title: Fluorescence of a histidine-modified enhanced green fluorescent protein (EGFP) effectively quenched by copper(II) ions.
abstract: 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.
authors.affiliation: Department of Inorganic Substances Technology and Environment Protection, Politechnica University of Bucharest, Polizu street No. 1-7, 011061 Bucharest, Romania.
authors.forename: Emese-Éva
authors.initials: EÉ
authors.lastname: Bálint
authors.forename: Judit
authors.initials: J
authors.lastname: Petres
authors.forename: Mária
authors.initials: M
authors.lastname: Szabó
authors.forename: Csongor-Kálmán
authors.initials: CK
authors.lastname: Orbán
authors.forename: László
authors.initials: L
authors.lastname: Szilágyi
authors.forename: Beáta
authors.initials: B
authors.lastname: Ábrahám
mesh_terms: D003300:Copper / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D003300:Copper