Zinquin (ZQ) is a fluorescent probe that was initially used to quantify labile zinc in biological systems due to its fluorescence emission at ~500 nm that appears upon formation of [Zn(ZQ)2]2- complexes. Subsequent studies in complex biological matrices revealed a blue shift in fluorescence emission for ZQ in the presence of Zn-binding proteins cells and peptides. The distinct interaction of ZQ with these biological components and the nature of the interactions driving the blue shift in fluorescence emission is not fully understood. In this work the metallo-β-lactamase NDM-1 was used as a model to characterize the molecular interactions of ZQ with a zinc-binding protein. NDM-1 has two well-characterized zinc ions bound in its active site enabling experimentation to detect zinc stripping quantify enzyme activity and characterize structure. Results from fluorescence spectroscopy enzyme activity assays inductively-coupled plasma mass spectrometry X-ray absorbance spectroscopy and nuclear magnetic resonance reveal two sources of ZQ fluorescence for this protein: formation of [Zn(ZQ)2]2- by stripping the weaker-bound zinc and a zinc-mediated ternary complex formed by NDM-1 Zn2+ and ZQ. This 1:1:1 complex is situated around the higher-affinity zinc binding site in NDM-1 with ZQ interacting with both protein-bound zinc and the non-polar residues of the NDM-1 active site the latter of which causes the blue shift in fluorescence. This study characterizes the ternary complex giving rise to a fluorescence shift and highlights the importance of understanding the complex interactions that occur with fluorescent probes within cells.
Supporting Data contains plasmid sequencing results Bruker NMR data peak annotations for holo 15N-NDM-1 and monoZn NDM-1:ZQA HADDOCK structure.
Supporting Data contains plasmid sequencing results Bruker NMR data peak annotations for holo 15N-NDM-1 and monoZn NDM-1:ZQA HADDOCK structure.