Metal ion accessibility of histidine-modified superfolder green fluorescent protein expressed in Escherichia coli

Abstract

Green fluorescent protein (GFP) is frequently utilized for metal ion detection and quantification. To improve the metal binding potential of GFP, three residues (N146, F165, and L201) were substituted to histidines. Each variant responded differently upon interaction with metal ions. More than 80% of N146H, having the most accessible surface area, could bind to immobilized metal ions. However, only F165H exhibited significant differences in quenching by soluble metal ions (22% fluorescence decrease) in comparison with the template protein (12%). These findings can be utilized for designing GFP variants for metal binding and sensor applications.

Development of a Biosensor for Copper Detection in Aqueous Solutions Using an Anemonia sulcata Recombinant GFP

“…In this set of experiments, we observed a similar trend of fluorescence reduction using lead, cadmium and nickel as quenchers, suggesting that those metals interact with the proteins but with different and probably non-specific effects. These data were in line with findings in other investigated FPs [6,7,10,12].…”

“…For instance, fluorescent proteins (FPs) from different marine organisms may become potential candidates for sensor development. In fact, FPs, such as red and green (GFP) proteins, were used to evaluate the effects of heavy and essential metals on fluorescence intensity [5][6][7][8][9][10][11][12].…”

“…Moreover, the introduction of metalbinding sites onto the GFP surface places metals in close proximity to chromophore resulted in fluorescence quenching probably induced by energy transfer, at low concentrations (micromolar) of copper ions [10]. Additional mutations on the histidines at different positions have been performed resulting in increased Cu 2+ -based quenching [12,13]. Moreover, a dramatic absorbance and fluorescence change upon mercuration in mutant Aequorea victoria GFP was recorded [6].…”

GFP-Based Biosensors

“…Making a few modifications can make GFP sensitive to the concentration of other ions.For example, superfolder GFP can be made sensitive to copper ions by mutating the arginine at position 146 to a histidine, which, as previously mentioned, coordinates well with metal ions [79]. GFP can also become sensitive to ions by creating channels in the structure through which small molecules can pass through and access the chromophore (Figure 9).…”

Expanding the chemistry of fluorescent protein biosensors through genetic incorporation of unnatural amino acids

“…Single GFP-based in vitro Cu 2+ sensors have been developed by the introduction of histidine and cysteine residues as copper-binding sites, which results in fluorescence quenching upon binding to Cu 2+ . [51][52][53][54] To further improve the sensitivity and selectivity towards Cu 2+ , Ayyadurai and co-workers 10 constructed a novel FP-based sensor by replacing all tyrosine residues in the GFP with metal-chelating L-DOPA. 10 The resulting GFPdopa mutant displayed good selectivity towards Cu 2+ over other metal ions, including K + , Mg 2+ , Ca 2+ , Na + , Cd 2+ , Co 2+ , Mn 2+ , Ni 2+ , and Zn 2+ .…”