Fluorescent probes for zinc metalloproteins to study cellular zinc homeostasis

Zinc and other metals in cells exist in two main forms: the labile or ‘free’ metal ion pool, and as metalloproteins where the metal ion is ‘tightly bound’ to the protein and necessary for structural, catalytic and regulatory functions in cells. Moreover, metalloprotein activity, localization and quantity vary in different cell types and in diseased states. While many fluorescent probes have been reported for mapping labile zinc, there are few reported for tracking zinc tightly bound to metalloproteins. Thus, the focus of my doctoral work was aimed at synthesizing fluorescent probes that can selectively and reversibly bind holo metalloproteins to allow dynamic monitoring of metals in these metalloproteins. Our fluorescent probes consist of two components: (a) a metal-binding group that binds specifically and reversibly to zinc in the protein active site and (b) a fluorophore that elicits a fluorescence turn-on upon protein binding. Following synthesis, we characterized and employed our fluorescent probes in tracking two different metalloproteins and the effect of zinc perturbations via live-cell confocal imaging. First, we studied carbonic anhydrase (CA) that catalyzes the reversible conversion of carbon dioxide to bicarbonate and hence, is essential for respiration, ion transport and pH balance. Our probe 1a interacted reversibly and selectively with CA and its isoforms showing up to 12-fold increase in fluorescence. Confocal imaging studies in HeLa cells showed that perturbations in cellular zinc did not affect the 1a-CA fluorescence, likely due to its strong zinc binding and narrow active site pocket. We further developed a library of CA-targeted fluorophores to explore structure-function relationships that govern fluorescence response in both human and bovine CAII. In bacteria, we studied New Delhi metallo-β-lactamase (NDM) that catalyzes the hydrolysis of the β-lactam moiety of commonly used antibiotics, rendering them inefficacious, and eventually leading to antibiotic resistance. Our probe 4D is the first reversible, selective fluorophore reported for tracking holo NDM and its isoforms, showing up to a 17-fold increase in fluorescence when bound to the protein. In vitro and in vivo studies showed significant effects of zinc chelators on 4D-NDM fluorescence owing to its weak Zn2 binding and open active siteChemistr