Fluorescence of a histidine-modified enhanced green fluorescent protein (EGFP) effectively quenched by copper(II) ions.

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.