Determining protein:protein interaction partners

 

Lab rotation project description

ABCG2 is a multidrug transporter and much research has been described regarding its pharmacology. Comparatively little is known regarding the proteins that ABCG2 interacts with. In order to explore this, His-tagged ABCG2 will be immobilised on metal affinity chromatography resins and putative interacting proteins isolated using affinity purification. Identities of proteins will be confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and western blotting. Comparisons will be made with detergent solubilised, versus, SMALP-solubilised ABCG2. The list of putative interacting proteins will be subject to bioinformatic and/or literature analysis to determine the significance of putative hits, and identify candidate proteins for validation and further characterisation. Training will be provided in affinity chromatography and analysis of LC/MS/MS datasets.

Fact file

Research theme

MCO

Location

School of Life Sciences

Rotation

Contact

Main Supervisor: Robert Layfield

robert.layfield@nottingham.ac.uk

2nd supervisor

Ian Kerr and Neil Oldham

BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

Drug export from cells is a major factor in the acquisition of cellular resistance to antimicrobial and cancer chemotherapy, and poses a significant threat to future clinical management of disease. Many of the proteins that catalyse drug efflux do so with remarkably low substrate specificity, a phenomenon known as multidrug transport. For these reasons we need a greater understanding of drug recognition and transport in multidrug pumps to inform research that attempts to circumvent their action.

One family of proteins that are capable of multidrug export is the ATP binding cassette (ABC) transporter family; bacterial, yeast, plant and mammalian ABC transporters can all contribute to multidrug (MDR) export. The standard model for ABC multidrug pumps is that the binding and hydrolysis of ATP at two intracellular nucleotide binding sites, results in the binding and transport of drug molecules from allosterically linked sites in the membrane spanning domains of these proteins. In this project, you will attempt to determine the drug binding sites on bacterial and human MDR pumps using a novel detergent-free, membrane protein purification technique known SMALPs, and a cutting edge mass spectrometric labelling technique, known as carbene footprinting.

Bacterial ABC transporters BmrA and Sav1866 will be expressed in E. coli C41.DE3 cells and human ABCB1 and ABCG2 will be expressed in mammalian HEK293T cells. The function of each transporter will be confirmed with drug export assays using flow cytometry (ABCG2 and ABCB1) or fluorescence spectroscopy (BmrA and Sav1866). Transporters will be isolated from the membrane using styrene maleic acid co-polymer (SMALP) solubilisation, which results in the extraction of the protein still in a patch of the native lipid bilayer. Given that the membrane bilayer is essential for the function of membrane proteins, SMALP confers significant advantages over conventional detergent solubilisation which removes the lipid bilayer. Purification of each transporter by Ni or Co-affinity chromatography will result in the production of purified ABC transporters suitable for mass spectrometric interrogation.

The objectives for this project are as follows:

  • expression and purification of ABC transporters at concentrations and purities suitable for carbene footprinting
  • structural biology has already confirmed where nucleotides bind to ABC transporters and carbene footprinting of Sav1866, BmrA, ABCB1 and ABCG2 in the presence and absence of nucleotides will confirm that the technique can distinguish the binding sites of small molecules
  • the great unknown is precisely where on these transporters are the drug binding sites; determination of carbene footprinting sites masked by the interaction of known drug substrates of ABC multidrug transporters will determine the structural basis of MDR binding and transporter
 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk