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Overexpression, purification and characterization of potential iron-trafficking proteins from a phenol-degrading pseudomonad

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Overexpression, purification and characterization of potential iron-trafficking proteins from a phenol-degrading pseudomonad

Wong, Amy Ho Yee (2006) Overexpression, purification and characterization of potential iron-trafficking proteins from a phenol-degrading pseudomonad. Masters thesis, Concordia University.

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Abstract

Frataxin is a small protein implicated in intracellular and mitochondrial iron trafficking. Some studies of frataxin have suggested that it can act as an iron donor for iron-sulfur cluster assembly and heme assembly. We are interested in iron-binding proteins involved in phenol degradation by Pseudomonas sp. strain CF600, and the possible involvement of frataxin and other proteins in their assembly. One of the goals of this research was to overexpress, purify, and to characterize the stability, metal iron binding properties, and subunit structure of CyaY, the frataxin homologue from Pseudomonas sp. strain CF600. CyaY was successfully overexpressed and CyaY purified to homogeneity using Fast-Flow DEAE-Sepharose, Sephacryl S-300HR Gel Filtration, and High-Performance Phenyl Sepharose chromatographies. Analytical ultracentrifugation studies indicated that CyaY exists mainly as a monomer and that it does not aggregate in the presence of Fe(II). Isothermal titration calorimetry results were consistent with the binding of 1 Fe(II) or 1 Mn(II) per monomer of CyaY, with Fe(II) binding with a higher affinity. These and other results were similar to the reported iron-binding properties of CyaY from E. coli . Additional studies showed that CyaY significantly retarded oxidation of Fe(II), helping to keep it available for incorporation into other proteins. A second goal was to overexpress and purify a putative iron-sulfur protein (CyaZ) that is encoded in the Pseudomonas putida genome adjacent to CyaY. This was successfully accomplished, and the resulting preparation appears to contain an iron-sulfur cluster. As was demonstrated for CyaY, CyaZ significantly retarded oxidation of Fe(II) in the presence of both proteins. CyaY and CyaZ are currently being tested for their ability to affect assembly of iron-containing proteins involved in phenol degradation

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Wong, Amy Ho Yee
Pagination:xvi, 118 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry and Biochemistry
Date:2006
Thesis Supervisor(s):Powlowski, Justin
ID Code:8979
Deposited By:Concordia University Libraries
Deposited On:18 Aug 2011 14:41
Last Modified:18 Aug 2011 15:00
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