Iron is essential for life and growth in most organisms. Although it is abundant, iron exists mostly as insoluble iron-oxyhydroxide. Bacteria secrete siderophores to chelate iron and transport it into the cell via specific outer membrane receptors. The FhuA receptor protein transports ferrichrome, a siderophore produced by Ustilago sphaerogena. We determined the binding affinity of variants from the conserved 'lock region' of FhuA and also created and characterized variants of the highly conserved R452 to determine its role in ferrichrome transport. We hypothesize that during transport the plug domain of FhuA does not leave the barrel; rather it undergoes a conformational change to form a channel. We mutated selected amino acids to cysteine to form disulfide bonds to tether the plug, preventing its displacement or unfolding during transport. The tetra-cysteine mutant 72/615/109/356C was able to bind and transport radiolabeled ferrichrome. One double-cysteine mutant, 104/149C, was purified for crystallization.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:etd-3144 |
| Date | 09 May 2009 |
| Creators | Cooke, Jennifer K. |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
| Rights | Copyright by the authors. |
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