For Escherichia coli, uptake of iron into the cells require FhuA, a TonB-dependent outer membrane receptor. The periplasmic protein FhuD then binds TonB and transfers iron to the cytoplasmic membrane-associated permease FhuB/C. Phage display results predict complementary binding sites on both TonB and FhuD, advocating the need for direct evidence of these interactions. Analytical ultracentrifugation results indicated that both FhuD and TonB were predominantly monomeric in Hepes-NaCl buffer. SEDFIT analysis agreed with their theoretical molecular weights of 32 kDa and 25 kDa, respectively. TonB sedimented at 1.7 S and FhuD at 2.2 S. Frictional ratios confirmed that TonB is elongated while FhuD is globular in solution. For surface plasmon resonance experiments, TonB with a cysteine residue introduced at its N-terminus was immobilized using thiol or amine coupling methods on Biacore sensor chips. Kinetic modeling of FhuD interactions with immobilized TonB indicated that this is a high-affinity interaction (ie. low nanomolar KD) with 1:1 binding stoichiometry. In the presence of ferricrocin, the degree of binding was not altered significantly. Overall, the present data provide the first direct evidence of a specific binding interaction between FhuD and TonB. FhuD has a higher affinity for TonB in absence of siderophores. This suggests that FhuD might be released from TonB upon binding of siderophores, thereby efficiently shuttling ligands to FhuB/C complex on the cytoplasmic membrane.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.101722 |
| Date | January 2007 |
| Creators | Lee, Jongchan, 1981- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Microbiology and Immunology.) |
| Rights | © Jongchan Lee, 2007 |
| Relation | alephsysno: 002598753, proquestno: AAIMR32837, Theses scanned by UMI/ProQuest. |
Page generated in 0.0017 seconds