Date of Award

Fall 2022

Document Type

Open Access Thesis

Department

Chemistry and Biochemistry

First Advisor

Caryn E. Outten

Abstract

Iron is an essential metal cofactor required for a variety of biochemical pathways in most living organisms. In recent years, the nonpathogenic fungi Schizosaccharomyces pombe has been used to study iron regulation and uncover the molecular mechanisms for controlling intracellular iron levels. Some of these regulation pathways are conserved with those of pathogenic fungi, making these studies applicable for the future development of antifungals that target iron homeostasis. In S. pombe, the GATA-type transcriptional repressor Fep1 is responsible for repressing iron uptake genes during times of iron abundance. Previous reports have proposed that the binding of an iron-sulfur (Fe-S) cluster at a conserved cysteine-rich motif in Fep1 confers its repressor activity, although the exact cofactor bound has yet to be elucidated. Literature reports a mixture of cluster types binding the protein. We look to characterize these Fe-S clusters bound by Fep1 using spectroscopic techniques, as well as determine if Fep1-DNA interactions are mediated by the presence of the Fe-S cluster using fluorescence anisotropy.

UV-visible and CD absorption spectroscopic analysis of purified, overexpressed Fep1 from E. coli indicates the presence of at least one species of Fe-S cluster. Fluorescence anisotropy assays done with holo and apo proteins suggest that binding of an Fe-S cluster allows for DNA interaction. These studies will provide further insight into mechanisms for regulating Fep1 activity in response to iron that will lay groundwork for future studies targeting iron homeostasis pathways in pathogenic fungi.

Rights

© 2022, Allison Nicole Kimsey

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