Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Timothy L. Stemmler

Abstract

Iron-induced free radical damage has been implicated in the pathology of diseases of iron overload such as Friedreich's Ataxia, a genetic disorder characterized by an accumulation of iron in actively metabolizing tissues ultimately leading to cardio- and neuro- degeneration and cell death. It is caused by an inability to synthesize the mitochondrial protein, frataxin. Frataxin has been shown by numerous groups to be a part of the iron-sulfur cluster (ISC) multicomplex, where it functions in the capacity of a potential iron provider and an allosteric modulator of both the cysteine desulfurase and scaffold protein ISU. My research has been focused on studying the interaction between frataxin and ISU, particularly the mechanism of iron binding/ iron acceptance by ISU from frataxin. Using XAS, the Stemmler laboratory characterized the ISU-Fe(ll) bound complex in four orthologs and found that ISU appears to bind iron in a 6 coordinate oxygen/nitrogen ligand environment. The absence of any thiol ligation to metal indicates iron loading onto ISU occurs at a site distinct from its cysteine rich active site. Using site directed mutagenesis, followed by biochemical and biophysical analysis of the mutant proteins in the yeast system, we demonstrate that charged residues E144A and D145A possibly form the interface between iron loaded frataxin and ISU and

are involved in initial iron acceptance by ISU.

In this thesis, we also describe the crystal structure of Frataxin in D. melanogaster (Dfh). Dfh is well-folded with an α-β sandwich motif comprising two α-helices and six β-strands sandwiched between the two helices like its yeast and human homologs. The Dfh protein does not have a long unstructured N-terminal region like that seen in the yeast and human proteins and it has a considerably longer C-terminal tail; two factors that could contribute to its extraordinary stability. 15N HSQC experiments performed on Dfh in the presence of iron show that the majority of the perturbed residues lie on the α helix-1 and β-strands 1 & 2 region; this is in the region of the conserved acidic patch shared by frataxin proteins indicating an iron binding/ chaperone role for Dfh. As the Dfh protein has been found to be much more stable than its yeast and human counterparts, it would serve as a great starting point to attempt to crystallize the cysteine desulfurase/Isd11/Isu/frataxin (SDUF) proteins in the Drosophila system considering the poor stability of the yeast proteins.

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