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Applying Mass Spectrometry to the Investigation of Protein Structure

Beeston, Helen Sarah (2015) Applying Mass Spectrometry to the Investigation of Protein Structure. PhD thesis, University of Leeds.

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Complete thesis corrections 120216.pdf - Final eThesis - complete (pdf)
Restricted until 1 March 2036.

Abstract

Understanding protein structure is vital for our understanding of protein functionality and in aiding the design of effective therapeutics. The study of protein conformation by solution-phase hydrogen/deuterium exchange (HDX) of backbone amide protons is well documented. Solution-phase HDX provides details concerning the protein's tertiary structure. However, undesired back-exchange during post-HDX analyses can result in the loss of structural information. Here, gas-phase HDX-mass spectrometry (MS), during which labile hydrogens on amino acid side chains are exchanged in sub-millisecond time scales, has been employed to probe changes within protein structures and to distinguish co-populated protein conformers. Dysregulation of kinase function has been implicated in many diseases, including cancer, and a number of targeted therapies comprising kinase inhibitors are in development. There are two major classes of kinase inhibitor, Type I and Type II. Type II kinase inhibitors bind selectively to an inactive kinase conformation and are thought to have advantageous drug properties, including improved selectivity and specificity for their target kinase. High-throughput screens are not able to identify Type II kinase inhibitors as they are not amenable to phosphorylated kinase binding used in the screens. Here, collision induced unfolding-ion mobility spectrometry-mass spectrometry (CIU-IMS-MS) has been used to identify the binding of Type II inhibitors to an inactive FGFR1 kinase conformation and as a result has been used to identify two novel Type II inhibitors of FGFR1. Intrinsically disordered proteins such as myelin basic protein are common. MS and IMS-MS are well suited to the analysis of IDPs and have been used to characterise MBP. Here, Cu2+ metal ion binding to MBP has been found to stabilise MBP’s conformation, requiring increased CIU energies to produce unfolding. The addition of the membrane mimetic detergent (DDM) to MBP allows the observation of a compact MBP conformation under MS analysis.

Item Type: Thesis (PhD)
Keywords: Mass Spectrometry, Ion mobility spectrometry-mass spectrometry, Gas-phase HDX,
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Miss Helen Beeston
Date Deposited: 17 Feb 2016 09:45
Last Modified: 17 Feb 2016 09:45
URI: http://etheses.whiterose.ac.uk/id/eprint/11969

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