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Functional assays for purified hormone transport proteins.

Primary Supervisor: Professor Richard Napier, School of Life Sciences

Secondary supervisor: Dr Mark Wall, Life Sciences, Dr Charo del Genio, University of Coventry

PhD project title: Functional assays for purified hormone transport proteins.

University of Registration: University of Warwick

Project outline:

Background: 

Biology background: Auxin transport proteins establish auxin gradients and help determine plant form and size. We express a number of these transport proteins and have developed purification protocols for them. We also have structural models for the proteins. This project will develop and use assays for transporter activity, coupling hormone transport to structure and mechanism. Mechanisms will be evaluated from a series of structure-informed mutations.

Project: The Napier group express auxin transport proteins using insect cells in tissue culture for crystallography. Such structural biology will help explain how these vital transporters work. It is essential that the proteins expressed in insect cells are shown to be active and we have also established a set of activity assays which will help elucidate their mechanism of action. The Wall group use electrophysiology to study synaptic plasticity and communication in nerve cells and you will use their whole cell voltage-clamp technique to measure auxin currents through transport proteins. You will also use thermal shift assays and other biophysical techniques for activity measurements. The del Genio group use molecular dynamics to develop simulations of protein structure and help identify residues to be prioritised using site-directed mutagenesis. The resulting structure-activity relationships for selected mutants will reveal the mechanism of transport and its gating.

References:

  1. Lee et al. 2014. Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)-Aux/IAA co-receptor complex formation. ACS Chem Biol. Mar 21;9(3):673-82. doi: 10.1021/cb400618m.
  2. Hoyerova K, Hosek P, Quareshy M, Li J, Klima P, Kubes M, Yemm AA, Neve P, Tripathi A, Bennett MJ, Napier RM.(2017) Auxin molecular field maps define AUX1 selectivity: many auxin herbicides are not substrates. New Phytologist217(4):1625-1639, doi 10.1111/nph.14950
  3. Hill, Emily, Wall, Mark J., Moffat, Kevin G., Karikari , T., 2020. Understanding the pathophysiological actions of tau oligomers : a critical review of current electrophysiological methods. Frontiers in Molecular Neuroscience, 13

 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.

    Contact: Professor Richard Napier, University of Warwick