Date of Award

6-2014

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Dr. David Huffman

Second Advisor

Dr. Blair Szymczyna

Third Advisor

Dr. Gellert Mezei

Fourth Advisor

Dr. Douglas Coulter

Keywords

Wilson disease protein, metal-binding domains

Abstract

Wilson disease protein (WLNP) is a P1b-type ATPase crucial for maintaining copper homeostasis in humans. Mutations in this protein result in the autosomal recessive disorder Wilson disease, a condition characterized by copper accumulation in the liver and brain. WLNP provides copper for incorporation into cuproproteins and exports excess copper into the bile for excretion. There are six metal-binding domains (MBDs) in WLNP, found within the first 650 amino acids of this 1,465 amino acid protein. Though each MBD has a different amino acid sequence, all MBDs possess a similar ferredoxin fold with a conserved hydrophobic core and a MXCXXC metal-binding motif.

The manner in which the six MBDs communicate with each other and how they affect other cytosolic-facing domains of WLNP is not understood. There is a long linker between the fourth and fifth MBDs that provides spatial separation between the first four and the last two MBDs. To better understand how the first four MBDs function, I pursued a detailed biophysical characterization of these domains. Strikingly, when MBD4 is expressed by itself, it is highly resistant to both chemical and thermal denaturation: 50% of its structure is retained in 5.9 M guanidine hydrochloride (GuHCl) and it has a melting temperature of 78˚C. In contrast, when MBDs1-3 are expressed together as a single protein, 50% of its structure is retained at 2.3 M GuHCl and the melting temperature is 58˚C. Furthermore, the unusual stability of MBD4 is preserved when it is expressed in a protein construct that contains all four MBDs (MBDs1-4). In MBDs1-4, denaturation by GuHCl produced a double sigmoidal curve in which the second sigmoid correlated with that of MBD4 while the first one correlated with MBDs1-3. MBD4 also influenced the thermal denaturation of MBDs1-4, albeit in a more complicated manner. MBDs1-4 did not display a thermal unfolding transition and instead underwent a structural rearrangement that resulted in soluble aggregation. Though the extreme conditions that MBD4 can withstand are never experienced in vivo, the enhanced stability of this domain may play a role in its ability to serve as a primary target of copper acquisition.

Access Setting

Dissertation-Open Access

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