Principal Supervisor: Prof Richard NapierLink opens in a new window

Co-supervisor: Prof Alex Cameron (Warwick, Life Sciences) Dr Andrew Quigley (Membrane Protein Laboratory, Diamond Light Source).

PhD project title: Cryo-electron microscopy of the hormone transport protein AUX1

University of Registration: University of Warwick

Project outline:

Biology background: Auxin signalling is quintessentially important for higher plant life and auxin transport proteins establish gradients of this hormone to help determine plant form and size. We express these transport proteins in tissue culture systems and have developed a purification protocol which gives high yields of the auxin uptake carrier AUX1. The plan is to take AUX1 into cryo-electron microscopy for structural resolution. We have structural models and nanobodies will also be available. The structure of AUX1 will enable us to couple hormone activity to the molecular mechanism of auxin transport and provide a new target for use in structure-led agrochemical design.

Project: The Napier group express auxin transport proteins using insect cells in tissue culture for structural biology. Prof Cameron and Dr Quigley will direct the cryo-electron microscopy and structural biology. We also work with the del Genio group (University of Coventry) who use molecular dynamics to develop simulations of protein structure and will help identify residues to be prioritised for site-directed mutagenesis. In combination, these approaches will help explain how these vital transporters work. You will select and produce a set of site-specific mutants, express, purify and assess their activity. You will use thermal shift assays and other biophysical techniques to measure transport activity in the purified proteins. The resulting structure-activity relationships for selected mutants will reveal the how transport of auxin works at a molecular level. For students interested in pharmacology, libraries of natural products (phytochemicals) will be tested to evaluate AUX1 as a site for next-generation agrochemicals.

You will be registered as a visiting student at Diamond, subject to a signed studentship agreement, giving you access to facilities and a support network while on the Diamond site.

References:

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 Phytologist 217(4):1625-1639, doi 10.1111/nph.14950

Fukui, K et aL., (2022) Chemical inhibition of auxin inactivation pathway uncovers the roles of metabolic turnover in auxin homeostasis. Proceedings of the National Academy of Sciences, 119 (32). e2206869119. doi:10.1073/pnas.2206869119

Qi, L et al., (2022) A denylate cyclase activity of TIR1/AFB auxin receptors for root growth. Nature

Xu, et al., (2022) Mode of action of a novel synthetic auxin herbicide, halauxifen-methyl. Agronomy, 12 (7). 1659. doi:10.3390/agronomy12071659

Figueiredo, M. et al., (2022) An in-frame deletion mutation in the degron tail of auxin coreceptor IAA2 confers resistance to the herbicide 2,4-D in Sisymbrium orientale. Proceedings of the National Academy of Sciences of the United States of America, 119 (9). e2105819119. doi:10.1073/pnas.2105819119 .

BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology, and Plant Science, and Sustainable Agriculture and Food - Plant and Crop Science.

 

Techniques that will be undertaken during the project:

Cloning and tissue culture; Protein expression, purification and assays for protein activity (e.g. structure-activity relationship assays); cryo-electron microscopy, structural molecular biology and, depending on interest, molecular dynamics, computational chemistry.

 

Contact: Prof Richard NapierLink opens in a new window