Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.698277
Title: EM as a tool to study structure and function to guide inhibitor design
Author: Rawson, Shaun Deane
ISNI:       0000 0004 5990 2826
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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Abstract:
Rational drug design is underpinned by structural biology, in particular X-ray crystallography. This work aimed to explore a simple question; can electron microscopy (EM) play a useful role in the drug discovery process? To this end, two biological systems, the V-ATPase and Imidazoleglycerol-phosphate dehydratase (IGPD), were studied via EM. A modest (~1 nm) structure of the V-ATPase was solved via cryo-EM, permitting existing high resolution crystal structures to be accurately fitted in the context of the whole 1 MDa complex. This allowed new mechanistic insights to be uncovered. The dissociated V1 domain was studied to see if the mechanism of ATP silencing, used as a regulatory control, could provide new inhibitor targets to be identified. Although the specific mechanism could not be identified the resulting structures have shown a much more complicated regulatory mechanism than previously thought. The ubiquitous nature of the V ATPase makes selectivity a significant challenge. To address this the binding mode of the selective V-ATPase inhibitor, Pea Albumin 1 subunit b (PA1b), was studied at low resolution using negative stain EM. Combined with biochemical analysis this has successfully identified the subunits responsible for binding PA1b. Furthermore, IGPD was used as a model system for high resolution cryo EM studies to directly visualise inhibitor binding. A 3.1 Å reconstruction was obtained which allowed the de novo building of the atomic model and the identification of a small molecule inhibitor within the EM map. This work shows the potential of EM to provide valuable information for drug design at a wide range of resolutions, from rapid low resolution binding studies to direct visualisation of inhibitors at ~3 Å.
Supervisor: Muench, Stephen P. ; Bon, Robin ; Trinick, John Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.698277  DOI: Not available
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