Spectroscopic Analysis of Fe Ion-Induced Fluorescence Quenching of the Green Fluorescent Protein ZsGreen

Abstract

The fluorescence of fluorescent proteins (FPs) is quenched when they are exposed to certain transition metals, which makes them promising receptor materials for metal biosensors. In this study, we report the spectroscopic analysis of metal-induced fluorescence quenching of the fluorescent protein ZsGreen from Zoanthus sp. The fluorescence of ZsGreen was reduced to 2%, 1%, and 20% of its original intensity by Fe²⁺, Fe³⁺, and Cu²⁺, respectively. Metal titration experiments indicated that the dissociation constants of Fe²⁺, Fe³⁺, and Cu²⁺ for ZsGreen were 11.5, 16.3, and 68.2 μM, respectively. The maximum binding capacities of ZsGreen for Fe²⁺, Fe³⁺, and Cu²⁺ were 103.3, 102.2, and 82.9, respectively. Reversibility experiments indicated that the fluorescence of ZsGreen, quenched by Fe²⁺ and Fe³⁺, could be recovered, but only to about 15% of its original intensity, even at a 50-fold molar excess of EDTA. In contrast, the fluorescence quenched by Cu²⁺ could be recovered up to 89.47% of its original intensity at a Cu²⁺: EDTA ratio of 1:5. The homology model of ZsGreen revealed that the protein does not share any metal-binding sites with previously reported FPs, suggesting that ZsGreen contains unprecedented binding sites for fluorescence quenching metal ions.