Developing Analytical Tools to Study Copper Homeostasis in Biological Systems
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Nabatilan, Arielle Madison Montevirgen
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Abstract
Cellular copper concentrations are tightly regulated through a complex network of proteins and biomolecules that form thermodynamically stable yet kinetically labile complexes with Cu(I), the prevalent oxidation state of copper inside the reducing environment of cells. In order to shed new light on the mechanisms of cellular copper regulation and storage at the molecular level, selective ligands are required to manipulate copper availability and to identify and characterize the Cu(I) affinity of proteins and metallochaperones. To this end, we developed a family of high-affinity Cu(I) chelators and fluorescent probes based on phosphine-sulfide-stabilized phosphine (PSP) binding motifs featuring varying Cu(I) dissociation constants from the femto- to zeptomolar range with negligible affinity towards other biologically relevant trace metals such as Zn(II), Fe(II), and Mn(II). By immobilizing a PSP ligand on agarose, we devised a high-affinity sponge for selectively removing copper from growth media. Combined with size exclusion chromatography - inductively coupled plasma mass spectrometry (SEC-ICP-MS), we employed a suite of PSP ligands to probe the labile copper pool of mammalian cell lysates and determined the exchange kinetics, buffer depth, and average set point. In addition, we used a combination of spectrochemical titrations and electrochemical measurements to study the dynamics of competitive copper chelation in the presence of high-affinity Cu(I) ligands and to visualize TPEN-induced changes of Cu(I) levels in live mouse fibroblasts by two-photon excitation microscopy using the emission-ratiometric Cu(I) probe crisp-17. Altogether, these studies revealed that mammalian cells maintain a dynamic yet tightly controlled copper pool that is buffered at low attomolar levels through a polydisperse buffer with a broad molecular weight distribution.
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2023-08-28
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Dissertation