Reporting on project progress - Impacting Global Food Security

Lab experiments were essential for my project, and the Medical and Life Sciences Research Fund (MLSRF) award was utilized in purchasing lab consumables for large-scale cell culture, protein purification, protein quality assessment, crystallization trials and functional assays.

Large-scale cell cultures (3-5 liters) were used to produce AUX1 protein and initiatecrystallography trials by vapor diffusion and lipidic cubic phase. The quality of the purified protein was confirmed using circular dichroism spectroscopy. A total of 40 crystal plates were set up, each with 96 different crystallization conditions using a Mosquito nanoliter protein crystallization robot. A few crystals were taken for inspection to the synchrotron Diamond Light Source, Harwell, UK.

Unfortunately, the AUX1 protein remains reluctant to crystallize. To explore other options of structural determination, a high-throughput screen was carried out at the Membrane Protein Lab (MPL), Harwell, to find alternatives to detergent for solubilizing and stabilizing AUX1 protein in solution. As the AUX1 protein is too small for Cryoelectron microscopy, the current strategy is to send purified protein to the INSTRUCTERIC nanobody discovery center (VIB, Belgium). Nanobodies help to stabilize membrane proteins and assist in crystallization. They can also act as functional modulators.

MLSR support  has allowed me to explore every available option for structural biology of AUX1 protein, but this plant transporter is proving to be a challenging target. In addition to the crystallization experiments, several biophysical techniques have been employed to test the activity and affinity of the purified AUX1 protein. I have used microscale thermal melting assays using CPM dye and nano-DSF (at MPL), SPR, and ITC.

A collaboration with SLS Neurosciences has helped me to establish a patch clamping assay to confirm auxin transport activity in the tissue culture expression system which I developed for the AUX1 protein. A collaboration with Nanion (Munich, Germany) has allowed me to test solid supported membrane electrophysiology using SURFE2R N1 technology to establish that the purified AUX1 protein remains competent to auxin transport.

This multidisciplinary project to determine the novel structure of AUX1 protein and answer the question about the molecular mechanism of auxin accumulation in plant cells is ongoing. The challenges with membrane protein crystallization remain. Nevertheless, the experiments carried out with the support of the bursary award have been crucial in understanding more about the protein’s behavior and for designing alternative strategies for the accomplishment of the project objectives and the successful completion of my PhD. I am grateful to the MLSRF for supporting my research project.