Development of new peptide-based receptor of fluorescent probe with femtomolar affinity for Cu+ and detection of Cu+ in Golgi apparatus

Developing fluorescent probes for monitoring intracellular Cu+ is important for human health and disease, whereas a few types of their receptors showing a limited range of binding affinities for Cu+ have been reported. In the present study, we first report a novel peptide receptor of a fluorescent probe for the detection of Cu+. Dansyl-labeled tripeptide probe (Dns-LLC) formed a 1:1 complex with Cu+ and showed a turn-on fluorescent response to Cu+ in aqueous buffered solutions. The dissociation constant of Dns-LLC for Cu+ was determined to be 12 fM, showing that Dns-LLC had more potent binding affinity for Cu+ than those of previously reported chemical probes for Cu+. The binding mode study showed that the thiol group of the peptide receptor plays a critical role in potent binding with Cu+ and the sulfonamide and amide groups of the probe might cooperate to form a complex with Cu+. Dns-LLC detected Cu+ selectively by a turn-on response among various biologically relevant metal ions, including Cu2+ and Zn2+. The selectivity of the peptide-based probe for Cu+ was strongly dependent on the position of the cysteine residue in the peptide receptor part. The fluorescent peptide-based probe penetrated the living RKO cells and successfully detected Cu+ in the Golgi apparatus in live cells by a turn-on response. Given the growing interest in imaging Cu+ in live cells, a novel peptide receptor of Cu+ will offer the potential for developing a variety of fluorescent probes for Cu+ in the field of copper biochemistry.