The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni2+-NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6-Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.
Recently, the ‰uorometric detection of biomacromolecules has attracted much attention. In this paper, we report the development of two new techniques utilizing the chemical properties of amino acids or peptides: 1) a ‰uorescence assay for serine/threonine kinase activity; and 2)``turn-on'' ‰uorescent probes for protein labeling, which could be useful for bioimaging. To develop the novel kinase assay, we utilized the chemical reactivity of phosphorylated serine or threonine. Phosphorylated peptide on resin was successfully labeled ‰uorescently via base-mediated b-elimination, followed by Michael addition with novel coumarin derivatives. Protein kinase A and casein kinase I activities were detectable with our method. Also, this method was confirmed to be applicable for kinase inhibitor screening. For the development of the novel protein labeling technique, the selective interaction between``His-tag (His 6 )'' and``metal ion nitrilotriacetic acid (NTA) complex'' was utilized. This interaction is useful for protein puriˆcation and immobilization. We designed ‰uorescent probes composed of a ‰uorophore and Ni 2+ or Co 2+ -NTA complex. These probes were found to be weakly ‰uorescent as expected. When His-tag peptide was added, these probes became brightly ‰uorescent. On the other hand, these probes remained non ‰uorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-HisLeu-OH). These probes will be powerful tools for the bioimaging of target proteins.
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