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Doctoral Dissertation Announcement
Candidate: Joshua M. Muia
Doctor of Philosophy
Title: Characterization of the N-terminal Domains and Disease-causing Mutations of the Human Wilson Protein
Dr. David Huffman, Chair
Dr. Ekkehard Sinn
Dr. Dave Reinhold
Dr. James Kiddle
Dr. Wendy D. Ranson-Hodgkins
Date: Monday, February 22, 2010 9:00 a.m. - 11:00 a.m.
1710 Wood Hall
The human Wilson protein (ATP7B) is a copper transporting ATPase that is involved in copper trafficking and homeostasis. Unlike the other known P-type ATPases, such as the SERCA calcium ATPase, it possesses six homologous metal binding domains at the N-terminal end. Several mutations in the gene coding for this protein lead to Wilson disease, a hepatic disorder characterized by impaired excretion of copper in the bile and accumulation of copper in body organs such as the liver, brain, kidney, and eye cornea. Characterization of various regions of expressed and purified ATP7B has been hampered by its low stability, aggregation and degradation.
In this research, novel methods were developed to express and purify stable N-terminal copper binding domains (NTCBDs), a 577 amino acid construct. These domains were characterized by Gel Filtration, Dynamic Light Scattering and Circular Dichroism. The results indicate that the NTCBDs are polydisperse and have a larger hydrodynamic radius relative to a globular protein of the same molecular mass.
Y532H, a novel disease-causing mutation in domains 5 and 6 of the Wilson protein, was also expressed. Y532H could not be purified by established protocols. Therefore, the Y532H mutant was purified using two different methods: (1) insoluble recombinant inclusion bodies were extracted and a procedure was developed to purify the refolded protein; and (2) a lower temperature procedure was developed to express soluble recombinant protein, hereafter termed non-refolded. The Y532H mutant protein was characterized by Gel Filtration, Dynamic Light Scattering, Circular Dichroism and Nuclear Magnetic Resonance (NMR). The results reveal that the Y532H mutation does not affect the overall structural organization of the native WD5-6 and the conformational stability of the protein. However, the NMR data of the refolded Y532H mutant protein reveals structural perturbations in the area around the mutation.
This work represents an advance in the understanding of the function of the N-terminal region of the Wilson protein (ATP7B). The purification methods and strategies, key findings, and new developments presented in this work will be helpful not only to the individuals working in this field, but also to the scientific community at large.