Abstract
Proteins play an important role in all biological systems from bacteria to humans. The research of proteins is paramount to understanding the fundamentals of biology and for furthering medical knowledge. The mechanisms of these biological systems are underpinned by their protein conformational dynamics. While gold-standard structural techniques such as X-ray crystallography allow the study of snapshots of proteins, this does not paint the full picture. Structural mass spectrometry (MS) is a growing field of structural biophysics. Ion mobility (IM) and hydrogen-deuterium exchange (HDX) MS both allow probing of protein conformational dynamics in either the gas phase or in solution. This thesis focuses on the application of these structural MS techniques to investigate the dynamics and biological mechanisms of immunoglobulins and membrane proteins.The first part of this thesis focuses on immunoglobulins, specifically, IgG. Chapter 2 focuses on the gas-phase dynamics and collapse of IgG molecules. Ion mobility-mass spectrometry (IM-MS) was used to determine the collisional cross-section (CCS) of IgG molecules. The CCS of IgG molecules was much lower than the expected theoretical CCS. A combination of molecular modelling and molecular dynamics simulations were used to devise a workflow to explain the reduced experimental CCS. This IgG collapse workflow was published in Angewandte Chemie.
The second part of this thesis (chapters 3 and 4) concentrates on the application of structural MS to membrane proteins. Chapter 3 focuses on the ATP-binding cassette (ABC) transporter MacB. ABC transporters utilise ATP energy to drive efflux substrates through the cell membrane. MacB is one part of a tripartite membrane protein efflux pump capable of exporting antibiotics from cells. As such, MacB is an important drug target for the fight against antimicrobial resistance. HDX-MS was used to study and elucidate the unique efflux mechanism of MacB. Further, the binding sites of two substrates including a common antibiotic (erythromycin) were determined for the first time. Finally, chapter 4 focuses on the membrane proteins BAM and HTL. The BAM-HTL complex transports nascent bacterial proteins through the inner membrane and inserts the folded protein into the outer membrane of gram-negative bacteria. The interaction and mechanism of these two proteins were investigated by HDX-MS.
Overall, the research presented here successfully utilised HDX-MS and IM-MS to probe the conformational dynamics of several protein systems, giving further insight into mechanisms of antibodies IgG, and membrane proteins MacB and BAM-HTL.
| Date of Award | 1 Oct 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Argyris Politis (Supervisor) & Brian Sutton (Supervisor) |