About the Project
There are few plant responses for which auxin and its movement are not vital. Our group studies the proteins which underpin all auxin responses, the auxin receptors such as TIR1 and the PIN proteins that dominate the establishment of auxin gradients and help determine plant form and size. There are crystal structures for the receptor complex and we have an activity assay for PINs. In each case there are projects which will use expression of these proteins in tissue culture, purification using affinity chromatography and a set of advanced techniques for evaluating how the chosen protein functions in detail.
Project 1: The Wall group use electrophysiology to study synaptic plasticity and communication in nerve cells. Preliminary experiments have shown that auxin currents can be measured using the patch-clamp technique on insect cells expressing both uptake and efflux carrier proteins. You will use patch-clamping to investigate properties of the efflux protein PIN5; sensitivity (concentration dependence), selectivity (substrate specificity, pharmacology) and mechanism (inhibitor pharmacology, possible pH dependence etc). You will also use site-directed mutagenesis guided by the molecular dynamics models generated by del Genio to learn about the mechanism of transport and its gating.
Project 2: A similar project is available to work on the auxin uptake carrier AUX1.
Project 3: We routinely purify the receptor TIR1 and its homologues using baculovirus expression and affinity chromatography and assay auxin binding using Biacore technology. You will use site-directed mutants and examine how specificity is conferred and lost, quantifying the ‘pharmacological costs’ of affinity vs specificity. You will use pharmacological software routines both for summarising the binding data and for predicting novel ligands.
BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Plant and Crop Science. Understanding the Rules of Life: Plant Science & Structural Biology
Techniques that will be undertaken during the project:
Cloning and tissue culture; Protein expression, purification;
Functional assays and pharmacology (e.g. structure-activity relationship assays);
Depending on interest: molecular dynamics, computational chemistry, Electrophysiology, patch-clamp technique, Biophysics (surface plasmon resonance, isothermal titration calorimetry, dynamic light scattering, thermal shift assays etc)
Hoyerova et al. (2017) Auxin molecular field maps define AUX1 selectivity: many auxin herbicides are not substrates. New Phytologist 217(4):1625-1639, doi 10.1111/nph.14950
Hill et al 2020. Understanding the pathophysiological actions of tau oligomers : a critical review of current electrophysiological methods. Frontiers in Molecular Neuroscience, 13.
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