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Title: A new detergent-free approach to solubilize membrane proteins to maintain their native environment
Author: Bada-Juarez, Juan Francisco
ISNI:       0000 0004 7654 344X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Biophysical characterisation of membrane receptors in their native environment is essential for drug development, as lipid removal may affect conformational freedom and ligand binding. Membrane proteins are commonly solubilised with detergent for structural and functional studies, although the resulting micelles may be polydisperse, prone to aggregation and occlude of the binding site. Recently a hydrolysed copolymer of styrene and maleic acid (SMA) has been shown to be highly effective in extracting membrane proteins from native membranes without the use of detergent and the resulting coin-shaped nanoparticles, called Lipodisq (or SMALPs) are suitable for use in a wide range of biophysical methodologies. Here we have been able to purify and characterize a range of membrane proteins from their native environment including bacteriorhodopsin (bR), archaerhodopsin 3 (AR3), Dopamine receptor 1 (D1, a human GPCR) and EcMATE (a bacterial transporter) using size-exclusion and affinity chromatography, circular dichroism and dynamic light scattering. Microscale thermophoresis and radio-ligand binding have been used to assess the activity of the different proteins. These nanoparticles have further allowed us to characterize the oligomeric state and to identify native ligands by native mass spectrometry. The crystal structure of AR3 was obtained in the absence of detergent directly from its native membrane and its structure was solved to 1.3 Å resolution, where post-translational modifications, such as the pyroglutamate residue (PCA) at the N-terminus of AR3, and several side-chain rotamers are resolved for some key residues in the proton translocation channel as well as multiple water molecules. Some of the water molecules have partial occupancy within the extracellular-facing half channel, consistent with the existing FTIR data. Native mass spectrometry with Lipodisq has characterised the post-translational modifications carried by the protein. Moreover, a first structural study of the AR3 photointermediates using time-resolved XFEL crystallography has been performed. Lipodisq nanoparticles were used to study the lipid and protein composition of C. elegans by comparing the wild-type N2 worm to a mutant strain (agmo-1 ), which lacks for an important enzyme in the lipid biosynthesis. Finally, Lipodisqs have been evaluated as a drug delivery agent for Doxorubicin, a potent anti-tumour drug, which is commercially available as the PEGylated liposomal formulation Doxil. Previous work on the delivery of the drug to intracellular compartments of HeLa cells followed by confocal microscopy and IC50 measurements show that, unlike in the case of encapsulated systems, there is no decrease in in vitro efficacy over the water-solubilised form of the drug. Here the fluorescent derivative of the SMA polymer has been synthesised and its biodistribution followed in vivo.
Supervisor: Watts, Anthony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biophysics--Research ; Structural Biology