Integral membrane transporters are responsible of the translocation of essential compounds and waste products from one side of the membrane to the other.
Boron is an essential micronutrient for plants, critical for cell growth, development and stability. The main focus of the PhD was Arabidopsis thaliana AtBOR1, responsible of active transport of B from root cells to the xylem. A structure of a C-terminally truncated AtBOR1 was solved at a resolution of 4.1 Å, lacking key details essential for understanding the transport mechanism. AtBOR1 WT was expressed, purified and crystallised, yielding crystals diffracting maximally to 7 Å. Mutants AtBOR1-M363F and -M363Y, presented enhanced stability and decreased activity compared to the WT, suggesting a reduction in conformational flexibility. AtBOR1-M363F and -M363Y crystals diffracted on average to a higher resolution than the WT (~5.5 Å).
A second project focused on the uric acid-xanthine/H+ symporter of Aspergillus nidulans (UapA). The structure of a non-functional mutant UapA-G411V1-11 was recently solved at 4.2 Å, in the inward facing conformation. Evidence suggested that UapA-Q408E is in the outward facing conformation.
UapA-Q408E1-11 was purified to high homogeneity and the crystals diffracted maximally to ~4.5 Å, allowing collection of a partial dataset. The protein was also reconstituted in nanodiscs for negative stain analysis revealing that the sample is monodisperse, forming the basis of possible future work using cryo-EM.
The third branch of the PhD focused on the development and characterisation of novel tools for membrane protein studies. AtBOR1 WT was employed to screen six different families of novel amphiphiles, proving to enhance the protein stability in solution. Finally, a novel high-throughput screen to facilitate identification of lipids stabilising membrane protein in detergent-based solution was designed, produced and tested using UapA-G411V1-11 in collaboration with Molecular Dimensions and Anatrace. Upon further optimisation this screen could become a commercially viable product.