Date of Award
Master of Science
Dr. David Huffman
Dr. Gellert Mezei
Dr. Susan Stapleton
Masters Thesis-Abstract Only
Wilson disease protein (ATP7B) is a Cu(I) P1B ATPase important for maturation of apo-enzymes in humans. Wilson disease, caused by mutations in ATP7B, leads to copper accumulation in liver and brain. The N-terminus of WLNP has six Cu(I) binding ferrodoxin folds (MBDs) with the sequence MXCXXC in the binding loop. In contrast, Dictyostelium discoideum (Dd) has three putative Cu(I) ATPases denoted DdATP1, DdATP7A, and DdATP3, as well as the copper chaperone DdATOX1. The N-terminus of DdATP1 contains two Cu(I) binding domains, DdATP1-MBD1 and DdATP1-MBD2. Herein is compared the Cu(I) binding and biophysical characterization of human ATP7B domain 4 (WLN4) to the chaperone DdATOX1, and the Dd MBDs DdATP1-MBD1 and DdATP1-MBD2.
WLN4, with 74-amino acids, is resistant to the guanidine hydrochloride (GuHCl) chemical denaturation; 50% of its structure is retained in 5.9 M guanidine hydrochloride (GuHCl). Cu(I) affinity studies of WLN4 reveal a KD of 1.85 x10-18 M, like that of WLN5-6, and the human chaperone Atox1. Amoebal DdATP1-MBD2, Cu(I) binding domain 2 of ATP1, gives a KD of 2.6 x10-18 M, only 50% higher than its human counterpart. It is anticipated that amoebal ligand exchange reactions are similar to those proposed for the human systems and are under kinetic control.
Circular dichroism studies with these proteins show that addition of Cu(I) to WLN4, DdATOX1, DdATP1-MBD1 and DdATP1-MBD2 led to an increase in molar ellipticity. The increase in ellipticity, due to the asymmetric nature of the copper binding site and not to the changes in the secondary structure, are attributed to the Pfeiffer effect.
Lastly, synthesis of the newly discovered metallophore Staphylopine and preparation of metal complexes are presented along with their properties.
AL-Hilfi, Aimen Sabah Ali, "Copper Binding and Biophysical Studies of Dictyostelium Discoideum PIB-Type Atpase, Wilson Protein Metal- Binding Domain Four, and Synthesis and Metal Binding Studies of Staphylopine" (2017). Master's Theses. 937.