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Title: Investigations on an iron transport pathway
Author: Bilton, Paul
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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In this study, an iron-uptake ATP-binding cassette (ABC) transporter, FbpABC, from the Gram-negative human pathogen Neisseria gonorrhoeae has been investigated. The periplasmic binding protein, FbpA, has been mutated to facilitate investigation of complex formation with the two other components of the transporter, FbpB and FbpC. FbpBC was PCR-amplified from N. gonorrhoeae genomic DNA, co-expressed in E. coli cells, and its purification optimised. FbpA residues R48 and V272 have been mutated to allow covalent modification with a linker molecule to allow attachment to a sensor surface and used as bait to pull-down the purified FbpBC complex, using surface plasmon resonance. This is the first report of the purification of FbpB, and the first demonstration of FbpABC association in vitro. A radioactive iron-uptake assay revealed that E. coli cells expressing the N. gonorrhoeae FbpABC operon take-up more iron than control cells. The FbpC gene was cloned using recombinant DNA technology and over-expressed in E. coli to perform biochemical characterisation of the enzyme. The expression and purification of selenomethionine-labelled FbpC allowed determination of the X-ray crystal structure of FbpC at Imperial College, London. Crystals of FbpC diffracted to a resolution of 2.7 Å, and the structure is currently being solved. Metal-free apo-FbpA was reloaded with ruthenium(III) and osmium(III) salts and characterised by various techniques. Chromatography and ICP-OES analysis of Ru- and Os-FbpA showed that there were two main species formed with each metal. Both species have four metal atoms bound and differ in that one species may be capped by a single phosphate anion, and the other by two phosphate anions. EXAFS was also performed on the ruthenium bound species.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available