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

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 pK(a) values indicative of histidine and cysteine residue involvement in Cu(2+)-binding.