Development Of A Novel Protein Sensor For The Intracellular imaging of Zinc.

Abstract

Zinc is the second most abundant transition metal in the human body. In recent years, research on zinc homeostasis in mammalian cells has revealed distinct pools of zinc. Zinc is tightly bound to metalloproteins as a cofactor or structural component including metallothioneins that may provide a specific labile pool of zinc. The zinc-binding protein, Carbonic Anhydrase II (CA(II)) can be compartmentalized in intracellular organelles such as lysosomes, endosomes, endoplasmatic reticulum, mitochondria and the Golgi apparatus or in specialized organelles like synaptic vesicles and secretory granules. However, the concentration of “free” zinc inside cells is estimated to be at or below thepicomolar level. Disruption of cellular Zn2+ homeostasis is implicated in several major disorders including Alzheimer's disease, diabetes, and cancer.

The molecular mechanisms of Zn2+ physiology and pathology are insufficiently understood, owing in part to the lack of tools for measuring changes in intracellular Zn2+ concentrations with high spatial and temporal fidelity. To address this critical need, we have designed, characterized, and applied an intracellular protein-based sensor expressed in BL21(DE3) cells for the ratiometric imaging of Zn2+ based on the carbonic anhydrase (CA(II)) platform. FRET excitation ratiometric detection of intracellular Zn2+ occurs through Zn2+- mediated energy transfer between dapoxylsulfonamide (Dps) bound to carbonic anhydrase and the fluorescent protein, mCherry fused to the C-termini of (CA(II)).

The series of probes developed are expressible in various cell types, feature visible excitation and emission profiles, and show excellent selectivity responses for Zn2+ at intracellular concentrations. Furthermore, the ratio of the binding affinity for Zn2+/Cu2+ can be varied by mutations at Q92 and T199. We demonstrate the value of the (CA(II))_mCherry sensor for biological applications by imaging induced changes in intracellular [Zn2+] in E. coli.. We anticipate that (CA(II))_mCherry and related probes will prove useful for elucidating the biology of Zn2+ in a number of in vivo processes.