Structural and functional study of the nitrate transporter, NrtA
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Membrane proteins play a crucial role in most cellular processes. Transporter integral proteins carry a whole variety of solute molecules across biological membranes. While the structure of thousands of soluble proteins has been determined, very few integral membrane proteins have been solved. The structures of several major facilitator superfamily (MFS) proteins that have been determined revealed structural similarities but these proteins exhibit strict substrate specificity and the structural and functional basis of this specificity is poorly understood. In this study, a membrane protein from the saprotrophic ascomycete Aspergillus nidulans NrtA, which is involved in the nitrate transport was investigated. This high-affinity nitrate transporter is a member of the MFS and shares similar structural features to other MFS proteins that have been fully characterised. In order to reach a better understanding of the function and the structure of NrtA certain amino acids - that could play a critical role in nitrate transport - were changed using oligonucleotide mediated site-directed mutagenesis. NrtA homologue sequences facilitated the identification of certain conserved and highly conserved amino acids for mutational studies. Alanine scanning mutagenesis was used to analyse highly conserved glycine residues in the conserved NRT2 motif, which were found to play important roles but not essential as mutants were able to uptake nitrate. Three other residues located adjacent to this highly conserved motif were also analysed, revealing that a phenylalanine at position 457 probably play an important, albeit not crucial, structural role, an asparagine at position 459 is critical for NrtA function, whereas a leucine at position 460 is not essential. A comparative study of NrtA residues that have been replaced by their residue-equivalent in NrtB showed that a slight change in the hydrophobicity around TM2 highly conserved arginine at position 87 and also around TM8 highly conserved arginine at position 368 can potentially be the cause of differences between NrtA and NrtB 6-fold rate of transport and 10-fold affinity to nitrate. A variety of conserved and highly conserved aromatic residues were analysed revealing a variety of roles. A residue located at the proposed nitrate binding site was also analysed and substitutions to other residues were not tolerated and no alteration to enzyme kinetics in mutant N364Q was found when using the radioactive tracer ¹³NO₃⁻. Understanding the role of certain amino acids on nitrate uptake should in turn enable development of improved methods for the prediction of NrtA’s structure.
Thesis, MPhil Master of Philosophy
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