DsRed (DrFP583) is a red fluorescent protein cloned from reef corals [101]. Eli and Chakrabartty investigated the fluorescent emission changes in response to metal ions in DsRed and its mutant proteins (gRF, Rmu74, Rmu80, Rmu162, and Rmu13) [97]. DsRed, Rmu13, and gRF showed a high percentage of quenching of Cu²⁺, whereas the mutants Rmu74, Rmu80, and Rmu162 showed modest quenching of Cu²⁺, indicating that the quenching effect of the metal can differ among the mutants despite using red fluorescence of the same origin. Rmu13 was characterized by dual-fluorescence emission peaks at 500 and 580 nm. The titration of DsRed (blue- and red-shifted mutants) and Rmu13 with Cu²⁺ yielded binding constants of 15 and 11 mM, respectively. Moreover, Sumner et al. reported that copper in the binding constant of DsRed is 0.5 mM [102], which differs from that reported by Eli and Chakrabartty. This apparent discrepancy is due to differences in the formulas for calculating the binding constants, which indicates the need for formalization of the binding constant calculation method applied when developing FP-based metal biosensors (see Section 3 Discussion). The dissociation constant (K_d) of the copper-binding site of DsRed and Rmu13 was approximately 14.80 and 10.90 μM, respectively.

Although structural evidence for quenchable metal binding to DsRed has not been reported yet, biochemical and spectral analyses clearly show a Cu²⁺-binding site on DsRed. Eli and Chakrabartty investigated copper binding after chemical modification using iodoacetamide and diethyl pyrocarbonate (DEPC) of cysteine and histidine residues, respectively, to trace the Cu²⁺-binding site of DsRed [97]. As a result, K_d increased by >100 μM when treated with iodoacetamide, whereas no remarkable difference was observed upon treatment with DEPC [97]. This result indicates that the cysteine residue of DsRed is involved in Cu²⁺-binding [97]. Rahimi et al. investigated the mechanism of DsRed fluorescence quenching: far-UV CD spectral analysis showed no structural changes in the entire protein, even when DsRed was bound to a metal ion [103]. In addition, the UV-visible spectra and Stern–Volmer constant showed a static quenching mechanism during the interaction between DsRed and Cu²⁺ [103]. The crystal structure of DsRed showed a tetramer with 222 non-crystallographic symmetry [104] (Figure 3a).

To understand the Cu²⁺-binding site of DsRed, the deposited crystal structure of DsRed (PDB code 1GGX) was analyzed. The Cys117 residue, which can bind Cu²⁺ of DsRed, was located on the β7-strand of the β-can surface (Figure 3a,b). The closest distance between the sulfhydryl of Cys117 and the imidazolinone ring of the chromophore was approximately 15.4 Å (Figure 3c). Because the DsRed monomer contains only one cysteine residue, the tetrameric DsRed can bind four Cu²⁺ ions (Figure 3a). Moreover, as Cys117 of DsRed was located on the surface of β-can, the chelator can easily remove the Cu²⁺ bound to Cys117, which is consistent with the previous spectral results [97].

BFPms1 is an engineered fluorescent protein that uses a specific metal to bind GFP and alter its fluorescence signal [35]. To create the chromophore of BFPms1 as a metal ligand, such as porphyrin, Tyr66 (corresponding to the chromophore) was substituted with a histidine residue. In particular, Barondeau et al. have developed an H148G mutant to generate holes between β-strands for metal access (Figure 4a). Moreover, additional amino acid substitutions were performed to increase quantum yield (Y145F), improve solubility (F64L, F99S, M153T, and V163A), and promote rapid chromophore formation (S65T). The metal ion screening results showed that Cu²⁺ quenched the fluorescence emission whereas Zn²⁺ enhanced it. BPFms1 has a K_d of 24 μM and 50 μM for Cu²⁺ and Zn²⁺, respectively, whereas K_D is greater than 2 mM for other transition metal ions [35]. The crystal structures of BFPms1 complexed with Zn²⁺ and Cu²⁺ were determined at a resolution of 1.44 and 1.50 Å, respectively. In Zn-bound BFPms1 (PDB code 1KYS), Zn²⁺ exhibited a trigonal bipyramidal geometry distorted by the chromophore Glu222 and water molecules (Figure 3b). In Cu-bound BFPms1 (1KYR), a square-planar geometry was exhibited by the amino acid Glu222 and the porphyrin of the chromophore (Figure 3c). The histidine ring of BFPms1 exhibited a metal bond shift and rearrangement of Glu222. Structural changes around the chromophore have been shown to occur depending on the type of metal ion bonded to the chromophore. However, it has not been clarified whether the fluorescence emission is changed by the conformational change of the chromophore or whether a metal is bound to the chromophore and has an effect. In this review, additional metal ions binding to the surface of β-can from coordinates deposited in PDB (PDB code 1KYR and 1KYS) were observed. In the BFPms1-Zn²⁺ structure, three additional Zn²⁺ ions interacted with the His25, Glu142, and Glu172 residues, whereas in BFPms1-Cu²⁺, Mg²⁺ ions interacted with Glu142 in the final model structure. In a structural study of BFPms1, only the metal around the chromophore was described [35], and further studies are required to determine whether the metal bound to the surface of BFPms1 affects fluorescence emission.

Two surface-exposed histidine residues separated by one residue of the β-sheet (i and i+2) create robust transition metal ion binding sites in proteins [95,105]. Using this protein engineering concept, Yu et al. have generated mutant constructs of mEmerald-1H (His147), mEmerald-2H (His202 and His204), and mEmerald-3H (His147, His202, and His204). In mEmerald-2H, two histidine residues are located on the same β10-strand [9]. In Emerald-3H, the His147 residue is located at the β7-strand, which is the closest neighbor position of the two histidine residues of mEmerald-2H. mEmearld-2H and mEmerald-3H showed strong fluorescent quenching for Cu²⁺ with a K_d of 0.3 and 0.2 μM, respectively, whereas wild-type mEmerald and mEemrald-1H showed weak (at high concentration) or low affinity (1–10 μM), respectively. Accordingly, Yu et al. have generated the most robust and sensitive mEmerald-3H mutant (iq-mEmerald) for Cu²⁺-induced fluorescence quenching. Physiological concentrations of Ca²⁺ (1 mM) and Mg²⁺ (10 mM) did not affect the fluorescence quenching behavior of iq-mEmerald. The engineered histidine motif of iq-mEmerald can bind specifically to other transition metal ions, such as Co²⁺, Ni²⁺, and Zn²⁺. The crystal structure of iq-mEmerald complexed with Ni²⁺ (PDB code 4KW8) and Zn²⁺ (4KW9) ions were determined at a resolution of 2.45 Å, and 1.80 Å, respectively (Figure 5a). Both Ni²⁺ and Zn²⁺ interacted with His202 and His204 of iq-mEmerald; however, the conformations of the side chains of the metal-interacting histidine residues differed (Figure 5b). In native iq-mEmerald, the side chains of His202 and His204 were in the direction opposite to the metal-binding site. In iq-mEmerald-Ni²⁺, the side chains of His202 and His204 were rotated toward the metal-binding site, and Ni²⁺ was coordinated by His202 and His204 at a distance of 2.08 and 2.04 Å, respectively. In contrast, in iq-mEmerald-Zn²⁺, the conformation of His202 was similar to the conformation of His202 in metal-free iq-mEmerald, whereas the side chain of His204 was rotated to Zn²⁺ ion. Zn²⁺ was coordinated by His202 and His204 at a distance of 2.01 and 2.00 Å, respectively. The closest distance between the imidazoline ring and metal-binding site for Ni²⁺ and Zn²⁺ was approximately 15.7 and 13.4 Å, respectively (Figure 5c). This result indicates that the metal-binding configuration and the distance between the metal ion and chromophore differ depending on the type of metal ion, although the metal-binding sites are the same.

In contrast, in iq-mEmerald-Zn²⁺ (PDB code 4KW8) deposited in PDB, one Cl⁻ was modeled at a position 2.24 Å away from Zn²⁺ interacting with His202 and H204. Moreover, in the final model structure of iq-mEmerald-Zn²⁺, four Zn²⁺ were observed, which interacted with Glu124, His139, His147, and His169. However, the effect of the addition of metal ions has not yet been described in related structural studies [9]. Accordingly, it is necessary to investigate the metal-binding sites observed at nonengineered locations and further study their effects on fluorescence emission. This structural information is useful for understanding the metal-binding moiety for further engineering of FP-based metal biosensors. In addition, because the crystal structure of iq-mEmerald complexed with the most effective quencher Cu²⁺ has not yet been determined, further crystallographic studies of Cu²⁺-bound iq-mEmerald are required to better understand the molecular mechanism of Cu²⁺ binding to iq-mEmerald.

Dronpa is a photoswitchable green fluorescent protein isolated from the Echinophyllia sp. SC22 [85]. The Dronpa chromophore consists of a CYG tripeptide with maximum excitation and emission wavelengths of 503 and 518 nm, respectively [85]. The cysteine residue of the Dronpa chromophore is critical for its photoswitchable function [106]. The optical properties of metal-induced fluorescence quenching of Dronpa were screened using divalent ions such as Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Co²⁺ [98]. Cu²⁺ clearly exerted the strongest quenching effect on the fluorescence emission of Dronpa, and Co²⁺ reduced the fluorescence intensity, whereas other metal ions did not significantly affect the fluorescence intensity of Dronpa. The fluorescence intensity of Dronpa quenched by Cu²⁺ and Co²⁺ was reversible and recovered by 97% and 95%, respectively, upon the addition of an EDTA chelator. Dronpa naturally has three histidine residues (His194, His210, and His212) located on the surface of the β-barrel. The crystal structures of Dronpa complexed with Co²⁺, Ni²⁺, and Cu²⁺ had a resolution of 2.20, 1.90, and 2.85 Å, respectively. In the Dronpa-Co²⁺, the Co²⁺ interacted with the His194 and His212 residues at a distance of 2.21 and 2.26 Å, respectively, and showed distorted octahedral coordination along with four water molecules. In Dronpa-Ni²⁺, the Ni²⁺ interacted with the His194 and His212 residues at 2.12 and 2.19 Å, respectively, and showed distorted octahedral coordination along with four water molecules. Accordingly, the metal coordination features of Dronpa-Co²⁺ and Dronpa-Ni²⁺ were similar but distinct from those of the surrounding water molecules. The distance between Co²⁺/Ni²⁺ and the imidazoline ring of the chromophore was approximately 14.4 Å. In Dronpa-Cu²⁺, Cu²⁺ metal interacts with His210 and His212 at distances of 2.34 and 2.54 Å, respectively. However, the Cu²⁺-binding coordination was not clear because of the poor electron density map. The side chains of His210 and His212 were rotated to the Cu²⁺-binding site by 90° and 115°, respectively, compared with the native Dronpa structure. Accordingly, when Dronpa interacts with a metal, the histidine residue at the binding site undergoes a conformational rearrangement, indicating that the metal has a geometrically stable configuration. The distance between Cu²⁺ and the imidazoline ring of the chromophore is approximately 14.9 Å. In this study, the metals around the chromophore were described [98]; however, additional metal ions were present at the bottom of the β-barrel in the deposited crystal structure. In Dronpa-Co²⁺, -Cu²⁺, and -Zn²⁺, additional metal ions were bound to His200, and the side chain was exposed to the solvent (Figure 6a). Moreover, the distance between the imidazoline ring of the chromophore and His200 was approximately 19 Å. Accordingly, further experiments are required to determine whether the metal bound to His200 affects the fluorescence-quenching ability of Dronpa.