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Date of Award
Biochemistry and Molecular Biology
All living organisms required trace metals for different purposes, e.g. some metals are important as a cofactor in different proteins. Although trace metals are important for living system, in excess amounts they can be toxic. Deficiency or excess amount of these metals can lead to health problems in humans; therefore, maintaining homeostasis of these metals is very crucial. Homeostasis of trace metals is maintained by different transporter proteins. One of the important metal transporter families is the ZIP (Zrt-Irt like proteins) family of proteins. ZIP transporters are present in all kingdoms; they transport metal ions from the lumen of the intracellular organelles to the cytoplasm. They can transport many different metals such as zinc, copper, cadmium, iron, manganese, cobalt. The first characterized bacterial ZIP protein is ZupT.
In this study, ZupT purification was performed. To find out a good expression system for ZupT, different E. coli strains were used. The best expression system for ZupT was determined and conditions for expression were optimized. Partial purification of the protein was successful, though due to overall poor expression, it could not be purified further.
Homology alignment with other ZIP proteins showed conserved residues (which may contribute to the metal binding site) and these residues were replaced by other amino acids; these mutants were used further for metal uptake assays. Conserved amino acid residues at position 152, 148, 119, 123 were replaced with other amino acids. Protein sequence for wild type and mutants were confirmed. Metal uptake assays were performed for wild type ZupT, and the mutants H148NZupT and E152QZupT. Metal uptake assays were performed for zinc, copper, cadmium, cobalt and manganese. Wild type ZupT showed uptake of zinc, copper, manganese whereas for cadmium and cobalt no consistent uptake was observed. Uptake of zinc and copper was impaired in case of E152QZupT; H148N ZupT showed no zinc uptake and reduced copper uptake. Also, no uptake activity was observed for cadmium, manganese and cobalt. Mutation of histidine at position 148 may be important for uptake of divalent metal ions in general whereas glutamic acid at position 152 might be necessary for uptake of zinc and copper.
Joshi, Pallavi Bhagwat, "Investigating The Metal Selectivity Of Zupt Using Site Directed Mutagenesis" (2017). Wayne State University Theses. 623.