dc.title: Exploring environment-sensitive fluorescent turn-on probes for Zn²⁺-containing metalloenzymes dc.description.abstract: Zinc is an essential micronutrient that is widely integrated into many cellular processes, serving structural, catalytic, and regulatory roles. In cells, it exists largely in two tightly regulated pools: labile and sequestered zinc. To date, most methods for studying cellular zinc can detect labile or total zinc content, but there are few tools available for studying sequestered zinc. Herein, we report the design and application of new fluorescent molecules for the study of Zn²⁺-containing metalloenzymes. Our tools are environment-sensitive fluorescent probes, where the fluorophore is conjugated to some metal-binding pharmacophore (MBP). Although the probes are weakly fluorescent when free in solution, upon MBP coordination to active site Zn²⁺ ions in metalloenzymes, fluorescence increases dramatically. Coordination is reversible, allowing us to monitor displacement of the probe in situ in real time by treatment with Zn²⁺-coordinating enzyme inhibitors or chelators. The central challenges we explore are (1) the design of molecules that yield a sufficiently large increase upon metalloenzyme binding and (2) their application to report metalloenzyme inhibitor engagement and metalation state. First, we expanded upon the design of a tool that was previously reported for visualizing the dynamic metalation state of New Delhi Metallo-β-lactmases (NDMs). We found that minor structural modifications near the MBP did not enhance fluorescence increases upon binding structurally similar enzymes, Verona Integron-encoded Metallo-β-lactamase-2 (VIM-2) or Imipenemase-1 (IMP-1). However, one modification did decrease the extent of off-target fluorescence we observed in mammalian cells. Next, we developed a new fluorophore based on a molecular rotor, whose unique fluorescent properties make it more likely to exhibit a fluorescence increase upon binding a metalloenzyme. We were able to create probes that yielded ≥15-fold fluorescence increases for seven metalloenzymes representing four different families. These molecules were selective enough to be applied in live cells, and we demonstrated their ability to detect inhibitor engagement in live E. coli and HeLa cells as well as report enzyme metalation state in live E. coli. Finally, we applied our new fluorescent probes for the study of metallo-β-lactamases (MBLs), periplasmic antibiotic resistance enzymes. Our experimental plan is to use our probes, along with a biarsenical dye, to understand the relationship between MBL metalation state, kinetic stability, and resistance conferred under conditions that mimic the site of infection.