Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.

The metal sensitivity and selectivity of two fluorescent proteins was investigated. The studied proteins are mutant forms of the red fluorescent protein (DsRed); according to our results, both proteins showed sensitivity to the presence of copper[II] and nickel ions. The fluorescence intensity of the proteins has decreased significantly in the presence of copper ions in the micromolar range. Metal binding by these proteins is a reversible process, as addition of a chelating agent liberates the bound ions. Considering these advantageous properties, the studied proteins can be used as copper ion biosensors.