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Solubilisation and characterisation of G-protein-coupled receptors using styrene maleic acid polymer

Charlton, Jack (2016)
Ph.D. thesis, University of Birmingham.

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Abstract

Detergent-free solubilisation using a polymer of styrene maleic acid (SMA) has proven useful in the study of membrane proteins. SMA was employed to solubilise G-protein-coupled receptors (GPCRs) from mammalian cells, into SMA lipid particles (SMALPs). Optimal SMALP solubilisation conditions were determined to be 2 % (w/v) SMA, at 37 °C for 1 h retaining wildtype (WT)-like pharmacological profiles and conferring improved stability on GPCRs over detergent micelles.

Endoplasmic reticulum (ER)-retention motifs –KDEL and –KHILFRRRRRGFRQ were found not to be applicable to all proteins. Study of SMALP-solubilisation of intracellular membranes was prevented by the inability to retain the adenosine 2a receptor (A2aR) in the ER. A cysteine-nul A2aR construct was produced containing two reporter groups and behaved as WT-A2aR. This construct is ready for use in fluorescence studies to further understanding of A2aR and the use of SMALPs in biophysical techniques.

A range of GPCRs, from different GPCR subfamilies, were SMALP-solubilised with retention of ligand binding capability. Methods were successfully developed to reduce non-specific binding arising from ionic interactions of ligand and SMA. Finally, in a world first, the SMA analogue styrene maleimide, was shown to solubilise GPCRs with retention of ligand binding capability.

Type of Work:Ph.D. thesis.
Supervisor(s):Wheatley, Mark
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QH301 Biology
Institution:University of Birmingham
ID Code:6525
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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