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

Yasmeen Rahimi, Ann Goulding, Suresh Shrestha, Sweetie Mirpuri, Sapna K. Deo

Research output: Contribution to journalArticle

48 Scopus citations

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 Cu2+ is rapid, selective, and reversible upon addition of a copper chelator. DsRed has been employed as an in vitro probe for Cu2+ determination by us and other groups. It is also envisioned that DsRed can serve as an intracellular genetically encoded indicator of Cu2+ concentration, and can be targeted to desired subcellular locations for Cu2+ determination. However, no information has been reported yet regarding the mechanism of the fluorescence quenching of DsRed in the presence of Cu2+. In this work, we have performed spectroscopic investigations to determine the mechanism of quenching of DsRed fluorescence in the presence of Cu2+. We have studied the effect of Cu2+ addition on two representative mutants of DsRed, specifically, DsRed-Monomer and DsRed-Express. Both proteins bind Cu2+ 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 Cu2+. 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 Cu2+. Circular dichroism (CD)-spectroscopic studies revealed no effect of Cu2+-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 pKa values indicative of histidine and cysteine residue involvement in Cu2+-binding.

Original languageEnglish (US)
Pages (from-to)57-61
Number of pages5
JournalBiochemical and biophysical research communications
Volume370
Issue number1
DOIs
StatePublished - May 23 2008

Keywords

  • Copper-binding
  • DsRed
  • DsRed-Express
  • DsRed-Monomer
  • Fluorescence quenching
  • Static quenching

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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