Doctor of Philosophy (PhD)
Department of Chemistry - Faculty of Science
Watt, Stephen J, Use of electrospray ionization mass spectrometry to study protein conformation and protein-protein interactions, PhD thesis, Department of Chemistry, University of Wollongong, 2005. http://ro.auo.edu.au/theses/582
The polymerisation of a polypeptide chain from an encoded genetic sequence allows the formation of structured molecules, known as proteins. These are essential components for a range of processes including molecular recognition, DNA replication and enzymatic functions. In this thesis, the ability of electrospray ionization (ESI) mass spectrometry (MS) to be used as a tool to determine functional properties of proteins has been explored. The coupling of ESI-MS to hydrogen deuterium exchange has been used to show how restriction of the C- and N-termini by cyclization of the polypeptide backbone can affect the ability of a protein to sample unfolded or partially unfolded states. The development of appropriate methodologies for analysis of linear (uncyclized) and cyclized systems identified a slowing of the rate of unfolding due to cyclization. The implication for the unfolding processes of proteins are discussed. An increased thermal stability of the cyclized protein was also demonstrated. This property was used to analyse the ability of ESI-MS to identify changes in protein structure from shifts in ion distributions. Important observations regarding the polarity of ionization used in these experiments are highlighted. The effect opposite polarity ionization has on the ability to detect conformational changes in proteins and interactions with small ligands was explored using the well-characterized calmodulin-calcium-antipsychotic drug system. Important considerations regarding the binding of metal ions to protein structures are discussed in relation to the ability to unequivocally identify a conformational transition in protein structure from ESI mass spectra. An inability to detect complexes of calcium loadedcalmodulin with the antispychotic drug trifluroperazine in the negative ion mode was observed, a result believed to be due to the Coulombic repulsions between acidic residues of calmodulin. Finally, the non-covalent complex and interactions of the E. coli helicase (DnaB) were probed by nanoESI-MS and MS/MS studies. Development of suitable conditions allowed for identification of a previously unresolved heptamer in addition to the expected hexamer. The interaction of DnaB with its loading partner DnaC and the possible roles of ATP and ADP in this interaction were also probed with findings being related to the biological functions of these proteins.
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