Project Outline

Biology background

Transport proteins are quintessentially important for establishing hormone gradients, but they remain challenging proteins for structural biology. We have established purification for the auxin transporter AUX1 and have selected a set of nanobodies against it. In this project you will use cryo-electron microscopy to solve the structure of AUX1.

Project

We express transport proteins using insect cell cultures. You will be taught our purification protocol and screen for the best nanobodies before preparing the complex for cryo-electron microscopy. You will then screen the protein for suitability for cryo-EM and collect and process cryo-EM data where appropriate. Additional options include producing a set of site-specific mutants and developing thermal shift assays or other biophysical techniques to measure transport activity in the purified proteins. The resulting structure-activity relationships will reveal the how transport of the hormone auxin works at a molecular level.

References

Hatton, C, Brotherton, D, Spencer, M and Cameron, A. (2022) Structure of cytosine transport protein CodB provides insight into nucleobase‐cation symporter 1 mechanism. The EMBO Journal, 41 (16). e110527

Brotherton, D, Savva, C, Ragan, T, Dale, N and Cameron, A. (2022) Conformational changes and CO2-induced channel gating in connexin26. Structure, 30 (5). 697-706.

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) Adenylate cyclase activity of TIR1/AFB auxin receptors for root growth. Nature (611). pp. 133-138

Techniques

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, computational chemistry