Mobile Zn2+ ions act as signaling agents in critical biological processes, including those located within the brain. To elucidate the actions of mobile Zn2+, we have been examining hybrid fluorescent sensors. These tools combine the tunability of small molecule probes with the targetability of protein-based sensors through covalent linkage of derivatized synthetic fluorophore substrates to genetically encodable protein tags such as HaloTag, which react with specific functional groups. We report here zinc-selective reaction-based sensors that are prepared by acetylation of probes having an N3O binding motif and high dynamic ranges in cells and brain tissue slices. Probe acetylation leads to quenching of background fluorescence that arises from inefficient photoinduced electron transfer. Zinc-promoted deacetylation yields high fluorescence turn-on, and there is minimal background produced by spontaneous hydrolysis. The engineered enzyme HaloTag reacts rapidly with Zinpyr1- and di-acetylated Zinpyr1-modified chlorocarbon substrates to form covalent protein-small molecule hybrids that retain the zinc-selective sensing properties of the parent synthetic sensors. This system can detect exogenous Zn2+ and endogenous mobile Zn2+ in response to reactive nitrogen species in live HeLa cells. HaloTag fused with protein sequences targets specific subcellular locales, including the nucleus and mitochondrial outer membrane, resulting in hybrid systems that are not only highly sensitive and zinc selective but which can be predictably localized to or on subcellular organelles in live cells. This work was supported by a grant from the National Institute of General Medical Sciences and by a postdoctoral fellowship to M.L.Z. from the National Institute of Biomedical Imaging and Bioengineering.

Targetable, reaction-based small molecule-protein hybrid sensors for detecting mobile zinc

251st National Meeting (2016)

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