Kerr, RA; (2013) Ion mobility & mass spectrometric studies of macromolecules required for organism viability. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Studies of gas phase protein complexes using mass spectrometry and ion-mobility mass spectrometry are becoming increasingly commonplace in the field of structural biology. These studies apply the combined speed, mass sensitivity and structural resolution of a single instrumental method, that of mass spectrometry. Work presented here has used a range of gas, and solution phase methods. These methods have made it possible to investigate the oligomers and structural conformations of three proteins required, within their respective organisms, to ensure viability. Mutations of the human serine protease inhibitor, α1-antitrypsin, are known to promote polymerogenic intermediates under biologically relevant conditions. Using ion-mobility mass spectrometry we have characterised the structure and stability of the K154N slow polymerisation mutant. The results obtained have shown that this mutant populates an increased stability structural intermediate upon incubation at biologically relevant temperatures. Saccharomyces cerevisiae Sgt1 dimers mediate binding between Hsp90 and Skp1, to initiate chromosome separation. Mutations of the Sgt1 dimerization domain are known to inhibit Sgt1: Skp1 binding, arresting the cell cycle at the G2/M interface. Work here has shown that this dimer is stabilised by an Ascomycota specific structural loop within the dimerisation domain, with potential contributions from other domains. Using tandem mass spectrometry, we have shown that Sgt1 dimers do not represent the structural minimum for Skp1 binding. The Escherichia coli DNA binding protein, CbpA, promotes chromosome compaction that arrests the cell cycle during the stationary phase, and phosphate starvation conditions, producing structural aggregates exceeding 60 nm. The organisation of both CbpA and DNA within these aggregates remains currently unresolved therefore, mass spectrometry combined with ion-mobility mass spectrometry has been applied to resolve these interactions. Although unsuccessful in observing gas phase CbpA-DNA oligomers using a variety of conditions and methods, investigations have produced ion-mobility constraints for computational modelling of the biologically relevant CbpA dimer.

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