Principal Supervisor: Prof Richard Napier - SLS
Co-supervisor: Dr Alex Cameron - SLS
PhD project title: Structural biology of transporter protein PIN1
University of Registration: Warwick
- The goal is to express, purify and determine the structure by crystallography of the auxin efflux carrier PIN1, a member of a family of eukaryotic membrane transport proteins for which no structure exists currently. Yet these transporters determine some of the most profound morphogenic events in biology, such as polarity in the plant embryo.
Background: Almost 30 % of all sequenced genomes encode membrane proteins. These play vital roles in many areas of cell biology such as signalling, the selective uptake or removal of molecules from the cell and the maintenance of cellular pH. PIN proteins are in the same family as the human apical sodium-dependent bile acid transporter (ASBT), but determine morphogenic gradients of the hormone auxin by acting as efflux transporters. This transporter family has fundamental importance across the eukaryotic kingdom and knowledge of their structure will illuminate many important features of their mechanisms of action. Correct delivery of auxin by PIN proteins deteremines plant productivity making these transporters intriguing targets for improving food security.
Work Plan: We have expression constructs of a series of PINs in E. coli vectors, but you will also generate recombinant baculovirus lines for expression in a eukaryotic host. Given the premium of crystal data for informing all later parts of the project, initial work will be focussed production and purification of the proteins. Focussing on the candidate that proves more amenable, you will evaluate detergents for solubilisation, finding conditions which give good yield, clean protein and retains biological activity. Once protein is produced, you will set up crystallisation trials.
Less protein is required for many biophysical assays and so you will explore ligand gating, substrate recognition and selection etc using a variety of the advanced instruments for studying molecular interactions. For example, biotinylated ligand analogues are available and can be used for structure-activity profiling using SPR (Biacore T200), thermal shift or radiotracer assays.
Once diffraction data are available you will focus on solving the structure of the protein, making use of homology modelling or molecular dynamics (depending on interests) to help promote greater understanding of these important transport proteins.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Cloning and tissue culture, protein expression, purification, solubilisation.
- Biophysics (surface plasmon resonance, isothermal titration calorimetry, dynamic light scattering, thermal shift assays etc)
- Functional assays (e.g. radiolabel accumulation assays, structure-activity relationship assays)
- Crystallography screens and techniques, Data collection at synchrotrons.
- Depending on interest, molecular dynamics, cheminformatics
Contact: Professor Richard Napier, University of Warwick