Dissertation

Structural and functional studies of the xanthine transporter uapa and the boric acid transporter bor1

Leung, James S. K

2012

Overview

The nucleobase/ascorbate transporters (NAT) are responsible for the uptake of nucleobases in all kingdoms of life, and for transportation of ascorbic acid (vitamin C) in humans. Nucleobase analogues are often used as antimetabolites in the treatment of a range of diseases. A detailed understanding of nucleobase transport will increase understanding of the mechanism of action of these important proteins and facilitate design of drugs with enhanced efficacy and specificity. Owing to the extensive genetic and biochemical analysis, UapA from A. nidulans is the best studied example of the NAT family. The aim of this project was to produce pure and stable UapA from A. nidulans for structural studies by X-­‐ray crystallography. In this study, it was possible to express wild type UapA in S. cerevisiae as a GFP fusion to 1.91 mg/L. Following purification, the protein was extremely unstable, degrading almost completely after 48 hours at 4°C. A number of single point mutants previously shown to exhibit reduced transport activity but correct targeting to the membrane were generated in an attempt to increase UapA stability. hFSEC and FSEC allowed successful identification of the thermostable mutant G411V, with preserved xanthine binding. G411V expressed well as a GFP fusion and was purified to homogeneity with a yield of 2.8 mg from 10 L culture. The purified protein was stable at 4°C for at least 2 days. Removal of the 11 amino acid residues at the N terminus, G411V12-­‐574, improved purification yield to 4.2 mg from 10 L culture and resulted in protein that was stable at 4°C for at least 6 days. Optimised crystals of this construct currently diffract to 7.16 Å resolution. Boron (B) toxicity and deficiency in plants are worldwide agricultural problems. Understanding the mechanism of boron homeostasis in plant will provide route for the design of crops tolerant to soils with suboptimal B concentrations. We aimed to solve the structure of a boric acid transporter from A. thaliana. Preliminary crystals diffracted to 7.6 Å. Significant optimisation screening including the use of structure specific antibodies failed to improve the diffraction limit. A breakthrough came with the use of a novel type of amphiphile, undecyl-­‐MNG, which significantly improved the diffraction limit to 5.0 Å. Crystal optimisation is underway to further improve the resolution.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry