Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.658961
Title: Mass spectrometry-based studies for the investigation of protein structure and dynamics
Author: Edgeworth, Matthew
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2014
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Abstract:
The use of mass spectrometry to study of proteins and their non-covalent complexes has grown during the last few decades thanks to the introduction of soft ionisation techniques capable of preserving the weak molecular bonds present in higher order protein structure. The integration of the shape selective technique ion mobility with mass spectrometry has allowed the study of conformation and dynamics of native proteins. The work presented in this thesis focuses on the use of ion mobility mass spectrometry to investigate both protein conformation, and conformational differences induced by mutation. Prolyl oligopeptidase family enzymes are proteases characterised by their unique function; these enzymes are only capable of digesting short amino acid sections of no more than thirty amino acids. These proteins have been implicated in a number of neurological disorders and have been targeted as potential drug candidates. PREP is an 80.7 kDa monomeric protein that has been targeted as a potential drug candidate. PREP has been crystallised in two distinct conformations, open and closed, and it has been suggested that the protein exists in equilibrium between the two states in solution, with only the open conformation allowing substrate entry via a domain separation mechanism. Ion mobility mass spectrometry has been used to confirm the presence of both conformations, and to investigate the effect of ligand binding on conformation. It was found that, at lower energy states, PREP was only capable of adopting a single conformation, and that more extended conformations were only present following activation of the protein. Binding of ligand appeared to increase the relative stability of the protein. DPP IV, is a larger, dimeric, protein from the same family. Unlike PREP, DPP IV has only been crystallised in a single conformation and it was proposed that small loop movements, rather than domain separation, allowed substrate entry. Ion mobility measurements show only a single conformation of DPP IV, consistent with no large conformational changes, supporting this hypothesis. Haemoglobin is the main protein involved in gas transport in mammalian systems, taking oxygen from the lungs to the tissues of the body. Disorders of haemoglobin represent the most common of all inherited disorders, with an estimated 7% of the global population being carriers for a haemoglobin disorder. A previous study by Scarff et al used ion mobility mass spectrometry to identify conformational differences between normal HbA and HbS, the haemoglobin mutant responsible for sickle cell disease. Recent experimental improvements in sample preparation, data collection and data processing have been used in this research to provide improved experimental information. HbA, HbS and HbC, a third haemoglobin variant known to form crystals within erythrocytes, were investigated using ion mobility mass spectrometry. Calibration of ion mobility data using native protein standards indicated that the structural differences between HbA and HbS were smaller than previously reported, and that the CCS measurements of the two proteins were similar for the native charge states. The HbC molecule does however adopt a smaller conformation. All three proteins unfold as a factor of increased protonation, with HbS and HbC showing evidence of adopting a more extended conformation at lower charge states when compared to HbA, suggesting possible differences in protein stability. These stability differences were investigated using collision-induced activation of the protein, the results suggesting that HbA is more resistant to unfolding that either HbS or HbC. Monoclonal antibodies represent a new generation of biotherapeutic capable of high specificity and selectivity, with diminished risk of inducing a host immune response. Antibodies therapeutics have an added benefit of interacting with host cellular systems, promoting host immune response. Engineering of antibodies has become well established to improve or diminish antibody-receptor binding in order to increase or abolish this interaction. Four antibody Fc variants have been studied using a combination of ion mobility mass spectrometry and hydrogen deuterium exchange mass spectrometry; a wild type, a TM mutant, a YTE mutant and a TMYTE double mutant. Previous studies have shown that the introduction of both TM and YTE mutations leads to a decrease in the thermal stability of the protein, and it was an aim of the study to provide structural information to explain this thermal destabilisation. Ion mobility mass spectrometry measurements suggest that there is little change in global conformation between the four variants. HDX results show that mutation introduces changes in local conformation across the protein, with increases in deuterium uptake observed at sites distant to the mutation sites. One region, located between the TM and YTE mutation sites, showed a greater than additive increase in deuterium uptake in TMYTE mutant compared with either TM or YTE mutants, indicating a region that hade become destabilised in the double mutant. These changes could be responsible for the observed loss of thermal stability.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.658961  DOI: Not available
Keywords: QD Chemistry
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