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FOR THE RECORD

Variants of DsRed fluorescent protein: Development of a copper sensor

Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada

(RECEIVED March 24, 2006; FINAL REVISION May 30, 2006; ACCEPTED July 24, 2006)

The fluorescence quenching of drFP583 (DsRed) protein by metal ions was investigated. CuSO₄ reversibly and pH dependently quenched the red emission at 583 nm of drFP583. The copper binding constant was 15 mM. Following random mutagenesis, blue- and red-shifted mutants of drFP583 were generated, and their metal sensing properties were examined. Mutant gRF possessed properties similar to green fluorescent protein and had a 18 mM copper binding constant. Mutant Rmu162 had an extraordinary red-shifted emission peak at 620 nm. A third mutant, Rmu13, had dual emission peaks at 500 nm and 583 nm and possessed the properties of a copper sensor with a binding constant of 11 mM.

Keywords: red fluorescent protein; fluorescence quenching; copper ion; random mutagenesis; drFP583, DsRed

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