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Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions
1College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, P.R. China
2Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Nanjing Forestry University, Nanjing 210037, P.R. China
3National Positioning Observation Station of Hung-tse Lake Wetland Ecosystem in Jiangsu Province, Hongze, Jiangsu 223100, P.R. China
J. Microbiol. Biotechnol. 2021; 31(2): 233-239
Published February 28, 2021 https://doi.org/10.4014/jmb.2009.09048
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Cyanobacteriochromes (CBCRs) are distantly phytochrome-related photoreceptor biliproteins that contain several GAF (cGMP-specific phosphodiesterase/adenylate cyclase/FhlA) domains that act as sensory modules in cyanobacteria [1]. A single GAF domain can bind covalently linear tetrapyrroles and serve as a chromophore. Tetrapyrrole-binding proteins have great spectral diversity and can sense a wide range of wavelengths, from the near-ultraviolet to the near-infrared [2-7]. Most CBCRs utilize phycocyanobilin (PCB) tetrapyrrole as a chromophore and show reversible photoconversion, which is triggered by
Fluorescent proteins show fluorescence quenching by specific metal ions, and can be used in metal biosensing applications [15-17]. The fluorescence intensity of fluorescent proteins may be reduced, depending on the specific transition metal environment,
In this article, we identified a novel red/green CBCR GAF domain, SPI1085g2 from
Materials and Methods
Cloning and Plasmid Construction
The SPI1085g2 (encoding the apoprotein SPI1085g2) gene was amplified from
Protein Expression and Purification
The constructed plasmids, pETDuet-SPI1085g2-ho1::pcyA and pETDuet-SPI1085g2-ho1::pebS, were separately transformed into
SDS-PAGE and Zn-Induced Fluorescence Assay
Protein samples (50 μl each) were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS. The SDS-PAGE gel was composed of a 10% resolving gel and a 5% stacking gel. The samples were boiled with 2×SDS sample buffer containing 30 mM β-mercaptoethanol for 5 min. The purified proteins were visualized by staining with Coomassie blue, and the bilins in the samples were detected by Zn2+-induced fluorescence. Fluorescence was visualized through a 630-nm filter upon excitation at 530 nm (GenoSens1850, China).
Spectral Analysis
All experiments were conducted at room temperature. The absorbance spectra were obtained on a Perkin Elmer Lambda 365 spectrophotometer. After denaturing the proteins in 8 M urea at pH 2.0 in the dark, extinction coefficients were determined by the absorption at: 662 nm (ε = 35,500 M−1 cm−1) for PCB; 550 nm (ε = 42,800 M−1 cm−1) for PEB [23]. Red light was provided by a cold fiber optic light source with a 150 W halogen lamp (Bocheng, Nanjing, China) through a bandpass filter with a peak wavelength of 653 nm and a 20-nm half-bandwidth (Rayan, China), while green light was provided by a light source with a peak wavelength of 535 nm and a 40-nm half-bandwidth (Rayan). The light intensity at the sample plane for photoconversion was 15 μmol m−2 s−1. Samples were irradiated for 3 min for photoconversion. Fluorescence spectra were recorded with a model LS 55 spectrofluorometer (Perkin Elmer, USA). The fluorescence quantum yields were determined in KPB (pH 7.2) using the known ΦF = 0.98 of the biosynthetically obtained CpcB(C-84)-PEB as standards [23]. The biliprotein concentrations were calculated using the Beer-Lambert Law (A = εlc).
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 (CuSO4, NiCl2, MnCl2, ZnSO4, CrCl3, C°Cl2, PbCl2 ,CdCl2) 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 Cu2+ on the fluorescence of SPI1085g2-PEB, 10 μl of a Cu2+ 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 Cu2+ 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 Cu2+ using the equation as follows:
Here,
The dissociation constant for copper (
where
Results
Chromophorylation of SPI1085g2
SPI1085, a hypothetical signal transduction protein from
-
Fig. 1.
Domain position and chromophorylation of SPI1085g2. (A ) Domain architecture of SPI9445_RS0121085 (SPI1085). GAF: cGMP-specific phosphodiesterase/adenylate cyclase/FhlA; HisKA: histidine kinase acceptor; HATPase: histidine associated ATPase. The underline indicates the SPI1085g2 position. (B ) SDS-PAGE analyses of SPI1085g2 chromophorylated with PCB (phycocyanobilin), PEB (phycoerythrobilin) and BV (biliverdin): Coomassie brilliant blue staining (upper gel image) and zinc-induced fluorescence (lower gel image). (C ) Absorbance spectra of native (heavy lines) and acid-urea-denatured (thin lines) SPI1085g2-PCB. The dashed lines correspond to the 15E state obtained after irradiation with 653/20-nm light, and the solid lines correspond to the 15Z state obtained after irradiation with 530/20-nm light. (D ) Absorbance spectra of native (heavy line) and acid-urea-denatured (thin line) SPI1085g2-PEB. (E ) The structure of phycoerythrobilin (PEB).
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.
-
Table 1 . Spectral properties of reconstituted biliproteins.
Biliprotein Absorbance Fluorescence λmax[nm] εvis [M-1·cm-1] × 10-4 λmax [nm] ФF SPI1085g2-PEB 520 7.4 555 0.996 SPI1085g2-PCB 642 8.3 662 0.007 Spectra were obtained in potassium phosphate buffer (20 mM, pH 7.0). Extinction coefficients and fluorescence yields were averages of two independent experiments.
-
Fig. 2.
Fluorescence properties of chromophorylated SPI1085g2. Fluorescence excitation (dashed line) and emission (solid line) spectra of SPI1085g2-PCB, excited at 600 nm (A ), and SPI1085g2-PEB, excited at 480 nm (B ). (C ) Plot of fluorescence emission vs. excited absorbance for the dilution series of SPI1085g2-PEB (solid line) and CpcB(C-84)-PEB (dashed line). The slopes of these lines are proportional to the quantum yields for fluorescence.
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 Cd2+, Co2+, Cr3+, Mn2+, Ni2+, Pb2+, and Zn2+. However, the addition of 0.04 mM Cu2+ exerted a maximum fluorescence quenching of approximately 75% (Fig. 3A). Therefore, among the tested heavy metal ions, only Cu2+ caused selective fluorescence quenching of SPI1085g2-PEB. Once the Cu2+ 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 Cu2+. A large instantaneous decrease in fluorescence intensity was observed within less than 5 sec after adding Cu2+ and the intensity continued to decline slowly over time (Fig. 3B). This indicates that SPI1085g2-PEB exhibited very short response time to Cu2+ and provided an advantage for biosensor applications.
-
Fig. 3.
Selectivity and response time of SPI1085g2-PEB. (A ) Effects of different heavy metal ions on the fluorescence emission of SPI1085g2-PEB. (B ) Response time of SPI1085g2-PEB in the presence of Cu2+.
Next, the responding properties of SPI1085g2-PEB for Cu2+ were analyzed by incubating equimolar amounts of purified fluorescent proteins with 0-40 μM Cu2+. The fluorescence emission spectrum of SPI1085g2-PEB indicated a reduction of fluorescence intensity with increasing Cu2+ 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 Cu2+ 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.
-
Fig. 4.
Response of SPI1085g2-PEB in a concentration-dependent manner. (A ) Effect of the presence of different copper concentrations on the fluorescence emission of SPI1085g2-PEB (0.25 μM). (B ) Reversibility of the Cu2+-mediated quenching of SPI1085g2-PEB. (C ) Dose-response curve of SPI1085g2-PEB against Cu2+ in a broad range from 0.01 to 100 μM. (D ) Dose-response curve generated for SPI1085g2-PEB with different concentrations of Cu2+ in a narrower range from 2 to 10 μM.
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 Cu2+ was added, yet Cu2+ 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 Cu2+ 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
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
-
Fig. 5.
Linear relationship of SPI1085g2-PEB against Cu2+ in the micromolar range. (A ) Stern–Volmer plot generated for SPI1085g2-PEB by the addition of Cu2+ at different concentrations. (B ) The plot of the normalized fluorescence intensity of SPI1085g2-PEB vs. Cu2+ concentration (8-40 μM). Measurements were repeated 3 times and data were fitted linearly.
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
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 Cu2+ in a concentration-dependent 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 Cu2+ 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 Cu2+, 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 N-terminus 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 Cu2+-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.
Supplemental Material
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Province, China (No. BK20150884), the National Natural Science Foundation of China (No. 41977354 and No. 41471191), China Postdoctoral Science Foundation (No. 14ZR1401500), Jiangsu Planned Projects for Postdoctoral Research Funds (No. 1501020A), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(2): 233-239
Published online February 28, 2021 https://doi.org/10.4014/jmb.2009.09048
Copyright © The Korean Society for Microbiology and Biotechnology.
Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions
Su-Dan Jiang1, Yi sheng1, Xian-Jun Wu1,2,3*, Yong-Li Zhu1,2,3, and Ping-Ping Li1,2,3*
1College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, P.R. China
2Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Nanjing Forestry University, Nanjing 210037, P.R. China
3National Positioning Observation Station of Hung-tse Lake Wetland Ecosystem in Jiangsu Province, Hongze, Jiangsu 223100, P.R. China
Correspondence to:X. Wu, xjwu@njfu.edu.cn
P. Li, ppli@njfu.edu.cn
Abstract
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.
Keywords: Cyanobacteriochrome, phycoerythrobilin, fluorescence quantum yield, biosensor, copper ions, fluorescence quenching
Introduction
Cyanobacteriochromes (CBCRs) are distantly phytochrome-related photoreceptor biliproteins that contain several GAF (cGMP-specific phosphodiesterase/adenylate cyclase/FhlA) domains that act as sensory modules in cyanobacteria [1]. A single GAF domain can bind covalently linear tetrapyrroles and serve as a chromophore. Tetrapyrrole-binding proteins have great spectral diversity and can sense a wide range of wavelengths, from the near-ultraviolet to the near-infrared [2-7]. Most CBCRs utilize phycocyanobilin (PCB) tetrapyrrole as a chromophore and show reversible photoconversion, which is triggered by
Fluorescent proteins show fluorescence quenching by specific metal ions, and can be used in metal biosensing applications [15-17]. The fluorescence intensity of fluorescent proteins may be reduced, depending on the specific transition metal environment,
In this article, we identified a novel red/green CBCR GAF domain, SPI1085g2 from
Materials and Methods
Cloning and Plasmid Construction
The SPI1085g2 (encoding the apoprotein SPI1085g2) gene was amplified from
Protein Expression and Purification
The constructed plasmids, pETDuet-SPI1085g2-ho1::pcyA and pETDuet-SPI1085g2-ho1::pebS, were separately transformed into
SDS-PAGE and Zn-Induced Fluorescence Assay
Protein samples (50 μl each) were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS. The SDS-PAGE gel was composed of a 10% resolving gel and a 5% stacking gel. The samples were boiled with 2×SDS sample buffer containing 30 mM β-mercaptoethanol for 5 min. The purified proteins were visualized by staining with Coomassie blue, and the bilins in the samples were detected by Zn2+-induced fluorescence. Fluorescence was visualized through a 630-nm filter upon excitation at 530 nm (GenoSens1850, China).
Spectral Analysis
All experiments were conducted at room temperature. The absorbance spectra were obtained on a Perkin Elmer Lambda 365 spectrophotometer. After denaturing the proteins in 8 M urea at pH 2.0 in the dark, extinction coefficients were determined by the absorption at: 662 nm (ε = 35,500 M−1 cm−1) for PCB; 550 nm (ε = 42,800 M−1 cm−1) for PEB [23]. Red light was provided by a cold fiber optic light source with a 150 W halogen lamp (Bocheng, Nanjing, China) through a bandpass filter with a peak wavelength of 653 nm and a 20-nm half-bandwidth (Rayan, China), while green light was provided by a light source with a peak wavelength of 535 nm and a 40-nm half-bandwidth (Rayan). The light intensity at the sample plane for photoconversion was 15 μmol m−2 s−1. Samples were irradiated for 3 min for photoconversion. Fluorescence spectra were recorded with a model LS 55 spectrofluorometer (Perkin Elmer, USA). The fluorescence quantum yields were determined in KPB (pH 7.2) using the known ΦF = 0.98 of the biosynthetically obtained CpcB(C-84)-PEB as standards [23]. The biliprotein concentrations were calculated using the Beer-Lambert Law (A = εlc).
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 (CuSO4, NiCl2, MnCl2, ZnSO4, CrCl3, C°Cl2, PbCl2 ,CdCl2) 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 Cu2+ on the fluorescence of SPI1085g2-PEB, 10 μl of a Cu2+ 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 Cu2+ 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 Cu2+ using the equation as follows:
Here,
The dissociation constant for copper (
where
Results
Chromophorylation of SPI1085g2
SPI1085, a hypothetical signal transduction protein from
-
Figure 1.
Domain position and chromophorylation of SPI1085g2. (A ) Domain architecture of SPI9445_RS0121085 (SPI1085). GAF: cGMP-specific phosphodiesterase/adenylate cyclase/FhlA; HisKA: histidine kinase acceptor; HATPase: histidine associated ATPase. The underline indicates the SPI1085g2 position. (B ) SDS-PAGE analyses of SPI1085g2 chromophorylated with PCB (phycocyanobilin), PEB (phycoerythrobilin) and BV (biliverdin): Coomassie brilliant blue staining (upper gel image) and zinc-induced fluorescence (lower gel image). (C ) Absorbance spectra of native (heavy lines) and acid-urea-denatured (thin lines) SPI1085g2-PCB. The dashed lines correspond to the 15E state obtained after irradiation with 653/20-nm light, and the solid lines correspond to the 15Z state obtained after irradiation with 530/20-nm light. (D ) Absorbance spectra of native (heavy line) and acid-urea-denatured (thin line) SPI1085g2-PEB. (E ) The structure of phycoerythrobilin (PEB).
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.
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Table 1 . Spectral properties of reconstituted biliproteins..
Biliprotein Absorbance Fluorescence λmax[nm] εvis [M-1·cm-1] × 10-4 λmax [nm] ФF SPI1085g2-PEB 520 7.4 555 0.996 SPI1085g2-PCB 642 8.3 662 0.007 Spectra were obtained in potassium phosphate buffer (20 mM, pH 7.0). Extinction coefficients and fluorescence yields were averages of two independent experiments..
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Figure 2.
Fluorescence properties of chromophorylated SPI1085g2. Fluorescence excitation (dashed line) and emission (solid line) spectra of SPI1085g2-PCB, excited at 600 nm (A ), and SPI1085g2-PEB, excited at 480 nm (B ). (C ) Plot of fluorescence emission vs. excited absorbance for the dilution series of SPI1085g2-PEB (solid line) and CpcB(C-84)-PEB (dashed line). The slopes of these lines are proportional to the quantum yields for fluorescence.
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 Cd2+, Co2+, Cr3+, Mn2+, Ni2+, Pb2+, and Zn2+. However, the addition of 0.04 mM Cu2+ exerted a maximum fluorescence quenching of approximately 75% (Fig. 3A). Therefore, among the tested heavy metal ions, only Cu2+ caused selective fluorescence quenching of SPI1085g2-PEB. Once the Cu2+ 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 Cu2+. A large instantaneous decrease in fluorescence intensity was observed within less than 5 sec after adding Cu2+ and the intensity continued to decline slowly over time (Fig. 3B). This indicates that SPI1085g2-PEB exhibited very short response time to Cu2+ and provided an advantage for biosensor applications.
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Figure 3.
Selectivity and response time of SPI1085g2-PEB. (A ) Effects of different heavy metal ions on the fluorescence emission of SPI1085g2-PEB. (B ) Response time of SPI1085g2-PEB in the presence of Cu2+.
Next, the responding properties of SPI1085g2-PEB for Cu2+ were analyzed by incubating equimolar amounts of purified fluorescent proteins with 0-40 μM Cu2+. The fluorescence emission spectrum of SPI1085g2-PEB indicated a reduction of fluorescence intensity with increasing Cu2+ 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 Cu2+ 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.
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Figure 4.
Response of SPI1085g2-PEB in a concentration-dependent manner. (A ) Effect of the presence of different copper concentrations on the fluorescence emission of SPI1085g2-PEB (0.25 μM). (B ) Reversibility of the Cu2+-mediated quenching of SPI1085g2-PEB. (C ) Dose-response curve of SPI1085g2-PEB against Cu2+ in a broad range from 0.01 to 100 μM. (D ) Dose-response curve generated for SPI1085g2-PEB with different concentrations of Cu2+ in a narrower range from 2 to 10 μM.
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 Cu2+ was added, yet Cu2+ 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 Cu2+ 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
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
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Figure 5.
Linear relationship of SPI1085g2-PEB against Cu2+ in the micromolar range. (A ) Stern–Volmer plot generated for SPI1085g2-PEB by the addition of Cu2+ at different concentrations. (B ) The plot of the normalized fluorescence intensity of SPI1085g2-PEB vs. Cu2+ concentration (8-40 μM). Measurements were repeated 3 times and data were fitted linearly.
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
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 Cu2+ in a concentration-dependent 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 Cu2+ 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 Cu2+, 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 N-terminus 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 Cu2+-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.
Supplemental Material
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Province, China (No. BK20150884), the National Natural Science Foundation of China (No. 41977354 and No. 41471191), China Postdoctoral Science Foundation (No. 14ZR1401500), Jiangsu Planned Projects for Postdoctoral Research Funds (No. 1501020A), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

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Table 1 . Spectral properties of reconstituted biliproteins..
Biliprotein Absorbance Fluorescence λmax[nm] εvis [M-1·cm-1] × 10-4 λmax [nm] ФF SPI1085g2-PEB 520 7.4 555 0.996 SPI1085g2-PCB 642 8.3 662 0.007 Spectra were obtained in potassium phosphate buffer (20 mM, pH 7.0). Extinction coefficients and fluorescence yields were averages of two independent experiments..
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