Large-Scale Proteomic Analysis of Metal-Protein Interactions

Abstract

Metal-protein interactions are extensively found throughout biology. However, there are detrimental effects when strictly regulated metal homeostasis is disturbed. One mechanism attributed to metal toxicity, specifically copper, is protein aggregation and precipitation. Metal cations have been exploited for their protein precipitation properties in a wide variety of areas but despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated nor the susceptibility of individual proteins to aggregation upon exposure to copper ions (Cu). The work in this dissertation is focused on the identification and characterization of proteins throughout proteomics that are sensitive and tolerant to Cu-induced precipitation and on understanding the mechanism behind copper-induced protein precipitation. Initially described is a proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli (E. coli) proteome to Cu-induced aggregation. A metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom–up proteomics is used to define the metal concentration–dependent precipitation properties of Escherichia coli proteins on a proteomic scale. The results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. Analysis of the MiPP data allowed for the investigation of underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation.The MiPP protocol was also used to compare the Cu-induced precipitation properties of proteins in cell lysates generated from three cell lines from three different species: Escherichia coli, Candida albicans, and the human prostate cancer cell line 22Rv1. The human cell line was the most sensitive to Cu-induced protein precipitation, while C. albicans was the most tolerant. The unique susceptibilities of these proteomes to precipitation by Cu were examined to identify factors that influence a protein’s relative sensitivity to this effect. Identified were intrinsic factors such as frequency and solvent accessibility of known metal-binding amino acids, as well as external factors related to the molecular composition of their native cell lysates. The mechanism of copper-induced protein precipitation was also investigated using histidine hydrogen-deuterium exchange (His HDX) and limited proteolysis (LiP). His HDX analysis of copper-histidine binding in proteins throughout the E. coli proteome revealed that throughout E. coli there are specific histidines that are binding copper more strongly than others, these histidines were more likely to be found within proteins previously established to be Cu-sensitive in MiPP. Furthermore, using LiP it was found that Cu-sensitive proteins in the MIPP experiment were found to be more susceptible to proteolytic digestion in the presence of Cu suggesting Cu-induced protein unfolding events occur. Through this work a few variables involved in the aggregation and precipitation mechanism of Cu-toxicity were identified. Finally, a protein stability methodology was developed that would allow for a SPROX-like analysis of metal-protein interactions, as well as other oxidation sensitive ligands and their protein targets. The model protein results demonstrated the methods ability to gather precise thermodynamic properties of proteins in a similar fashion to SPROX, however without the use of hydrogen peroxide. Nevertheless, due to complications with modification lability during bottom-up proteomic sample preparation further experimentation is needed for implementation into a large-scale proteomic workflow.