Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants.

The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.