The biophysical properties, biological activity and function of macromolecular systems are highly dependent on their structure. Structure-activity relationships of proteins and their binding partners are critical for drug discovery, biochemical and medical research. While the gas-phase environment might present as an unusual venue from which to explore protein structure, for over the past two decades, nano-electrospray ionization (nESI) coupled to mass spectrometry (MS) has been recognized as having great potential for analysis of protein structure and protein non-covalent complexes. In conjunction with related technique of ion mobility (IM), mass spectrometry (IM-MS) provides insights into protein native-like conformations and any structural changes in may undergo upon ligand binding or alternations induced via physical parameters such as temperature, pressure or solution conditions. As most proteins tend to exist as multiple domains; from the distribution of oligomeric states in the Protein Data Base (PDB) 86% of proteins exist as oligomers; the work presented in this thesis focuses on application of MS techniques to probe the tertiary and quaternary structure of various large and multimeric protein complexes, their dynamics and/or conformational changes. Wherever relevant, the gas-phase studies reported here are complemented by other techniques, such as hydrogen deuterium exchange MS (HDX), molecular modelling (MD) and analytical ultracentrifugation (AUC). Firstly, the dynamics of intact monoclonal antibodies (mAbs) and their fragments are explored with IM-MS. Variations observed in conformational landscapes occupied by two mAb isotypes are rationalized by differences in disulfide linkages and non-covalent interactions between the antibody peptide chains. Moreover, mAb intrinsic flexibility is compared to other multimeric protein complexes in terms of collision cross section distribution span. Secondly, variable temperature MS (VT-MS) and variable temperature IM-MS (IM-MS) are used to probe unfolding and dissociation of four standard multimeric protein complexes (TTR, avidin, conA and SAP) as a function of the of analysis environment temperature. VT-MS is found to allow for decoupling of their melting temperature (Tm) from the protein complex dissociation temperature (TGPD). Whereas, VT-IM-MS is used to investigate structural changes of these protein complexes at elevated temperatures and provide insights into the thermally induced dissociation (TID) mechanism, as well as strength of the non-covalent interactions between subunits. Thirdly, VT-(IM)-MS methodology is applied to study behaviour of three mAbs: IgG1, IgG4 and an engineered IgG4 of increased thermal stability. Such analysis shows to be promising for comparative thermal stability studies for proteins of therapeutic interest. Lastly, the structure of ATP-phosphoribosyltransferase (MtATPPRT), an enzyme catalysing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, is explored. Conformational changes occurring upon feedback allosteric inhibition by L-histidine are probed with MS, IM-MS, HDX-MS and AUC. Reported results serve as the basis for IM-MS/HDX-MS based screening method to be used for screening of a library of novel and promising anti-tuberculosis agents.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:712270 |
| Date | January 2015 |
| Creators | Pacholarz, Kamila Jolanta |
| Contributors | Hulme, Alison ; Barlow, Paul |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/21084 |
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