Defense: Amy Robison Springfield- Analysis of the Effects of Copper Ions on Protein

Amy Robison Springfield

Analysis of the Effects of Copper Ions on Protein

In order for the proteostasis network to function properly, metallostasis must be maintained. Up to a third of proteins within a proteome are metalloproteins and the metal is integral to the proteins catalytic activity, structure, or regulation. If proteostasis is dysregulated it often coexists with the dysregulation of metallostasis and vice versa. Within a class of diseases known as proteinopathies that are characterized by protein aggregation researchers have also found increased copper levels. On the other hand, when the cell enters copper-toxicity, proteins within the cell begin to aggregate. The exact effect of copper (Cu) on this protein aggregation still requires further research. The goal of this work is to gain insight into the mechanism of Cu-induced protein precipitation thereby helping further understand the effects of Cu ions on protein aggregation. This work focuses on exposing proteins to Cu ions outside of the cells biological regulatory pathway and determining the biophysical characteristics of their interactions. This allows for further understanding of the inherent properties that increase or decrease a proteins susceptibility to Cu-induced precipitation. Bottom-up proteomics techniques are used to assess individual proteins susceptibilities to precipitation. This data allows for determination of the biophysical characteristics affecting a protein's susceptibility. Electron paramagnetic resonance and inductively coupled plasma mass spectrometry were used to determine the oxidation state and location of copper ions post exposure to proteins. To compare between cell lines, bottom-up proteomic methodologies were employed and key intrinsic and extrinsic factors determining the differential susceptibilities of the cell lines were determined. To provide insight as to how Cu's redox activity affects precipitation, bottom-up proteomic methodologies were run on proteins precipitated by Cu2+, Cu1+, and Zn2+. This showed that for both oxidation states and Zn the same proteins precipitated with the same relative susceptibilities, but the overall concentration of metal differed. Additionally, reversibility assays showed that this protein precipitation was reversible for all three metals and in two different cell lines. Lastly, a bottom-up proteomics methodology utilizing iodoacetamide-alkyne was developed for identification of cryptic metal binding sites on cysteines