Fluorescent chemosensors for divalent zinc based on zinc finger domains. Enhanced oxidative stability, metal binding affinity, and structural and functional characterization

The design, synthesis, and characterization of a family of peptides modeled after the zinc finger domains, which has led to the production of a fluorescent peptidyl sensor for divalent zinc with enhanced oxidative stability, are reported. The chemosensor design comprises a synthetic peptidyl template and a covalently attached fluorescent reporter which is sensitive to metal-induced conformational changes in the polypeptide construct. The modular synthetic approach employed for the construction of these chemosensors allows independent modification of the metal coordination sphere and the fluorescent reporter group. The structural, fluorescence, and zinc binding properties of these peptides and the effects of integrating various environment sensitive fluorophores, 4-(dimethylamino)-benzamide, 5-(dimethylamino)naphthalenesulfonamide, and 3-carboxamidocoumarin, are described. Manipulation of the Ligand sphere, by removal of one of the pair of thiolate ligands, was undertaken to enhance the oxidative stability of the chemosensor. For each of these peptides, the apparent dissociation constant of the peptide-zinc complex has been determined by spectroscopic methods. High-affinity binding, with dissociation constants ranging from 7 pM to 65 nM, is observed.