Mechanism of copper induced fluorescence quenching of red fluorescent protein, DsRed

Abstract

The red fluorescent protein, DsRed, and a few of its mutants have been shown to bind copper ions resulting in quenching of its fluorescence. The response to Cu 2+ is rapid, selective, and reversible upon addition of a copper chelator. DsRed has been employed as an in vitro probe for Cu 2+ determination by us and other groups. It is also envisioned that DsRed can serve as an intracellular genetically encoded indicator of Cu 2+ concentration, and can be targeted to desired subcellular locations for Cu 2+ determination. However, no information has been reported yet regarding the mechanism of the fluorescence quenching of DsRed in the presence of Cu 2+. In this work, we have performed spectroscopic investigations to determine the mechanism of quenching of DsRed fluorescence in the presence of Cu 2+. We have studied the effect of Cu 2+ addition on two representative mutants of DsRed, specifically, DsRed-Monomer and DsRed-Express. Both proteins bind Cu 2+ with micromolar affinities. Stern–Volmer plots generated at different temperatures indicate a static quenching process in the case of both proteins in the presence of Cu 2+. This mechanism was further studied using absorption spectroscopy. Stern–Volmer constants and quenching rate constants support the observation of static quenching in DsRed in the presence of Cu 2+. Circular dichroism (CD)-spectroscopic studies revealed no effect of Cu 2+-binding on the secondary structure or conformation of the protein. The effect of pH changes on the quenching of DsRed fluorescence in the presence of copper resulted in p K a values indicative of histidine and cysteine residue involvement in Cu 2+-binding.