-terminal motif known as the Ton box. The TonBdependent transporter FecA is responsible for the transport of ferric citrate as well as transcriptional auto-upregulation. Previous work performed by Ferguson et al. has identified several key residues in FecA that may constitute as a signal transduction pathway. Mutating several of these key residues results in lower levels of FecA transcription. In this study, in vitro analysis on these mutants was performed to determine whether these transcription-defective mutations will affect the substrateinduced unfolding of the Ton box region. Using site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR), spin labels were placed in the Ton box region; and the EPR line shapes were analyzed for each transcription-defective mutant. The results of the experiment indicate that the mutations that disable transcription also have an inhibiting effect on the unfolding of the Ton box. Therefore, the unfolding of the Ton box is coupled to the transcriptional activation of FecA.

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MS (Master of Science)
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