Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582277
Title: Investigating the interactions and conformations of metalloproteins by means of mass spectrometry
Author: Kondrat, Frances D. L.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2012
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Abstract:
The use of mass spectrometry for the study of proteins and their non-covalent interactions has expanded rapidly since the introduction of ionisation techniques capable of transferring intact complexes into the gas phase. The coupling of the shape selective technique of ion mobility to mass spectrometry has advanced the ustility of these techniques for structural biology studies. The work presented in this thesis showcases the use of mass spectrometry for the study of proteins and their interaction partners including metal ions, proteins and DNA. SmtB is a homodimeric, DNA binding protein capable of binding zinc ions with high affinity. SmtB functions as a transcriptional repressor in the absence of zinc ions but has the ability to reduce its DNA binding affinity upon a quaternary conformational change induced by the binding of zinc ions. In this fashion SmtB acts as a zinc sensor allowing the zinc chelator SmtA to be produced under zinc stress conditions. Mass spectrometry has been used to probe the zinc binding stoichiometry and monomer/ dimer equilibrium of a recombinant SmtB sample expressed in the presence of added zinc ions. These experiments confirmed that, in the presence of zinc ions, SmtB predominantly existed as a homodimer with four zinc ions bound. The complementary technique of ICP-OES confirmed that the zinc binding stoichiometry of the SmtB dimer was clearly higher than the value which had been presented in the literature. A variety of different zinc ion removal techniques were implemented to investigate the removal of zinc ions from SmtB together with any associated change in monomer/dimer equilibrium and dimeric conformation. These were characterised by using mass spectrometry and ion mobility mass spectrometry respectively. The removal of zinc ions was found to promote expansion and unfolding of the dimer resulting in the production of monomeric species. The locations of the zinc binding sites were then studied using tandem mass spectrometry. The interaction of apo-SmtB, produced by dialysis under anaerobic conditions, with a double stranded DNA oligomer containing a SmtB binding site, was successfully transferred into the gas phase. The resulting complex was found to exist in two different stoichiometries, SmtB2:DNA and 2SmtB2:DNA, with no indication of a stepwise monomer binding mechanism. The conformational changes associated with zinc ion removal from the metallothionein SmtA were also investigated utilising ion mobility mass spectrometry. These results revealed an increase in the flexibility of SmtA during zinc ion removal along with an increase of the conformational space occupied.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council (EPSRC) ; Royal Society of Chemistry (Great Britain)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582277  DOI: Not available
Keywords: QC Physics ; QP Physiology
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