Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.736758
Title: Development of methods to study oriented membrane proteins using synchrotron radiation circular dichroism spectroscopy
Author: Evans, Luke Sean
ISNI:       0000 0004 6500 7983
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2017
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Abstract:
Membrane proteins are important targets for structural biology. They account for a disproportionately large percentage of pharmacological targets, but only a minority of available protein structures. The intractability of membrane proteins to recombinant expression and purification has been a limiting factor in their structural characterisation. Of those structures available the majority rely on detergent solubilisation of the protein. This removes the native lipid environment which is established to have structural and functional implications. A number of techniques allow the investigation of membrane proteins in lipid bilayers which mimic the cellular membranes. In this thesis, two of these approaches, oriented synchrotron radiation circular dichroism (OSRCD) and solid-state nuclear magnetic resonance (NMR) spectroscopies have been used to investigate the orientation and structure of the transmembrane domain of a putative glycosyltransferase fukutin. We have developed a method for OSRCD based on the conventional means of sample preparation, where aligned bilayers are prepared on a support substrate. This allowed the orientation of the fukutin transmembrane domain to be determined in bilayers of different thickness which model membranes of different cellular compartments. Both OSRCD and NMR results indicate that the protein adapts by changing its tilt angle and forming large oligomers in thicker membranes, potentially providing a model for retention of fukutin in the thinner Golgi apparatus membranes, which appears to be essential for its function. As the preparation of well oriented samples can be challenging, a novel method of OSRCD using magnetic alignment of lipid phases has also been developed. The goal of this work was to align lipid phases in a magnetic SRCD instrument. Extensive characterisation of lipid mixtures using NMR, electron paramagnetic resonance and conventional CD enabled identification of conditions to allow alignment at low magnetic fields such as those found in magnetic SRCD instruments. Although OSRCD measurements on magnetically aligned samples were not successful, a number of further modifications are suggested which may allow sample alignment under different conditions. With a view to expansion of the oriented techniques to a more complex protein, attempts were made to express and purify the pore region of the voltage-gated potassium channel expressed by the human Ether-á-go-go related gene (hERG). hERG is a particularly important target for structural characterisation as it has proclivity for non-specific drug binding, which can block the channel and result in drug induced long-QT syndrome, a phenomenon affecting a large number of newly developed pharmaceuticals.
Supervisor: Williamson, Philip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.736758  DOI: Not available
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