Abstract
Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu2+. During Cu2+-induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors.
Highlights
Fluorescent proteins are optical probes that are useful in cell and molecular biology for tracking target molecules [1,2,3]
Fluorescent proteins generally exhibit fluorescence quenching in the presence of divalent metal ions, the efficiency of fluorescence quenching varies depending on the type of metal and the fluorescent protein [16,17]
The addition of higher concentrations of ethylenediaminetetraacetic acid (EDTA) at 25 and 50 mM resulted in high fluorescence recovery of 80.0 and 90.2%, respectively. These results indicate that Cu2+ -induced fluorescence quenching of ZsYellow can be recovered by EDTA and that higher amount of EDTA than that of Cu2+ is required to achieve the high recovery of ZsYellow fluorescence (i.e., > 90%)
Summary
Fluorescent proteins are optical probes that are useful in cell and molecular biology for tracking target molecules [1,2,3]. Fluorescent proteins have been widely used to study the localization of target molecules and molecular interactions (e.g., protein-protein or protein-nucleic acid) in living cells using. One interesting optical property of fluorescent proteins is that their fluorescence intensity changes depending on the external factors, such as pH or the presence of metal ions [12,13,14,15,16,17,18]. The fluorescence quenching of fluorescent proteins by transition metal ions suggests their potential role as metal
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