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Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λabs.max = 520 nm, λfluor.max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.


Response Analysis to Heavy Metal
The effect of heavy metal ions on the fluorescence emission of SPI1085g2-PEB was determined by adding various metal ions (CuSO 4 , NiCl 2 , MnCl 2 , ZnSO 4 , CrCl 3 , CoCl 2 , PbCl 2 ,CdCl 2 ) at a final concentration of 40 μM to 0.25 μM biliprotein in KPB-containing 0.5 M NaCl. After 30 min of incubation at 25°C, the samples were excited at 500 nm, and the emitted fluorescence was measured at 555 nm on a Perkin Elmer LS-55 spectrofluorometer. The fluorescence intensities were measured at 30 sec intervals for 30 min to investigate the response time.
To further investigate the effect of Cu 2+ on the fluorescence of SPI1085g2-PEB, 10 μl of a Cu 2+ solution at varying concentrations (0~5 mM) was added to 490 μl of 0.25 μM biliprotein (in KPB-containing 0.5 M NaCl, pH 7.2). After 30 min of incubation at 25°C, the fluorescence at 555 nm of the samples and the emission spectra were recorded after excitation at 500 nm. To assess the reversibility of the fluorescence quenching of SPI1085g2-PEB, simultaneous addition of different concentrations of EDTA to the biliprotein solution with the addition of Cu 2+ was carried out. After 30 min of incubation at 25°C, the fluorescence emission spectra of the samples were recorded. All experiments were performed in triplicate at 25°C.

Stern-Volmer Plot and Dissociation Constant
A Stern-Volmer plot was generated for 0.25 μM biliprotein with different concentrations of Cu 2+ using the equation as follows: Here, F is the measured fluorescence intensity in presence of Cu 2+ , F 0 is the measured fluorescence intensity without Cu 2+ , [Q] is the concentration of Cu 2+ , and K SV is the Stern-Volmer constant. The curve for F/F 0 against copper concentration was linearly fitted using the equation, and the slope was determined to give the value of Ksv.
The dissociation constant for copper (K d ) was calculated by plotting the [ΔF/ΔF max ] against the different concentrations of Cu 2+ , fitted using the equation as follows [18]: where ΔF is the change in measured fluorescence, ΔF max is the maximum fluorescence change, K d is the dissociation constant, [P] is the protein concentration, and [Cu] is the concentration of Cu 2+ . The curve for ΔF/ ΔF max against copper concentration was fitted using the equation and K d was calculated. The strength of interactions between protein and a ligand is inversely related to the value of K d .

Chromophorylation of SPI1085g2
SPI1085, a hypothetical signal transduction protein from S. subsalsa, contains four GAF domains, as previously reported, one of which, SPI1085g3, can bind PCB as a chromophore and exhibits a red/dark fluorescence switch [21]. Here, we cloned the DNA segment encoding the second GAF domain of SPI1085 (SPI1085g2, residues 216-369) from the S. subsalsa FACHB351 genome (Fig. 1A). To evaluate the bilin-binding capability of SPI1085g2, we first tested the chromophorylation of SPI1085g2 with PCB. SPI1085g2 can covalently bind PCB, as demonstrated by the zinc-dependent fluorescence observed by SDS-PAGE (Fig. 1B), despite exhibiting a low bilin-binding rate, and by spectral and acid denaturation analysis of chromophorylated SPI1085g2 (Fig. 1C). The PCB-binding SPI1085g2 (SPI1085g2-PCB) showed reversible photoconversion between a red-absorbing Pr form at 642 nm and a green-absorbing Pg form at 535 nm (Fig. 1C), which was very similar to that of Slr1393g3 from Synechocystis sp. PCC 6803 [10]. We compared the primary sequence of SPI1085g2 with those of Slr1393g3 and another red/green CBCR GAF domain, All2699g3 [24]. Some residues conserved or enriched in typical red/green CBCR GAF domains appear in SPI1085g2 (Fig. S2). Therefore, SPI1085g2 belongs to the canonical red/green CBCRs. Next, chromophorylation with PEB instead of PCB as the chromophore was performed and unexpectedly resulted in highly efficient binding of PEB to SPI1085g2, as indicated by a band with strong zinc-dependent fluorescence (Fig. 1B) and spectral and acid denaturation analysis (Fig. 1D). Furthermore, we failed to obtain chromophorylated SPI1085g2 with BV as demonstrated by the zinc-dependent fluorescence (Fig. 1B). The PEB-binding SPI1085g2 (SPI1085g2-PEB) was not photochromic and exhibited maximum absorption at 520 nm (Fig. 1D). Although PEB also covalently binds to Slr1393g3 and All2699g3, they are partially converted into phycourobilin (PUB), which results in a lower yield [14]. The PEB bound to SPI1085g2 was not further modified. Therefore, SPI1085g2-PEB was more similar to the previously reported PEB-binding All2699g1 (All2699g1-PEB) but with a remarkably blueshifted absorption maximum compared to All2699g1 with absorption maximum at 575 nm [14]. Clearly, the PEBbinding GAF domains could be divided into two categories, Slr1393g3 and All2699g3 with isomerization of PEB to PUB and lower yield, and SPI1085g2 and All2699g1 with non-isomerization and higher yield, respectively. We compared the amino acid sequences of the four GAF domains based on multiple alignment (Fig. S2). SPI1085g2 and All2699g1 showed great differences in some conserved amino acid residues. It was speculated that some identical residues in SPI1085g2 and All2699g1 might play important roles for PEB incorporation.

Intense Fluorescence of SPI1085g2-PEB
To study the fluorescence properties of chromophorylated SPI1085g2, fluorescence spectra at room temperature were measured. The results showed that the thermostable forms of SPI1085g2-PCB exhibited only weak fluorescence with an emission maximum at 662 nm ( Fig. 2A), whereas SPI1085g2-PEB exhibited an intense orange fluorescence with an emission maximum at 555 nm (Fig. 2B). Furthermore, the fluorescence quantum yield of SPI1085g2-PEB was calculated to be very close to 1.0 (Table 1), which was comparable with that of some phycobiliproteins attached to PEB (Fig. 2C) but higher than that of All2699g1 (ΦF = 0.55) and Cph1 (ΦF = 0.72) [14,23,25]. Moreover, SPI1085g2-PEB also has a larger Stokes shift (35 nm) than PEB-binding phycobiliproteins All2699g1 and Cph1 for the reduction of scatter interference.

Response of SPI1085g2-PEB to Copper
To evaluate the potential of SPI1085g2 as a single fluorescent protein biosensor for heavy metal detection, we conducted spectroscopic studies to investigate whether SPI1085g2-PEB can be fluorescently quenched by various heavy metal ions. The results demonstrated that the fluorescence of SPI1085g2-PEB was not substantially affected by the addition of 0.04 mM heavy metal ions, such as Cd 2+ , Co 2+ , Cr 3+ , Mn 2+ , Ni 2+ , Pb 2+ , and Zn 2+ . However, the addition of 0.04 mM Cu 2+ exerted a maximum fluorescence quenching of approximately 75% (Fig. 3A). Therefore, among the tested heavy metal ions, only Cu 2+ caused selective fluorescence quenching of SPI1085g2-PEB. Once the Cu 2+ was added into SPI1085g2-PEB, the fluorescence intensity at 555 nm was recorded over time (0-30 min) to investigate the response time of SPI1085g2-PEB toward Cu 2+ . A large instantaneous decrease in fluorescence intensity was observed within less than 5 sec after adding Cu 2+ and the intensity continued to decline slowly over time (Fig. 3B). This indicates that SPI1085g2-PEB exhibited very short response time to Cu 2+ and provided an advantage for biosensor applications.
Next, the responding properties of SPI1085g2-PEB for Cu 2+ were analyzed by incubating equimolar amounts of purified fluorescent proteins with 0-40 μM Cu 2+ . The fluorescence emission spectrum of SPI1085g2-PEB indicated a reduction of fluorescence intensity with increasing Cu 2+ concentration, while the shape of the emission signal and its position did not change (Fig. 4A). To confirm that the fluorescence quenching we observed was reversible, we treated mixtures of 0.25 μM SPI1085g2-PEB and 40 μM Cu 2+ with 0.025-25 μM EDTA. We found that the fluorescence emission spectrum of the quenched fluorescent proteins gradually reverted close to the initial state with the increase of the EDTA concentration (Fig. 4B), demonstrating that the quenching effect was  due to the presence of the metal ion. A more detailed analysis of fluorescence quenching as a function of the copper concentration for SPI1085g2-PEB was performed over a broad concentration range from 0.01 to 100 μM. The results showed that the fluorescence intensity decreased with increasing copper concentration (Fig. 4C). Only a 10% decrease in emission was observed when 1 μM Cu 2+ was added, yet Cu 2+ in the range of 1 to 100 μM caused a remarkable decrease of fluorescence intensity. Furthermore, there was a sensitive dose-effect relationship between the fluorescence intensity and the concentration of Cu 2+ over a narrower range from 2 to 10 μM, and a decrease of fluorescence intensity with increasing copper concentration was clearly observed (Fig. 4D). The data were fitted to calculate the dissociation constant of SPI1085g2-PEB. The dissociation constant of the copper binding site on the rAsGFP (recombinant Anemonia sulcata green fluorescent protein) was identified as 14.18 ± 0.21 μM, which indicates the affinity of SPI1085g2-PEB for Cu 2+ . The copper dissociation constant of SPI1085g2-PEB was similar to that of DsRed mutant drFP583 [26], but higher than that of DsRed, GFPdopa, and CreiLOV [18][19][20].
To exhibit a linear dose-effect relationship, a Stern-Volmer plot was generated at room temperature and a linear plot was obtained with a correlation coefficient of 0.9936 (Fig. 5A). The K SV value was evaluated as 0.5 × 10 5 M −1 according to the slope of the fitted straight line. . Meanwhile, the dose response curve of Cu 2+ exposure (8-40 μM) demonstrated excellent linearity with a correlation coefficient of 0.9937 (Fig. 5B). This sensitivity covers the maximum allowed copper concentration in drinking water suggested by the World Health Organization, (31.15 μM) [27], above which aqueous solution may pose a threat to human health. These observations indicate that SPI1085g2-PEB has potential as a biosensor to be applied to the detection of copper ions.

Discussion
CBCRs chromophorylated with different tetrapyrrole chromophores exhibit a great diversity of spectral properties [6]. In the current study, we tested the chromophorylation of a novel red/green CBCR GAF domain of Spirulina, SPI1085g2. This GAF domain binds PCB but cannot bind BV. PEB chromophorylated SPI1085g2 exhibits a fluorescence quantum yield close to 1.0, higher than those of all reported biliproteins. SPI1085g2-PEB was not photochromic with intense orange fluorescence, similar to the previously reported All2699g1-PEB [14]. However, SPI1085g2-PEB exhibited blue-shifted absorption and emission spectra from 575 to 520 nm in absorption maximum and from 586 to 555 nm in emission maximum compared with All2699g1-PEB. Moreover, SPI1085g2-PEB had a larger Stokes-shift and higher fluorescence quantum yield close to unity than those of All2699g1-PEB. This novel GAF domain expands the spectral variation of biliprotein-based fluorescent constructs and can be used in combination with All2699g1 in flexible applications, such as dual-color labeling and FRET experiments. In terms of both the wavelengths of excitation and emission and fluorescence quantum yield, SPI1085g2-PEB was very similar to CpcB-PEB, a previously reported orange fluorescing phycobiliprotein [23]. However, the chromophorylation of CBCR GAF domains does not require the involvement of a lyase, facilitating chromophore assembly, compared to that of phycobiliproteins. SPI1085g2-PEB, which has the most intense fluorescence quantum yield reported to date, may be a promising developmental platform for fluorescent biosensor devices.
Direct conversion of fluorescent proteins into a sensor tool for detecting metal ions is currently attracting much interest. We investigated the possibility of using SPI1085g2-PEB as a single biosensor to detect heavy metal ions. The fluorescence of SPI1085g2-PEB can be selectively and instantaneously quenched by Cu 2+ in a concentrationdependent manner and recovered almost to the initial fluorescence with the addition of EDTA. SPI1085g2-PEB provided a much faster response time compared to other fluorescent proteins, such as His6GFP (chimeric green fluorescent protein harboring hexahistidine) [28], rAsGFP [29], and iLOV (variant derived through engineering of the light, oxygen or voltage sensing domain) [30], which suggests the potential of SPI1085g2-PEB as a biosensor for rapid detection of copper ions. In this case, the observed fluorescence changes may be due to the interaction between Cu 2+ and metal-binding sites, which presumably cause static quenching, as well as that of His6GFP, which bring copper close to the chromophore of the proteins to form a nonfluorescent ground state complex. Consistent with this hypothesis, the emission spectrum of SPI1085g2-PEB did not change in shape or position, but a decrease in intensity was clearly demonstrated. The PEB chromophore unit may be considered as a binding site for Cu 2+ , because previous studies tend to suggest the tetrapyrrole core as the probable binding site for heavy metal ions [31]. On the other hand, the intramolecular histidine and cysteine residues and the polyhistidine-tag at the Nterminus of SPI1085g2-PEB may also be responsible for binding copper since copper ions prefer to coordinate with nitrogen and thiolate groups typically present in the side chains of histidine and cysteine [32]. In brief, copper ions are bound to either the tetrapyrrole chromophore or the amino-acid residues, or both. This remains to be proved.
The fluorescence reversibility of SPI1085g2-PEB suggests that the continuous and reproducible monitoring of exchangeable copper ions is possible with the protein, which is important for cost-effective application of biosensor materials. The fluorescence recovery of Cu 2+ -binding SPI1085g2-PEB in the presence of some specific molecules could extend the applications of SPI1085g2-PEB into the detection of mercapto-biomolecules such as homocysteine [33]. SPI1085g2-PEB can be integrated into some immobilized carriers, such as Sol-gel [27], amine-coated glass surfaces [18], and nanocellulose matrices [34], enhancing its stability and facilitating reutilization of the systems. A very small amount of SPI1085g2-PEB is required for the response to copper due to its higher fluorescence quantum yield than those of GFP derivatives [15,27], iq-FPs [16], DsRed [19], HeRed [35], Dronpa [36], ZsYellow [37], and CreiLOV [20]. Furthermore, a large number of CBCRs have been identified and may provide a set of available libraries for single fluorescent protein biosensors due to their novel photophysical and biochemical properties.