Developing Polysulfide-Sensitive GFPs for Real-Time Analysis of

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Developing polysulfide-sensitive GFPs for real-time analysis of polysulfides in live cells and subcellular organelles Xin Hu, Huanjie Li, Xi Zhang, Zhigang Chen, Rui Zhao, Ningke Hou, Jihua Liu, Luying Xun, and Huaiwei Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04634 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Analytical Chemistry

Developing polysulfide-sensitive GFPs for real-time analysis of polysulfides in live cells and subcellular organelles Xin Hu1,2†, Huanjie Li1†, Xi Zhang1, Zhigang Chen1, Rui Zhao1, Ningke Hou1, Jihua Liu2, Luying Xun1,3, Huaiwei Liu1* 1State

Key Laboratory of Microbial Technology, Shandong University, Qingdao,

266237, People’s Republic of China. 2Institute

of Marine Science and Technology, Shandong University, Qingdao, 266237,

People’s Republic of China. 3School

of Molecular Biosciences, Washington State University, Pullman, WA,

99164-7520, USA. †These

authors contribute equally to this study

*Corresponding author Huaiwei Liu: [email protected]; Tel. +86 532 58631572. Fax. +86 532 58631572

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Abstract Polysulfides are newly discovered cellular content, and they are involved in multiple intracellular processes like redox homeostatsis and protein sulfhydration. The dynamic changes of polysulfides inside the cell are directly related to these processes. To monitor the intracellular dynamics and subcellular levels of polysulfides, we developed green fluorescent protein (GFP) based probes that were polysulfidespecific. A pair of cysteine residues was introduced near the GFP chromophore with the spatial distance between the cysteine residues being designed to allow the formation of intra –Sn– (n≥3) bond but not –S2– (disulfide) bond. We tested these probes in model microorganisms and found they displayed ratiometric change to intracellular polysulfides that had clear variations associated with the growth phases. The distribution of polysulfides in subcellular organelles are heterogeneous, suggesting polysulfides have multiple origins and functions in cells. These probes provided long-desired tools for polysulfides in vivo studies.

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Introduction Polysulfides (HSnH and RSnH, n≥2; RSnR, n≥3) are newly discovered cellular components, playing multiple roles in biological systems, including induction of Ca2+ inﬂux, regulation of tumor suppressor phosphatase, and maintenance of redox homeostatsis.1-5 They also serve as potent regulators of mitochondrial biogenesis and bioenergetics.6 Among them, hydrogen persulfide (HSSH) and glutathione persulfide (GSSH) are particularly important owing to their versatile activities and relatively abundance inside cells. HSSH and GSSH can react as both electrophiles and nucleophiles. As electrophiles, they can transfer sulfur atoms (the sulfane sulfur, S0) to protein thiols to generate protein persulfides (protein-SSH), a process termed as protein S-sulfhydration.7-9 According to a recent report, more than 40% of intracellular proteins are S-sulfhydrated.10 As nucleophiles, they can scavenge reactive oxygen species (ROS) and react with the autophagy regulator 8-NO2cGMP.3,11,12 Higher cellular levels of polysulfide should lead to more protein sulfhydration and confer cells with more resistance to oxidative stress. Although intense studies have been conducted on polysulfides in recent years, the progress is slow, partly due to the lack of suitable methods for their dynamics analysis and subcellular distribution analysis in living cells.13 Currently applied methods like MS-based polysulfidomics can detect and quantify endogenous polysulfides.10,14,15 Since this method needs to break cells, it cannot be used in either subcellular detection or real-time analysis.16 Some fluorescent probes like SSP4 and QS10 have been developed to detect polysulfides. Although these probes can be 3



applied to live cells, their localization to subcellular organelles is difficult to control.17-19 A new probe that can be specially deployed in desired subcellular organelles is required. Herein, we report a new-generation fluorescent probes that enables noneinvasive, real-time, and subcellular-level monitoring of polysulfides in living cells. These probes are green fluorescent protein (GFP)-based and specific to RSSH type of polysulfides. They can be easily deployed in desired subcellular organelles by fusing with organelle-targeting peptides, as shown in detecting the dynamics of polysulfides in Escherichia coli and Saccharomyces cerevisiae.

Experimental section Strains, plasmids and compounds Strains and plasmids used in this study were listed in Table S1. E. coli DH5α was used for plasmid construction. E. coli BL21 (DE3) was used for protein expression. E. coli BL21 and S. cereviase BY4742 were used for polysulfides in vivo analysis. E. coli strains were grown in Lysogeny broth (LB). Kanamycin (50 μg/ml) or ampicillin (100 μg/ml) were added when required. S. cereviase BY4742 was grown SD-uramedium (Yeast synthetic defined minimal medium without uracil20). Dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) were purchased from Sigma-Aldrich. Dimethyl trisulfide (Me-SSS-Me) was purchased from TCI (Shanghai) Company. HSSH was prepared by following a reported protocol.21 GSSH was prepared by following the protocol of Luebke.22 The obtained product was 4


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confirmed by HPLC analysis.

Rational structure-based design of reactive-sulfur-sensitive GFP variants The locations of two cysteine residues were designed near the chromophore of GFP and the spatial distance between their thiols were analyzed by SWISS-MODEL (http://swissmodel.expasy.org/) and PyMOL-1.5.0.3. The 3D structures of roGFP (PDB: 1JC0 and 1JC1) were used as templates for homologous modeling.

Protein expression, purifications, and reactions with polysulfides Amino acid mutations were introduced by a modified QuikChange™ method.23 For psGFP expression in E. coli BL21 (DE3), the encoding genes were introduced into the plasmid pET30a with an N-terminal His-tag. The recombinant E. coli was grown in LB at 30oC with shaking until OD600 reached about 0.6, 0.2 mM IPTG was added and the cells were further cultivated at 18oC for 20 h. Cell broken was conducted using crusher SPCH-18 (STANSTED), and protein purification was carried out by using nickel-nitrilotriacetic acid agarose resin (Invitrogen). Buffer exchange of the proteins was performed with PD-10 desalting column (GE Healthcare). The purified protein (1.5 mg/ml) was mixed with HSSH, GSSH, H2O2, or DTT in potassium phosphate buffer (10 mM, pH 7.0), and their concentrations were mentioned in the text. The mixture was incubated at room temperature for a desired period and then loaded onto PD-10 desalting column to remove unreacted small molecules. The fluorescence of reacted-psGFP was analyzed by RF-5301 PC 5



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spectrofluoro photometer or Synergy H1 microplate reader.

Redox Titration Using Fluorescence Spectroscopy Redox potential was determined using redox-oxidation titrations. The redox potential buffers were prepared using reduced (DTTred) and oxidized DTT (DTTox). The total concentration of DTT (DTTred + DTTox) was 10 mM and solution pH was set to 7.0. The ratio of DTTred to DTTox was adjusted to make eleven redox buffers with a series of redox potentials. The total oxidized psGFPox was prepared by reacting purified psGFP1.1 (about 3 mg/ml) with 200 μM HSSH for 30 min. Unreacted HSSH was then removed by using PD-10 desalting column. The obtained psGFPox (1.5 mg/ml) was dissolved into the redox buffers and incubated at room temperature for 1 h, then the 408/488 nm ratios were detected using Synergy H1 microplate reader. The redox midpoint potential calculation of psGFP1.1 was referred to that of roGFP.24 The Nernst equation was used: 𝑅𝑇

𝐸𝐷𝑇𝑇 = 𝐸0𝑚(𝐷𝑇𝑇) ― 𝑧𝐹 ln

[𝐷𝑇𝑇]𝑟𝑒𝑑 [𝐷𝑇𝑇]𝑜𝑥

𝑅𝑇

= 𝐸0𝑚(𝑝𝑠𝐺𝐹𝑃) ― 𝑧𝐹 ln

(

1 ― 𝑂𝑥𝐷𝑝𝑠𝐺𝐹𝑃 𝑂𝑥𝐷𝑝𝑠𝐺𝐹𝑃

)

(1)

In this equation, R is the gas constant (8.315 J K-1mol-1), T is the absolute temperature (298.15 K), z is the number of transferred electrons, and F is the Faraday constant (96,485 C mol-1). The standard redox potential of DTT at pH 7 (E0DTT) is -330 mV. OxDpsGFP is the percentage of oxidized psGFP (psGFPox). The value of OxDpsGFP was calculated by using the same algorithm as that for OxDroGFP calculation. The following equation was used: [𝑝𝑠𝐺𝐹𝑃𝑜𝑥]

𝑅 ― 𝑅𝑟𝑒𝑑

(2)

𝑂𝑥𝐷𝑝𝑠𝐺𝐹𝑃 = [𝑝𝑠𝐺𝐹𝑃𝑜𝑥] + [𝑝𝑠𝐺𝐹𝑃𝑟𝑒𝑑] = 𝑅𝑜𝑥 ― 𝑅𝑟𝑒𝑑 6


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In this equation, Rred and Rox refer to the 408/488-nm ratios of completely reduced and oxidized psGFP, respectively. R is the measured 408/488-nm ratio. The obtained OxDpsGFP data were plotted against the redox potential data generated by DTT redox buffers (EDTT), and the curve was fitted into the Nernst equation to calculate psGFP redox midpoint potential.

Protein LC-MS/MS analysis The purified protein (1.5 mg/ml) was mixed with 200 μM HSSH or 1 mM H2O2 in the desalting buffer (50mM NaH2PO4, 300 mM NaCl, pH 8.0). After incubated at 25oC for 30 min, the mixture was loaded onto PD-10 desalting column to remove unreacted small molecules. The re-purified proteins were reacted with iodoacetamide (IAM) and then digested with trypsin by following a previously reported protocol.1 The Prominence nano-LC system (Shimadzu) equipped with a custom-made silica column (75 μm × 15 cm) packed with 3-μm Reprosil-Pur 120 C18-AQ was used for the analysis. For the elution process, a 100 min gradient from 0% to 100% of solvent B (0.1 % formic acid in 98% acetonitrile) at 300 nl/min was used; solvent A was 0.1 % formic acid in 2% acetonitrile). The eluent was ionized and electrosprayed via LTQ-Orbitrap Velos Pro CID mass spectrometer (Thermo Scientific), which run in data-dependent acquisition mode with Xcalibur 2.2.0 software (Thermo Scientific). Full-scan MS spectra (from 400 to 1800 m/z) were detected in the Orbitrap with a resolution of 60,000 at 400 m/z.

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Detection of E. coli intracellular polysulfides with SSP4 probe. Middle-log phased E. coli cells (OD600=0.01) were inoculated into fresh LB medium and cultivated at 37oC for 24 hours with shaking (225 rpm). Cells were collected, suspended in 1 ml of 50 mM HEPES buffer (pH 7.4) at 1 OD600, and reacted with 20 μM SSP4 and 500 μM of CTAB. After reacting at 30oC for 10 min with shaking (200 rpm) in the dark, cells were washed twice with HEPES buffer and subjected to fluorescence analysis. The Synergy H1 microplate reader was used. Ex was set to 482 nm and Em was set to 515 nm.

In vivo analysis of intracellular polysulfides For E. coli cytoplasmic polysulfides analysis, psGFP was expressed from pET30psGFP plasmid (Table S1). The strain was cultivated in LB medium at 37oC with 225 rpm shaking until OD600 reached 0.6, then 0.2 mM IPTG was added and the temperature was turned to 18oC. After 10 h cultivation, cells (OD600=0.5) were collected and washed twice with potassium phosphate buffer (10 mM, pH 7.0), then incubated with HSSH, H2O2, or DTT at 37oC. Fluorescence was continuously detected with Synergy H1 microplate reader. Dynamic analysis of polysulfides during growth was performed with the same protocol except incubation temperature was constantly maintained at 37 oC. For S. cerevisiae in vivo experiments, YEPlac195 plasmid containing TEF1 promoter was used for psGFP1.1 expression and transformants were selected from SD-uramedium. To express psGFP1.1 in cytoplasm, the psGFP1.1 encoding gene 8


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was directly incorporated into YEPlac195. To localize psGFP1.1 into mitochondria, the MLSARSAIKRPIVRGLATV leading peptide was fused to the N-terminus. To localize psGFP1.1 into peroxisome, the SKL targeting peptide was fused to the Cterminus. The Localization of psGFP1.1 in mitochondria was tested with MitotrackerTM Red CMXRos and localization in peroxisome was tested with a fused PEX14-mChery (PEX14 is a natural peroxisome protein). The PEX14-mCherry was inserted into S. cerevisiae chromosome. The hygromycin B gene was co-inserted for transformant selection purpose. For analyzing the intracellular polysulfides, the recombinant S. cerevisiae strains were cultivated in SD-uramedium at 30oC with 225 rpm shaking. Middle-log phased cells (OD600=0.1) were collected and incubated with 1mM cystine or cysteine in fresh SD-uramedium at 30oC for 1 hour. After incubation, cells were washed twice with the potassium phosphate buffer then subjected to fluorescence analysis with Synergy H1 microplate reader. Dynamic analysis of polysulfides along with the growth cycle change was performed with the same protocol except with varied incubation time.

Statistical analysis The data in Figures 2, 5a, 5b, 6d, and 7a–c were from three independent replicates. The curves shown in Figures 3, 5c, 5d, and 6a–c were represents of respective technical triplicates.

Results and discussion 9



Structural based design of the polysulfides-sensitive GFP probes The design of polysulfide-sensitive GFP probes was inspired by the ROS sensitive GFP (roGFP). Wild-type GFP has two excitation maxima at about 400 nm and 475–490 nm, which are attributed to an internal equilibrium between the neutral (protonated) and anionic (deprotonated) forms of the chromophore.25 roGFP is constructed by adding two cysteine residues near the GFP chromophore.26,27 After reacting with ROS, roGFP forms an intra disulfide bond (-S2-) between these two cysteine residues. The bond alters the internal equilibrium of the GFP chromophore, reflected by increasing the 400 nm excitation peak (neutral form) while decreasing the 475–490 nm peak (anionic form). Hence, the 400/480 nm ratio indicates the proportion of oxidized/reduced roGFP. We speculated that a –Sn– (n≥3) bond, which has been discovered in several HSSH- or GSSH-reacted transcription factors,28 near GFP chromophore should alter the internal equilibrium between two forms of the chromophore in same way as a disulfide bond. To accomplish this, the distance between two cysteine residues should be longer to allow formation of a –Sn– (n≥3) bond but not a disulfide bond (2.05 Å). Using the 3D structures of roGFP (PDB ID: 1JC0 and 1JC1) as templates for homology modeling, we designed five polysulfide-sensitive GFPs (psGFPs); each contained a pair of cysteine residues near the chromophore, separated by a distance of 6.6 Å to 10.9 Å (Figure 1), larger than that in roGFP2 (3.4 Å).

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Figure 1. Design and structural analysis of the psGFPs. A pair of cysteine residues were placed near the chromophore and on the surface of GFP. The 3D structures were modeled by using SWISS-MODEL method and roGFP2 structures (PDB: 1JC0 and 1JC1) as templates. The distance between sulfur atoms of the cysteine residues was analyzed with Pymol (v1.5).

Experimental testing of the polysulfides-sensitive GFP probes The five designed psGFPs were expressed in E. coli and purified with a Nterminal His-tag. HSSH, DTT, and H2O2 were used as reactants to react with the purified psGFPs. After reaction, unreacted reactants were removed by passing a protein desalting column. For each psGFP, the excitation spectra of HSSH-reacted, H2O2-reacted, and DTT-reacted were compared. We used the 408/488-nm ratio to evaluate the response of a psGFP to HSSH treatment and H2O2 treatment, and the ratio of DTT-reacted psGFP (without disulfide or –Sn– bond) was used as the reference (Figure 2). psGFP1.2 and psGFP1.3 did not shown apparent response to HSSH or H2O2 treatments. psGFP1.5 and the control roGFP2 responded to both (Figure 2), suggesting they formed disulfide bonds. psGFP1.1 and psGFP1.4 11



responded to HSSH but not H2O2; they were likely sensors for only polysulfides and were subjected to further examination.

Figure 2. Ratiometric changes of psGFPs and roGFP2 in response to HSSH, DTT, and H2O2. 1.5 mg/ml of protein was mixed with 200 μM HSSH, 10 mM DTT, or 10 mM H2O2 in Kpi buffer (10 mM, pH 7.0). After reacting at room temperature for 1 hour, the modified protein was separated from small reagents by passing through a PD-10 desalting column and analyzed with RF-5301 PC spectrofluoro photometer.

The increase of 400-nm excitation peak was accompanied by a decrease of the 475–490 nm excitation peak in psGFP1.1 and psGFP1.4 (Figure 3), indicating the equilibrium between neutral and anionic forms of the chromophore was altered. However, HSSH can result in both –Sn– bond and cysteinyl persulfidation (cysteinylSSH), and the latter may also lead to equilibrium alteration. To test this, we constructed single-cysteine mutants (147C, 149C, 202C) and analyzed their excitation spectra after reacting with HSSH and DTT. None of them showed an apparent difference between HSSH-reacted and DTT-reacted (data not shown). Hence, the 12


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possibility that cysteinyl persulfidation in psGFP1.1 or psGFP1.4 caused such equilibrium alteration was excluded.

Figure 3. Excitation spectra of psGFPs. a) Both HSSH and GSSH reacted-psGFP1.1 showed increase at 400 nm excitation peak while decrease at 475–490 nm excitation peak. H2O2-reacted psGFP1.1 had no detectable Ex spectra change. DTT-reacted psGFP1.1 was the control. b) Only HSSH-reacted psGFP1.4 showed increase at 400 nm excitation peak. 1.5 mg/ml of protein was mixed with 200 μM HSSH, 10 mM DTT, 10 mM H2O2, or 200 μM GSSH in Kpi buffer (10 mM, pH 7.0) and reacted at room temperature for 1 hour. After passing through a PD-10 desalting column, the reacted-protein was analyzed with RF-5301 PC spectrofluoro photometer.

LC-MS/MS analysis of HSSH and H2O2-reacted psGFPs Both HSSH- and H2O2-reacted psGFP 1.1 and psGFP 1.4 were subjected to LTQ-Orbitrap Tandem MS analysis. For psGFP1.1, the trypsin-released peptide containing 147C and 202C was about 6439.08 Da, approaching detection limit of the equipment. We had difficulties to pin-point its MS2 data from MS1 file. For psGFP1.4, the trypsin-released peptide containing 147C and 149C was 1979.27 Da. We found this peptide had 2009.83 Da after HSSH treatment. Its MS2 data indicated the extra 30.56 Da (+S, -2H) was added between 147C and 149C, corresponding to a –S3– bond (Figure S1). For H2O2-reacted psGFP 1.4, a peptide fragment corresponding to C147-SOH and C149-SOH was found (Figure S2). In either HSSH or 13



H2O2-reacted psGFP1.4, no peptide fragment containing a disulfide bond was found. These results indicate that only the –Sn– bond can form in psGFP1.4.

Figure 4. Schematic representation of the reaction mechanisms. a) The first step of psGFPs + polysulfides reaction is the transfer of sulfane sulfur from polysulfides to psGFPs. Considering locations of the two cysteine residues (on the surface of GFP) and the space between them (> 6Å), this step may result in two intermediates (2a and 2b). The second step is intra-oxidation of psGFPs, which leads to the formation of a –S3– bond. The reaction of psGFP with H2O2 leads to a dead end. b) Considering the space limitation (3.4 Å), first step of roGFP2 + polysulfides reaction may result in only one thiol modification (intermediate 6), which is quickly reacted with the adjacent thiol to form a disulfide bond.

The formation of the –Sn– bond requires two sequential reactions. The first is sulfane sulfur transfer and the second is intra-oxidation (Figure 4). The first reaction can happen via two alternative mechanisms, resulting in intermediates 2a and 2b (Figure 4a), respectively. However, a peptide fragment corresponding to 2a or 2b was not found from LC-MS/MS data, indicating they are transient intermediates. This is very reminiscent of intermediate 8 that is formed during roGFP reacting with H2O2 14


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(Figure 4b). For all these intermediates, the intra-oxidation reaction occurs rapidly, probably due to the proximity effect between the two cysteine residues. Hence, as roGFP is stable as either reduced (roGFPred, 5) or oxidized (roGFPox, 7), psGFP also has two stable statuses, reduced (psGFPred, 1) and oxidized (psGFPox, 3) (Figure 4a).

psGFP1.1 reacts with both HSSH and GSSH while psGFP1.4 only reacts with HSSH GSSH is a pivotal intermediate in cellular polysulfide metabolism, and its intracellular content can be up to 100 μM.14 We examined the reaction of GSSH with psGFP 1.1 and psGFP 1.4. GSSH-reacted psGFP 1.1 showed increases of 400-nm excitation peak, but the increasing amplitude is obviously lower than that of HSSHreacted. On the other hand, GSSH-reacted psGFP 1.4 showed no detectable change; no fluorescence difference was found between its DTT-reacted and GSSH-reacted (Figure 3). These results indicated that psGFP1.1 can react with both HSSH and GSSH with the latter is preferred, whereas psGFP1.4 only reacts with the former. This is probably due to two reasons: first, HSSH is inherently more reactive than GSSH;9,13 second, as a tripeptide, GSSH has a steric hindrance issue when reacting with psGFPs. Other sulfur-containing chemicals including GSSG, Na2S2O3, and Me-SSS-Me were also tested; none could induce fluorescence change in either psGFP 1.1 or 1.4. For the same HSSH- or GSSH-reacted psGFP, the 408/488 nm data obtained with RF-5301 PC spectrofluoro photometer and Synergy H1 microplate reader were different but proportional. As the relative percentage of psGFPox (Oxidized%= psGFPox/(psGFPox+ psGFPred), see Methods) were equipment-independent, suitable 15



for comparative and quantitative analysis. We compared the sensitivities of psGFP1.1 toward GSSH and HSSH using psGFPox percentage data. psGFP1.1 showed apparent dose-dependent responses to both (Figure 5a). 40 μM GSSH caused about 40% of psGFPox, but further adding GSSH to 170 μM only led to a slight increase (< 60%). In comparison, 170 μM HSSH could result in >80% of psGFPox. HSSH also reacted with psGFP faster than GSSH did. The reaction between HSSH and psGFP finished in 20 min while the GSSH-psGFP reaction needed about 70 min (Figure 5b).

Figure 5. Oxidization of psGFP1.1 by GSSH or HSSH and reduction by GSH. a) psGFP1.1 showed dose-dependent response to both GSSH and HSSH. b) psGFP1.1 reacted faster with HSSH than with GSSH. c-d) Addition of GSH immediately reduced the GSSH and HSSH reacted-psGFP. 1.5 mg/ml psGFP was mixed with different concentrations of reactants in Kpi buffer (10 mM, pH 7.0). For a, the 408/488-nm data was obtained after 1 hour of reaction. For b, 100 µM reactants were used. For c and d, the 408/488-nm data was continuously acquired after 80 or 100 μM of GSSH/HSSH was added. Synergy H1 microplate reader was used and the obtained data were converted to psGFPox percentages using the equation described in the experimental section.

Redox potential and pH effects on psGFP1.1 16


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Using redox-oxidation titration and DTT (𝐸'0 𝑚 = -330 mV) as the calibrating redox buffer, we measured the redox midpoint potential (𝐸'0 𝑚) of psGFP1.1 to be -318 mV at pH 7.0 and 25oC. This value is somewhat more negative than that of roGFP (around -280 mV). Many studies have proved that RSSH is more active (easier gets oxidized) than RSH.13,29 The proximity effect between two cysteine residues may further accelerate the oxidation reaction (second reaction, Figure 4a). Hence, the lower 𝐸'0 𝑚 of psGFP is reasonable. It is worth noting that a recent study indicated roGFP is much more sensitive (>200-fold) to polysulfides than to H2O2.30 However, from redox potential perspective, sulfane sulfur is a weaker electron acceptor than H2O2 (0.144 mV vs 1.763 mV).31 We proposed this contradiction is because, similar to psGFP, roGFP also forms a Cys-SSH containing intermediate 6 when reacting with polysulfides (Figure 4b). The Cys-SSH is more reductive than Cys-SOH in intermediate 8. Hence, the second reaction in intermediate 6 is faster than that in intermediate 8, leading to that roGFP has higher sensitivity to polysulfides than to H2O2. We also tested the pH influence to psGFP1.1. Results showed that Em510 excited by Ex408 (510408) and Em510 excited by Ex488 (510488) were both sensitive to pH. At pH≤6.0, both 510408 and 510488 fluorescence was weak. At pH 7.0–10.0, 510408 and 510488 fluorescence intensified along with pH increase. At pH 7.0–9.0, the 408/488 ratio (510408/510488) was not obviously affected by pH due to the equal increases (Figure S3).

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GSH catalyzes the reverse reaction of psGFP1.1 oxidation GSH is a dominating reductant inside cells whose content is in the range of 1–15 mM depending on the cell status and the organism. In vitro experiments indicated it reduces polysulfides to H2S.32 To test whether it can reduce the oxidized psGFP, we reacted psGFP1.1 with GSSH or HSSH and real-timely monitored the psGFPox percentage using Synergy H1 microplate reader. After reacting for a period (55 min for GSSH and 15 min for HSSH), 10 mM GSH was added into the reaction mixture. The percentage of psGFPox showed an immediate and sharp drop, followed by a slower but continuous decrease. About 15–30 min after addition of GSH, psGFPox percentage was decreased to the status as that 0-min (Figure 5c and 5d). The sharp drop indicated oxidized psGFP 1.1 was directly reduced by GSH and the continuous slower decrease should be due to the gradually reduction of polysulfides by GSH in the mixture. These experiments demonstrated the oxidation of psGFP is reversible, which reflects the change of polysulfide contents in real time.

Analysis of E. coli cytoplasmic polysulfides The moderate 𝐸'0 𝑚 and reaction-reversibility properties make psGFP1.1 a good probe for intracellular polysulfides analysis. We first tested it in simple prokaryotic cells. E. coli BL21 harboring the psGFP1.1-encoding gene was used. The expression was induced by 0.2 mM IPTG. Middle-log phase cells were collected and prepared as cell suspensions, in which HSSH, DTT, and H2O2 were directly added. The cytoplasmic psGFP1.1 probe displayed rapid ratiometric change in response to extra 18


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added HSSH (Figure 6a). The increase of the psGFPox percentage reached the maximum (> 80%) at 10–20 min depending on the HSSH amount. High amounts of HSSH (> 100 μM) could keep the maximum for at least 90 min while a low amount (50 μM) did not. The psGFPox percentage of 400 μM HSSH-treatment was slightly lower than that of 100 and 200 μM, possibly due to its toxicity at high concentrations, as 400 μM HSSH could completely repress the growth of E. coli (Figure S4). In addition, we observed that after HSSH was removed by washing, the psGFPox percentage slowly decreased to untreated level, indicating psGFP1.1ox and the internalized HSSH were gradually reduced by intracellular redox system. As a control, DTT-treatment led to constantly low psGFPox percentage (< 10%, Figure 6b). High amount of H2O2 (> 10 mM) also led to apparent increase of psGFPox percentage. However, the increase maximum (< 40%) was much lower than that of HSSHtreatment (Figure 6c). This could be due to the interchange between ROS and polysulfides as mentioned in previous studies.4,5 Considering the endogenous H2O2 cannot be as high as 10 mM under normal physiological conditions, the ROS interference can be neglected during polysulfides analysis.

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Figure 6. Analysis of E. coli BL21cytoplasmic polysulfides. a) Incubation of E. coli cell with HSSH dramatically increased the content of its endogenous polysulfides. b) Incubation of E. coli cell with H2O2 slightly increased the content of its endogenous polysulfides. c) Incubation of E. coli cell with DTT decreased the content of its endogenous polysulfides. d) The dynamics of cytoplasmic polysulfides during growth in Lysogeny Broth (LB) medium. The ratios of 408/488-nm and SSP4 fluorescence were detected with Synergy H1 microplate reader.

We then monitored the dynamics of E. coli cytoplasmic polysulfides of a growth cycle in real-time. The endogenous polysulfides was relatively low in the early-log phase. It gradually increased and reached to the maximum at the end of the log-phase, then started to decrease in stationary phase. For confirmation, we also used the polysulfide-sensitive probe SSP4 to detect change of cytoplasmic polysulfides of E. coli and observed the same trend (Figure 6d). These results indicate that the content of intracellular polysulfides is growth-phase related in E. coli. The change from 20% to 60% of psGFPox is significant, suggesting that polysulfides are likely involved in global gene regulation and enzyme activities associated with growth phases. This speculation is in agreement with previously reports that polysulfides are the direct inducer for the activation of H2S-oxidizing genes in bacteria (FisR, CstR) and the H2S-oxidizing activity is higher for cells from the stationary phase than those from the 20


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log phase for most tested bacteria.33

Analysis of polysulfide distribution among subcellular organelles in S. cerevisiae Eukaryotes have relatively isolated organelles. How polysulfides are distributed among them has never been investigated. Using S. cerevisiae BY4742 as a model, we analyzed the polysulfides in its cytoplasm, mitochondria, and peroxisomes. For cytoplasm analysis, psGFP1.1 was expressed without any signal peptide and for mitochondria and peroxisomes analysis, psGFP1.1 was fused with targeting signal peptides. Subcellular localization experiments confirmed that psGFP1.1 was correctly deployed in desired organelles (Figure S5–S7). S. cerevisiae was cultivated in yeast synthetic defined minimal medium (SD medium), and cells of different growth phases were analyzed. Quite different from E.coli, S. cerevisiae had high content of polysulfides in the lag and early log-phases (Figure 7a and 7b). The content decreased along with growth and reached the minimum at the end of log-phase, and then started to increase in the stationary phase. The peroxisomes had lower contents than cytoplasm and mitochondria in the lag and early-log phases, but the situation reversed in the stationary phase. The mitochondria showed no obviously difference from the cytoplasm except for at 10 hours of growth. These results indicated the polysulfide distribution is not homogenous in subcellular organelles.

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Figure 7. Dynamics and distributions of polysulfides in S. cerevisiae organelles. a) Growth of S. cerevisiae BY4742 strains expressing peroxisomes-, cytoplasm-, or mitochondria-targeting psGFP1.1 in SD medium. No growth difference was observed from the three strains. b) The percentages of oxidized psGFP (psGFPox %) in different organelles and growth phases. c) The percentages of oxidized psGFP (psGFPox %) after cystine or cysteine treatment. Middle-log phase Cells (OD600=0.1) were incubated with 1 mM cystine or cysteine in SD medium for 1 hour. Controls were untreated cells. The ratios of 408/488-nm were detected with Synergy H1 microplate reader and converted to psGFPox percentages using the equation described in the Supporting Methods.

Some mammalian cancer cells have abnormal higher intracellular polysulfides and, CBS and CSE have been reported to be the main polysulfides-generating enzymes9,13,14. These enzymes use cystine to produce polysulfides, S. cerevisiae also has inherent CBS and CSE. When cystine was added into S. cerevisiae cell suspensions and incubated for 1 h, the levels of intracellular polysulfides were significantly increased in the cytoplasm, mitochondria, and peroxisomes (Figure 7c). Peroxisomes had the highest increase, which was surprising because CBS and CSE are supposed to be localized mainly in the cytoplasm and mitochondria instead of peroxisomes14. Recent studies indicated two house-keeping enzymes, 3-MST and cysteinyl-tRNA synthetase, also produce polysulfides from cysteine instead of cystine.6,34 We treated S. cerevisiae with cysteine and found that polysulfides did not change in peroxisomes but obviously increased in the cytoplasm and mitochondria. 22


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Considering the distribution of polysulfides in mammalian cells has never been investigated at subcellular level due to the lack of appropriate tools, psGFP provides a proper tool, which may reveal new physiological functions for polysulfides.

Conclusion We designed and constructed GFP-based, polysulfide-specific, and reactionreversible ratiometric probes that enable real-time analysis of polysulfides in live cells and subcellular organelles. ROS have minimal interference to the analysis because these probes do not respond to them. Compared with previously reported probes, our psGFPs have obvious advantages (Table 1), providing long-desired tools for in vivo studies of polysulfides. With these probes we found the content of cytoplasmic polysulfides are highly correlated with growth phases in E. coli. The distribution of polysulfides in subcellular organelles are heterogeneous in S. cereviase. These variations suggest that polysulfides participate in regulating gene expression and enzyme activities associated with growth phases and in subcellular organelles.

Table 1: Comparison of psGFPs with other polysulfide-sensing probes Probea

Property

Reaction

Real-time analysis

ROS disturbance

SSP4 and its derivatives QS10 roGFP psGFP

Chemical

Irreversible

No

No

Chemical Protein Protein

Reversible Reversible Reversible

Short termb Long term Long term

Unknown Yes No

[a] References [1, 17-19, 25, 26]. [b] Reported to be 4–12 min, for experiments involving no cell propagation.

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Acknowledgments The work was financially supported by grants from the National Natural Science Foundation of China (31770093, 91751207, 21477062), the Natural Science Foundation of Shandong Province (ZR2016CM03, ZR2017ZB0210).

Conflict of interest disclosure The authors declare no competing financial interests.

ORCID Luying Xun: 0000-0002-5770-9016 Huaiwei Liu: 0000-0002-0483-5318

Supporting Information Strains and plasmids, LTQ-Orbitrap Tandem MS analysis of psGFP, pH influence to psGFP, Subcellular localization of psGFP in S. cerevisiae.

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Developing Polysulfide-Sensitive GFPs for Real-Time Analysis of

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