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Investigating the structure and function of ABC transporters using polymer-lipid nanoparticles

Primary Supervisor: Dr Alice Rothnie, Life & Health Sciences

Secondary supervisor: Professor Brian Tighe

PhD project title: Investigating the structure and function of ABC transporters using polymer-lipid nanoparticles

University of Registration: Aston University

Project outline:

The ABC (ATP Binding Cassette) transporter superfamily is found in all organisms from bacteria to humans, and they utilise energy from ATP hydrolysis to power the transport of molecules across a membrane. The human transporters are involved in a wide range of functions including protection from toxins, metabolism, controlling drug distribution in the body, mediating inflammatory responses and transporting lipids. Several members are responsible for genetic diseases, such as cystic fibrosis and adrenoleukodystrophy, whilst others are involved in causing multi-drug resistance during cancer treatment. Of particular interest are these multi-drug transporters, which are able to recognise and transport a wide array of different substrates, which can vary in size, shape and chemistry. It is not yet understood how these proteins can facilitate this, where and how exactly the various substrates bind, or how this is coupled to the ATP hydrolysis.

Studying the function and structure of these important proteins has been hampered by difficulties in expressing, extracting and purifying them, due to their large size and membrane environment. Traditionally detergents were used to solubilise them from the membrane, but this can destabilise the proteins and strips away lipids which are important for function. However we have shown that ABC transporters can be effectively extracted and purified using a styrene-maleic acid co-polymer (SMA). The polymer inserts into the membrane, isolating small discs of lipid bilayer, encircled by the polymer, termed SMALPs (SMA lipid particle). SMALP purified ABC transporters are stable and amenable to many downstream applications, including ligand binding and structural studies. This project will utilise SMALP purification of a multi-drug ABC transporter to investigate the locations of the various substrate binding sites using approaches such as mutagenesis, fluorescence spectroscopy, EPR (electron paramagnetic resonance) and recognition imaging AFM (atomic force microscopy).

However there are limitations for the study of ABC transporters using SMA, including a sensitivity to Mg2+, which is needed for ATP hydrolysis, and an inability to measure transport. Thus this project will also investigate reconstitution from SMALPs to proteoliposomes and the application of novel polymer variants for the extraction, purification and downstream study of ABC transporters.

References:

  1. Gulati S, Jamshad M, Knowles TJ, Morrison KA, Downing R, Cant N, Koenderink J, Ford R, Overduin M, Kerr ID, Dafforn TR & Rothnie AJ* (2014) Detergent free purification of ABC transporters. Biochem. J. 461; 269-78
  2. Pollock NL, Lee SC, Patel JH, Gulamhussein AA, Rothnie AJ* (2018) Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer. BBA Biomembranes 1860(4); 809-817.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

  • Protein expression (insect cells and bacterial), solubilisation and purification
  • Molecular biology
  • Fluorescence spectroscopy
  • ATPase and transport assays
  • Biophysical techniques (EPR, AFM)
  • Polymer chemistry

Contact: Dr Alice Rothnie, Aston University