Purification and Functional Characterization of the Iron-Responsive Transcription Factor Aft1 from C. glabrata
Date of Award
Chemistry and Biochemistry
Director of Thesis
Due to its unique ability to serve as both an electron donor and acceptor, iron is utilized as a co-factor for many biological processes, including electron transfer, oxygen binding, and vitamin synthesis. Iron is also a key factor during fungal infections as the human host and invading pathogens battle over limited iron pools. The primary iron-responsive transcription factor Aft1 in the opportunistic pathogenic yeast Candida glabrata responds to iron deficiency by activating expression of iron acquisition genes. However, the mechanisms for sensing intracellular iron levels and regulating Aft1 activity in response to iron are unknown. The C. glabrata iron regulation system shares close homology to a similar system in the non-pathogenic yeast Saccharomyces cerevisiae, in which the monothiol glutaredoxins Grx3/4 and the BolA-like protein Bol2 form [2Fe-2S] binding complexes that deactivate Aft1 under iron replete conditions. To determine whether a similar mechanism controls C. glabrata Aft1 activity, we sought to analyze the in vitro interactions between Grx3, Grx4, and Bol2 from this yeast pathogen. For this project, we successfully subcloned the AFT1, GRX3, GRX4, and BOL2 genes from C. glabrata into Escherichia coli overexpression vectors allowing for expression of recombinant Aft1 and its presumed binding partners Grx3/4 and Bol2 alone or in complex. The overexpression conditions identified here will be used to purify these target proteins and characterize their structure and iron regulation function. In accordance with this aim, Aft1 and Grx4 were purified, and a fluorescence polarization assay was used to measure the DNA binding affinity of Aft1.
Ikahihifo-Bender, Jade, "Purification and Functional Characterization of the Iron-Responsive Transcription Factor Aft1 from C. glabrata" (2021). Senior Theses. 422.
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