Crystal structures of Dronpa complexed with quenchable metal ions provide insight into metal biosensor development

Abstract

Many fluorescent proteins (FPs) show fluorescence quenching by specific metal ions, which can be applied towards metal biosensor development. In this study, we investigated the significant fluorescence quenching of Dronpa by Co(2+) and Cu(2+) ions. Crystal structures of Co(2+) -, Ni(2+) - and Cu(2+) -bound Dronpa revealed previously unseen, unique, metal-binding sites for fluorescence quenching. These metal ions commonly interact with surface-exposed histidine residues (His194-His210 and His210-His212), and interact indirectly with chromophores. Structural analysis of the Co(2+) - and Cu(2+) - binding sites of Dronpa provides insight into FP-based metal biosensor engineering.

Spectroscopic and Structural Analysis of Cu2+-Induced Fluorescence Quenching of ZsYellow

“…A number of fluorescence characteristics of fluorescent proteins have already been reported in various application fields and their potential is far from being fully discovered [2,4,[9][10][11]. One interesting optical property of fluorescent proteins is that their fluorescence intensity changes depending on the external factors, such as pH or the presence of metal ions [12][13][14][15][16][17][18]. The fluorescence quenching of fluorescent proteins by transition metal ions suggests their potential role as metal biosensors [12,13,19].…”

“…The fluorescence quenching of fluorescent proteins by transition metal ions suggests their potential role as metal biosensors [12,13,19]. To elucidate the molecular mechanism behind their metal-induced fluorescence quenching and to utilize them as a metal biosensors, various fluorescent proteins such as eGFP, DsRed, mEmerald, Dronpa, AmCyan, and mOrange, have been already investigated [12][13][14][15][16][17]20,21]. Most fluorescent proteins exhibited a high degree of fluorescence quenching by Cu 2+ in a highly selective, reversible, and sensitive manner [12][13][14][15][16][17].…”

“…To elucidate the molecular mechanism behind their metal-induced fluorescence quenching and to utilize them as a metal biosensors, various fluorescent proteins such as eGFP, DsRed, mEmerald, Dronpa, AmCyan, and mOrange, have been already investigated [12][13][14][15][16][17]20,21]. Most fluorescent proteins exhibited a high degree of fluorescence quenching by Cu 2+ in a highly selective, reversible, and sensitive manner [12][13][14][15][16][17]. Fluorescence quenching by transition metal ions is induced by static quenching, energy transfer between a colored metal ion and the chromophore, or perturbations of the protein structure [15,[22][23][24].…”

“…Fluorescence quenching by transition metal ions is induced by static quenching, energy transfer between a colored metal ion and the chromophore, or perturbations of the protein structure [15,[22][23][24]. In order to understand the effector mechanism of metal ion-induced fluorescence quenching, several fluorescent protein-metal ion complex crystal structures have been determined [15,16,25]. Metal ions can be bound to fluorescent proteins in two ways.…”

“…First, as for BFPms1, the metal ion is directly bound to the chromophore through the engineered metal binding site within the β-barrel [25]. Second, the metal ion is bound to histidine residues outside the β-barrel as seen in mEmerald or Dronpa [15,16]. In the latter case, two or more histidine residues are required to coordinate the metal ion.…”