Expanding the metallomics toolbox: Development of chemical and biological methods in understanding copper biochemistry
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Copper is an essential trace element and required for various biological processes, but free copper is toxic. Therefore, copper is tightly regulated in living cells and disruptions in this homeostatic machinery are implicated in numerous diseases. The current understanding of copper homeostasis is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level. Intracellular copper is primarily present in the monovalent oxidation state. Therefore, copper(I) selective fluorescent probes can be utilized for imaging exchangeable copper ions in live cells, but these probes are often lipophilic and hence poorly water soluble. To address this problem, water-soluble fluorescent probes with greatly improved contrast ratio and fluorescence quantum yield are characterized in this work. This work also describes a novel application of water-soluble fluorescent probes, in-gel detection of copper proteins with solvent accessible Cu(I) sites under non-denaturing conditions. Knowledge of copper(I) stability constants of proteins is important to elucidate the mechanisms of cellular copper homeostasis. Due to the high affinity of most Cu(I)-binding proteins, the stability constants cannot be determined directly by titration of the apo-protein with Cu(I). Therefore, accurate determination of Cu(I) stability constants of proteins critically depends on the Cu(I) affinity standards. However, the previously reported binding affinity values of the frequently used Cu(I) affinity standards are largely inconsistent impeding reliable data acquisition for the Cu(I) stability constants of proteins. To solve this problem, a set of water-soluble ligands are developed in this work that form colorless, air-stable copper(I)-complexes with 1:1 stoichiometry. These ligands can be applied as copper(I) buffering agents and affinity standards in order to study copper biochemistry. Copper(I) binding proteins are an integral part of the copper homeostatic machinery and they work in conjunction to regulate copper uptake, distribution, and excretion. However, available evidence indicates the existence of putative copper-binding proteins that are yet to be characterized. Therefore, several proteomics-based methods are developed in this work by employing the strategy to label Cu(I)-binding cysteines in a copper-dependent manner which lays the foundation for the identification of new copper proteins from cellular extracts.