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

• Published in: Biochemical and biophysical research communications 2008-05, Vol.370 (1), p.57-61
• Main Authors: Rahimi, Yasmeen, Goulding, Ann, Shrestha, Suresh, Mirpuri, Sweetie, Deo, Sapna K.
• Format: Article
• Language: English
• Subjects: Animals; Circular Dichroism; Copper - chemistry; Copper-binding; Cysteine - chemistry; DsRed; DsRed-Express; DsRed-Monomer; Fluorescence; Fluorescence quenching; Histidine - chemistry; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Mutation; Protein Binding; Protein Structure, Secondary; Spectrophotometry, Ultraviolet; Static quenching
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2008.03.034

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.