Access Type

Open Access Thesis

Date of Award

1-1-2011

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Bharati Mitra

Abstract

In mammals zinc is the second most abundant essential trace metal. Since Zn2 is a small, hydrophilic, and a highly charged ion, it cannot be transported across the plasma or intracellular organelle membrane by passive diffusion. Different types of cells require a different constant concentration of zinc at all times. Presence of excess free Zn ions can be toxic to the cell. All cells must have tightly regulated homeostatic mechanisms in order to preserve healthy levels and proper compartmentalization of zinc. This is accomplished through the actions of specialized proteins that facilitate zinc uptake, efflux and compartmentalization. If the integrity of genes responsible for maintenance of zinc homeostasis is compromised by mutations or polymorphisms, this will likely result in complex genetic variations and even sensitivity to dietary zinc in health and disease. In E.coli, the uptake of zinc is mediated by two major types of transporters: ZnuACB, which belongs to the cluster C9 family of (TroA-like) ATP-binding cassette (ABC) transporters, and ZupT, which is a member of the ZRT/IRT-related proteins (ZIP) family of transporters. ZIPs are expressed amongst different organisms in order to maintain their metal homeostasis and thus contribute greatly to their growth and development. ZIPs have also been found to play key roles in bacterial infections, as well as the onset and progression of chronic diseases in humans. In comparison with mammalian cells, E.coli's lack of complex organelles, and ease of genetic analysis, allows for direct analysis of the protein's role in metal transport. In this study E.coli's ZupT was purified and characterized, and its expression was optimized with double-selection method. The binding specificity for ZupT with Cd2+, Fe2+, Pb2+, and Zn2+ were evaluated with fluorescence spectroscopy. Furthermore, UV-Visible spectroscopy was used to further explore the binding of ZupT with Fe2+. The binding stoichiometry between ZupT and Cd2+, Pb2+, and Zn2+ were determined utilizing ICP-MS (inductively coupled mass spectrometry) analysis. Finally, based on the results of sequence analysis of ZupT, four mutants were successfully created within transmembrane (TM) regions IV and V, which have been shown in other ZIP family members to be crucial for metal binding.

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