DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu+ and Cu2+ ions

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu+ and Cu2+, with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The Kd values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu2+ (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.