Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614586
Title: An efficient EPR method for characterising protein-protein interactions
Author: Bye, Morgan
ISNI:       0000 0004 5366 9568
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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Abstract:
Atomic resolution structures of protein-protein interactions were initially limited to X-ray crystallography; with unnatural buffers, pHs and forced lattice structures. NMR extended investigations to be able to determine solution based structures of small complexes, but proteins of any size or high dynamicity remain problematic, whilst ab initio investigation remains almost impossible. Using site-directed spin labelling (SDSL) across both docking partners, in combination with the electron paramagnetic resonance (EPR) technique pulsed electron-electron double resonance (PELDOR) allows for the precise measurement of intermolecular distances. Producing many mutant proteins with spin label sites across the protein allows for a 3-dimensional map to be built. Each EPR distance measurements is incorporated as a distance restraint into existing docking protocols with an aim to provide better quality docking models. The method outlined in this investigation presents EPR as a technique with immense potential. EPR only measures paramagnetic species, in this case introduced nitroxide spin labels, making it applicable to water-soluble and membrane proteins, without the protein size limitations as with NMR. Furthermore, incorporation with freeze-quench apparatus could yield future time-dependent modelling in a way unavailable to traditional biomolecular structural techniques. Using the well-characterised system of the DNase domain of colicin E9 and its inhibitor, colicin E9 immunity protein, Im9, a combination of structural NMR models and EPR long range distance restraints were able to present a structural solution with comparible resolution to high resolution NMR or X-ray crystallography. Looking forward, this method could be extended to molecules that have previously been available to study such as large proteins, proteins with high dynamicity, membrane proteins in lipids or even to build a structure in combination with in silico techniques.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.614586  DOI: Not available
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