Characterization of the D329H Disease Causing Mutation in a Truncated Form of the Human Wilson Disease Protein
Merritt, Jason S.
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The Human Wilson protein (WLNP) is needed to maintain copper concentrations within the cell at a homeostatic level. Mutations of this protein can prove to be detrimental in the function of the protein and can lead to Wilson’s disease. This study was conducted to investigate a novel D329H mutation found within WLNP. Since the mutation is located in the linker region between metal binding domains 3 and 4, it is unclear how the mutation causes Wilson’s disease. To analyze the mutation, the D329H mutation was induced in a plasmid that encoded for the expression of the first four metal binding domains of WLNP. The WLN1-4 D329H fusion protein was then expressed in E. coli and purified. The WLN1-4 D329H protein was characterized via high-resolution gel filtration and circular dichroism (CD). The results from the high-resolution gel filtration analysis showed that the construct has a calculated MW of 81,100 Da, which is double the expected MW of WLN1-4 D329H. Similar to WLN1-4, thermal unfolding CD analysis showed that WLN1-4 D329H exhibited a small change in molar ellipticity from 25 °C – 97 °C and exhibited a more negative molar ellipticity at smaller wavelengths after thermal unfolding. This suggested that the protein did not unfold, but was speculated to change conformation. Guanidine hydrochloride (GuHCl) unfolding CD analysis showed that WLN1-4 D329H exhibited two drastic changes in molar ellipticity over 0.0 M – 7.0 M GuHCl. When compared to the non-mutated WLN1-4 protein, the first major change in molar ellipticity for WLN1-4 D329H occurred at lower concentrations of GuHCl, which suggested that the WLN1-4 D329H construct is less stable. It is predicted that the destabilizing characteristic of the D329H mutation contributes to the development of Wilson’s disease.
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