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Exploiting polymer nano-encapsulation of membrane proteins for agrochemical target identification

Principal Supervisor: Professor Tim Dafforn, School of Biosciences & Dr Francisco Fernandez-Trillo, School of Chemistry

Non-academic partner: Syngenta

Project title: Exploiting polymer nano-encapsulation of membrane proteins for agrochemical target identification

University of registration: University of Birmingam

Project outline:

Identifying the proteins that agrochemicals target has the potential to help with the development of more effective compounds inhe future. Currently agritech companies use chromatographic separations combined with Mass spectrometry and data analysis to associate agents with protein targets. This works very well when the target protein is soluble, and hence amenable to chromatographic separations. However many target proteins are membrane bound making applying a similar methodology very challenging.

The key issue in applying the method to membrane proteins is extracting the protein from the membrane in a form that maintains activity during the separation process. Conventional approaches use detergent for extraction but the presence of detergent not only affects the separation process but also often causes inactivation of the target. In 2015 we carried out a set of proof of concept with an agrichemicals company to trial the use of an entirely new, detergent free, method for membrane protein extraction. The method uses a simple polymer (Styrene Maleic Acid Co-polymer, SMA) to extract the membrane protein, including local membrane environment as a self-contained nanoparticle. The SMA forming a stabilising belt around the lipid bilayer in which the protein is solubilised. The particle is called a SMALP. The results of the trial showed that the method performed better than conventional detergents allowing the chromatography process to be carried out successfully.

Project Plan: This project builds on this work and aims to extend it to produce a final protocol to extract, separate and identify potential agrochemical targets. The trial experiment showed that the method could work but also showed that the method needs optimising to improve separations and further increase stability. Since the trial we have developed a number of other polymer variants including polymers with different charges, functional groups, lengths, and compositions. The project will aim to assess these polymer variants as potential extraction agents for use by by our agritech partner.

WP1 (YEAR1) Each polymer variant will be assessed for its ability to solubilise a number of high value neuronal insecticide target proteins (e.g. nicotinic acetylcholine receptors). The activity of each target will be assessed using established binding assays. Polymers that do not solubilise will be discarded from the trial.

WP2 (YEAR2) Polymers that show solubilisation capability will be used in a range of chromatographic separations. The behaviour of the polymers will be examined to assess how well polymer particles containing membrane proteins are separated by each method.

WP3 (YEAR3-) we will develop a number of biochemical methods that take advantage of functionalisations of the polymer. For example we have used biotinylated SMA to enable ligand binding assays and fluorescently labelled polymer for FRET and FACS based assays. In this work package the use of these functionalised polymers in applications defined by our agritech partner will be assessed. Methods of interest include simplified/enhanced assay formats for functional characterisation of receptors and FACS-based purification of SMALP-isolated receptors to allow determination of subunit composition and presence of accessory proteins.

Closing date for applications: 7th January 2018

Interview dates: February 2018 TBC

Check eligibilty and apply