Stress-dependent conformational changes of artemin: Effects of heat and oxidant

Zeinab Takalloo; Zahra Afshar Ardakani; Bahman Maroufi; S Shirin Shahangian; Reza H Sajedi

doi:10.1371/journal.pone.0242206

. 2020 Nov 16;15(11):e0242206. doi: 10.1371/journal.pone.0242206

Stress-dependent conformational changes of artemin: Effects of heat and oxidant

Zeinab Takalloo ^1,^#, Zahra Afshar Ardakani ^1,^#, Bahman Maroufi ², S Shirin Shahangian ³, Reza H Sajedi ^1,^*

Editor: Reza Yousefi⁴

PMCID: PMC7668597 PMID: 33196673

Abstract

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer–oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Introduction

The brine shrimp Artemia is a micro-crustacean, well adapted to the extreme conditions such as desiccation, radiation, high and low temperatures and several years of anoxia. Populations of Artemia are found in many inland salt lakes and coastal salterns distributed all over the world [1]. This organism undergoes diapause by forming a unique structure called cyst enabling it to survive in the adverse ambient conditions, which can remain viable for decades [2]. Artemin is a stress protein of encysted Artemia embryos, representing about 10% of the soluble cellular proteins, but it is almost completely absent from nauplius larvae [3]. Due to its high structural stability, abundance and chaperone-like activity, artemin probably contributes to cyst stress resistance [4]. Artemin monomers consist of 229 amino acid residues and exhibit a molecular mass of 26 kDa, which are assembled into rosette-like oligomers of ~600–700 kDa consist of 24 monomer subunits [4, 5]. Artemin and ferritin, an iron storage protein, show a degree of homology in both sequence and structure, although artemin contains 45–50 extra residues compared to ferritin [3, 5–7]. In contrast, artemin fails to bind iron due to the extension of carboxyl terminal of the protein monomers, which suggests to have a role in its chaperone function [5, 8]. It is very heat stable and associates with RNA at elevated temperatures, suggesting protection of RNA [9].

Previous researches revealed a chaperone-like activity for artemin, responsible for protecting transfected cells and preventing protein aggregation in vitro [10]. Our previous computational and experimental studies on artemin from Artemia urmiana (Urmia Lake, Iran) also confirmed the potency of artemin in suppressing protein aggregation [5, 8, 11]. Besides, artemin was capable to protect proteins and cells against different stress conditions such as oxidant, cold and salt [12, 13]. These studies proposed that artemin binds to protein substrates via hydrophobic interactions. Artemin is a histidine/cysteine-rich protein and due to the high content of cysteines and their distributions, it was suggested that the oxidative state of cysteines modulates the redox-regulated activity of artemin [3, 12]. Therefore, in our recent study, the modification of cysteine residues revealed that the function of artemin is greatly dependent on the formation of intermolecular disulfide bonds suggesting artemin as a redox-regulated chaperone [14].

To date, artemin has been used for different purposes. For example, it could enhance the soluble production of some aggregation-prone proteins in bacterial cells such as aequorin [15] and luciferase [13]. Besides, the anti-amyloidogenic effect of artemin on α-synuclein [16] and blocking fibrillization of tau protein [17] were further confirmed. Despite such successes, there are no enough details on the protein activation mechanisms or structural modifications of this chaperone. Our suggestion is that conformational changes of artemin are responsible for its chaperone function upon exposing to stress conditions and interactions with the protein substrates. In fact, these modifications may change the surface hydrophobicity of the chaperone, which result in hydrophobic interactions with target proteins. Accordingly, in the present study, we tried to provide some basis on the conformational changes of artemin upon exposing to stress conditions and interaction with the protein substrates. The tertiary and quaternary structures of artemin under heat and oxidative stresses have been evaluated by intrinsic and extrinsic fluorescence measurements, protein cross-linking analysis, and dynamic light scattering (DLS). In addition, the activity of artemin was investigated under both stress conditions using dilution-induced aggregation assay of lysozyme. Notably, this is the first report in providing details on the conformational changes of artemin’s subunits. We believe that this information would help us to justify the protective function of artemin in Artemia cysts during stress conditions.

Materials and methods

Chemicals

Glycine, Tris, NaCl, sodium dodecyl sulfate (SDS), imidazole, kanamycin and Isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Bio Basic Inc. (Markham, Ontario, Canada). Peptone and yeast extract were provided by Micromedia Trading House Ltd (Pest, Hungary). AgNO₃ and 8-anilino-l-naph-thalene sulfonic acid (ANS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Nickel-nitrilotriacetic acid agarose (Ni-NTA agarose) was provided by Qiagen (Hilden, Germany). Chicken egg white lysozyme, dithiothreitol (DTT), glutaraldehyde, H₂O₂, glycerol, and all other chemicals were obtained from Merck (Darmstadt, Germany). Protein standard marker was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Expression and purification of recombinant artemin

pET28a encoding artemin from Artemia urmiana was provided [5] and protein expression was carried out in Escherichia coli BL21 (DE3) cells as previously described [11]. Purification of the His-tagged protein was carried out using Ni-NTA agarose column. Bound proteins were eluted with a buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, and 250 mM imidazole, pH 8.0. Aliquots of the eluted protein were taken, followed by dialysis against the phosphate buffer overnight at 4°C. The protein concentrations were determined using Bradford's method and BSA as standard [18].

Protein treatments

Heated artemin (H-artemin)

Purified artemin (in 50 mM phosphate buffer, pH 7.4) was placed in a heated circulating water bath at temperature range 25–80°C for 20 min followed by incubation at room temperature (RT) for 20 min before measurements. One standard thermometer was used for taking the temperature of the water in the bath. The temperature of the protein solution in the microtubes was further checked with the thermometer.

Oxidized artemin (O-artemin)

Purified artemin (in 50 mM phosphate buffer, pH 7.4) was incubated with 0–160 mM H₂O₂ for 6 h at 0°C in dark conditions before measurements.

Heated oxidized artemin (HO-artemin)

Purified artemin (in 50 mM phosphate buffer, pH 7.4) was incubated with 0–100 mM H₂O₂ for 6 h at 0°C in dark conditions, then placed in a heated circulating water bath at temperature range 25–70°C for 20 min followed by incubation at RT for 20 min before measurements.

Conformational analysis

Intrinsic fluorescence measurements

Intrinsic aromatic fluorescence measurements were performed at RT using a LS-55 fluorescence spectrometer (Perkin-Elmer, USA) in a quartz cell of 1-cm path length. Samples containing H-artemin, O-artemin, and HO-artemin in 50 mM phosphate buffer, pH 7.2 were used. The excitation wavelength was set at 280 nm, and the emission spectra were monitored in the wavelength range of 300–400 nm. Excitation and emission bandwidths were 10 nm. Protein concentration for intrinsic fluorescence measurements was about 3.7 μM.

ANS fluorescence measurements

Samples containing H-artemin, O-artemin, and HO-artemin in 50 mM phosphate buffer, pH 7.2 were used. The excitation wavelength was set at 380 nm and emission spectra were taken from 400 to 600 nm at RT by using the fluorescence spectrometer in a quartz cell of 1-cm path length. Excitation and emission slits were set at 10 nm. Protein concentration for ANS measurements was about 9.26 μM and ANS was added at a final concentration of 30 μM.

To investigate whether stress-induced conformational changes of artemin were reversible, the heated and oxidized artemin were kept at 4°C for 3 h and 48 h, then their intrinsic and extrinsic fluorescence were measured at RT. In the case of oxidized proteins, for elimination of hydrogen peroxide, catalase (8.33 nM) was added in the protein solution, followed by incubating the samples at 4°C.

Protein cross-link analysis by SDS-PAGE

H-artemin, O-artemin, and HO-artemin at final concentration of about 7.4 μM were used. Glutaraldehyde (0.5%) was added to all protein treatments and the reactions were terminated after 1 min by addition of 200 mM Tris-HCl, pH 8.0 at the final concentration. In the case of oxidized samples, catalase (8.33 nM) was added to decompose the hydrogen peroxide in the solution before addition of glutaraldehyde. The protein cross-linking were analyzed using 10% non-reducing SDS-PAGE stained with silver nitrate [19]. The non-heated/oxidized artemin (at 25°C/ 0 mM H₂O₂) was used as control. The areas of the bands regarding dimeric states of the protein on SDS-PAGE were calculated in each experiment by densitometry analysis using ImageJ software [20].

In addition, to check whether the dimerization of H-artemin, is dependent on disulfide bridges, DTT (30 mM at final concentration) was added to artemin (3.7 μM), then the samples were incubated at 25 and 60°C for 20 min followed by incubation at RT for 20 min. Dimerization of the protein was checked using 10% non-reducing SDS-PAGE with and without DTT and glutaraldehyde as the reducing and cross-linking agents, respectively.

DLS analysis

The size analysis of the H-artemin (1.85 μM) at 25–80°C was carried out using dynamic light scattering by Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, Worcestershire, UK) at RT.

Chaperone-like activity assay

Preparation of denatured reduced lysozyme

Denatured reduced lysozyme (10 mg/mL) was prepared by diluting appropriate volume of the enzyme in a denaturation buffer containing 6 M GdmCl and 40 mM DTT in 50 mM potassium phosphate buffer, pH 7.1. The sample was incubated at RT for a period of 12 hours to yield a fully reduced denatured enzyme. The denatured reduced lysozyme was used for refolding studies.

Aggregation accompanying refolding

Refolding of lysozyme was initiated after 50-fold rapid dilution of the denatured enzyme solution (10 mg/mL) with a refolding buffer (5 mM GSH, 5 mM GSSG in 50 mM potassium phosphate buffer, pH 8.5) containing artemin at the final concentrations of 0.5 and 1 μg/mL. The concentrations of the redox reagents were in the range of the optimum concentrations suggested by a previous report [21]. The final concentration of lysozyme was 13.89 μM in the buffer. The kinetic of refolding was recorded by monitoring light scattering at 400 nm at RT for 2 min using a microplate spectrophotometer (μQuant, BioTek, USA). The denatured lysozyme refolded without artemin was used as control. In these experiments, H-artemin (25–80°C), O-artemin (0–100 mM H₂O₂), and HO-artemin (25–100 mM H₂O₂, 25–70°C) were used.

Results

Temperature-dependent structural changes in artemin

Intrinsic fluorescence was monitored at 330 nm with 280 nm excitation to probe changes in tertiary structures of H-artemin (Fig 1A). The results indicated that when the temperature increased up to 30°C, the fluorescence intensity decreased sharply, and the lowest emission intensity was recorded for the heated protein at 80°C (Fig 1A). Besides, the wavelength of maximum emission (λ_max) did not show any shift.

The fluorescence of 30 μM ANS in the presence of H-artemin was also monitored at 470 nm after excitation at 380 nm (Fig 1B). ANS binding showed that the fluorescence intensity did not change considerably from 25 to 60°С. In contrast, a sharp emission peak was monitored at 470 nm for the heated proteins incubated at temperatures 70–80°C.

Glutaraldehyde cross-linking of artemin followed by SDS-PAGE was performed for the heated proteins (Fig 2A). Analysis of the areas of dimeric bands in each experiment using ImageJ software revealed that upon increasing temperatures from 25 to 50°C, the band corresponding to dimeric form of artemin around 54 kDa slightly strengthened and after that, it was reduced at 60°C, and finally completely disappeared at 70 and 80°C (Fig 2B). Besides, the dimeric band disappeared when artemin was incubated with DTT before heat treatments under both cross-linking and non-cross-linking conditions showing that induced dimerization of H-artemin is dependent on the formation of disulfide bond(s) (Fig 2C and 2D).

DLS measurement confirmed that the average size diameter of the heated chaperone increased upon enhancement of the temperature (Fig 3) and accordingly, the largest size was detected at 70 and 80°C. The larger size distribution of H-artemin at elevated temperatures can be explained by its ability to form oligomeric species as well as large aggregates as also indicated by SDS-PAGE (Fig 2A). All results showed that the conformational changes occur in artemin upon increasing temperature.

Oxidative-dependent structural changes in artemin

As depicted in Fig 4A, fluorescence emission maximum (λ_max), as well as the fluorescence intensity of protein samples, were influenced by increasing the oxidant concentration. The slight decrease in fluorescence at 332 nm was monitored with increasing the concentration of oxidant from 2.5 to 40 mM, followed by a considerable decline in fluorescence intensity for O-artemin with 80–160 mM H₂O₂. Besides, λ_max shifted to longer wavelengths, from 330 to 337 nm (Fig 4A).

Extrinsic fluorescence using ANS showed a two-state process. The O-artemin with lower concentrations of H₂O₂ (0–40 mM), showed slight intensity decreases, but the intensity was enhanced at higher concentrations of the oxidant (80–160 mM). As depicted in Fig 4B, a 10 nm red shift of the emission peak position of O-artemin was monitored when spectra at lower oxidant contents (0–40 mM, ~ 474 nm) are compared to those at higher oxidant concentrations (80–160 mM, ~ 484 nm).

SDS-PAGE and ImageJ analysis of cross-linked artemin showed that the band corresponding to dimeric form of O-artemin gradually strengthened upon increasing the concentration of H₂O₂ from 0 to 40 mM, and it was weakened at 100 and 160 mM H₂O₂ (Fig 5A and 5B).

Fig 5 — A) Protein cross-link analysis of oxidized artemin with various H₂O₂ concentrations by SDS-PAGE and B) the levels of the dimeric bands measured by ImageJ software. Purified artemin (7.41 μM) was incubated with 0–160 mM H₂O₂ for 6 h at 0°C in dark conditions. Before the samples were loaded on 10% non-reducing SDS-PAGE, a trace amount of catalase (8.33 nM) was added to the protein solutions to decompose the remaining H₂O₂. M; protein standard marker.

Structural changes of artemin under heat and oxidative conditions

To evaluate the influence of the two stressors, i.e. heat and oxidant, on protein structure, artemin was treated with 0–100 mM H₂O₂, followed by exposure to 50 and 70°C. Intrinsic fluorescence measurements showed that under both temperature incubations, the intensity declined gradually by increasing the oxidant concentrations from 0 to 100 mM (Fig 6A). Besides, ANS fluorescence indicated that the fluorescence intensity did not change considerably for HO-artemin incubated with 10–100 mM H₂O₂ at elevated temperatures (Fig 6B). This trend was not similar to those observed for the individual oxidant treatments (Fig 4B).

Fig 6 — Intrinsic (A) and extrinsic (B) fluorescence emission intensity of heated oxidized artemin. A) Intrinsic fluorescence emission of artemin was recorded by incubating artemin (3.7 μM in 50 mM phosphate buffer, pH 7.2) with 0–100 mM H₂O₂ for 6 h at 0°C in dark, followed by incubating the samples at 50 and 70°C for 20 min, then cooling down at RT. The fluorescence intensity was recorded with the excitation wavelength at 280 nm. B) Fluorescence emission intensity of ANS (30 μM at final concentration) was measured in the presence of HO-artemin (9.26 μM) with the excitation wavelength at 380 nm.

In a second approach, changes in the oligomerization state of HO-artemin were checked by SDS-PAGE (Fig 7A). Analysis of the areas of dimeric bands in each experiment by ImageJ software showed that the bands representing the dimeric forms of HO-artemin were clearly strengthened along with enhancement of the oxidant concentrations (Fig 7B).

Structural changes of artemin under stress conditions are irreversible

To determine whether the temperature/oxidation-dependent transition of artemin is reversible, artemin was incubated at elevated temperatures (H-artemin), high oxidant concentrations (O-artemin), and both heat and oxidant conditions (HO-artemin), followed by elimination of the heat and oxidant and keeping the samples at 4°C for 48 h before fluorescence measurements (Fig 8). The results showed that fluorescence intensity did not change after 48 h. In fact, the structure of artemin modified irreversibly upon exposing to the stressors.

Fig 8 — A) Artemin at final concentration of 3.7 μM was treated with 0–100 mM H₂O₂ for 6 h at 0°C in dark, followed by incubating at 50 and 70°C for 20 min. Then the samples were kept at 12°C for 48 hours and the fluorescence intensity was recorded with the excitation wavelength at 280 nm and the fluorescence intensity at λ_max was determined. B) ANS (30 μM) fluorescence intensity at λ_max in the presence of 9.26 μM HO-artemin with the excitation wavelength at 380 nm.

Chaperone-like activity of artemin under heat and oxidative stress

H-artemin

Denatured/reduced lysozyme was refolded by the dilution method and the kinetics of chaperone-assisted refolding was examined in the presence of 0.5 and 1 μg/mL artemin (Fig 9). As shown in Fig 9A and 9B, the heated artemin at 25 and 50°C was found efficient in suppressing aggregation of the enzyme. In contrast, 0.5 μg/mL chaperone incubated at elevated temperatures, 60 to 80°C, accelerated the aggregation of lysozyme (Fig 9A), and 1 μg/mL artemin showed no effect on the refolding yield at similar conditions (Fig 9B).

Fig 9 — Refolding of lysozyme in the absence ( $●$ ) and presence of ( $●$ ) heated (A, B) and ( $●$ ) oxidized (C, D) artemin. Denatured lysozyme (10 mg/mL) in a solution containing 40 mM DTT, 6 M GdmCl, and 50 mM potassium phosphate buffer, pH 7.1, was diluted with a mixing ratio of 1:50 by the refolding buffer. The diluted sample contained 13.89 μM lysozyme, 0.8 mM DTT, 0.12 mM GdmCl, and 50 mM potassium phosphate buffer, pH 8.5, 5 mM GSH, 5 mM GSSG, and 0.5 (A, C) and 1 μg/mL (B, D) artemin. The kinetic of refolding was recorded by monitoring light scattering at 400 nm at 25°C.

O-artemin

Results showed that the oxidized chaperone (0.5 μg/mL) with 25 and 50 mM H₂O₂ could partly suppress aggregation of lysozyme compared to the untreated control (Fig 9C). In contrast, the anti-aggregatory potency of O-artemin weakened by increasing the oxidant content, and the oxidized chaperone with 100 mM H₂O₂ did not show any effect on the enzyme refolding yield (Fig 9B and 9C).

HO-artemin

Fig 10 depicts the impact of HO-artemin (1 μg/mL) on refolding of lysozyme based on constant concentrations of H₂O₂ (25, 50 and 100 mM) (Fig 10A, 10B and 10C) and temperatures (25, 50 and 70°C) (Fig 10D, 10E and 10F). According to Fig 10A, the HO-artemin at 25 and 50°C showed a potency in suppressing the aggregation of lysozyme, but at the elevated temperature, 70°C, it could not efficiently prevent the aggregation of lysozyme (Fig 10A and 10B). These trends were similar to the graphs obtained for H-artemin (Fig 9A and 9B). In contrast, HO-artemin incubated with 100 mM H₂O₂ clearly exhibited an improved chaperone activity (Fig 10C).

We also checked the effect of HO-artemin on refolding of lysozyme based on constant temperatures (Fig 10D, 10E and 10F). Results clearly demonstrated an enhanced capacity of the chaperone along with the increased oxidant concentrations at 25°C (Fig 10A). At higher temperatures (50 and 70°C), oxidized artemin with 50 and 100 mM H₂O₂ showed a similar rate (Fig 10B and 10C). Comparing these observations with results from the effect of O-artemin on refolding yield (Fig 9C and 9D) reveals that the chaperoning function of HO-artemin was considerably improved in the presence of both stressors.

Discussion

Artemin is an abundant heat stable protein in Artemia encysted embryos and it was found that high regulatory production of artemin under harsh environmental conditions is probably relevant to stress resistance in this crustacean [4, 22]. Artemin is not only capable of suppressing heat-induced aggregation of different protein substrates such as citrate synthase, carbonic anhydrase, horseradish peroxidase and luciferase in vitro and in vivo, but also protecting nucleic acids as a potent chaperone [8–11, 13]. The intrinsic conformational properties of artemin seem to play crucial roles in its biological activities. Artemin showed a higher surface hydrophobicity when compared to other molecular chaperones, based on our previous studies [11]. Moreover, the chaperoning potency of artemin is greatly dependent on the presence of cysteine residues and intermolecular disulfide bond formation [14]. Accordingly, heat and oxidation are among the most important factors, which probably affect the chaperone capacity of artemin. In this report, the conformational transitions of artemin have been evaluated under heat and oxidative stress using different structural and functional analysis. The results have been summarized in Table 1. Our findings may shed light on the mechanisms by which small heat shock proteins (sHsps) could play their protective roles in various stress conditions. Notably, the structural changes of artemin under oxidative stress conditions have not been investigated to date.

Table 1. A summarize of the structural and functional changes of artemin upon heat and H₂O₂ treatments.

Stress Changes	Heat	H₂O₂	Heat & H₂O₂
Hydrophobicity	70–80°C: ↑	0–40 mM: ↓	50, 70°C; 10–100 mM: ↓
Hydrophobicity	70–80°C: ↑	80–160 mM: ↑	50, 70°C; 10–100 mM: ↓
Dimerization	25–50°C: ↑	0–40 mM: ↑	50, 70°C; 10–100 mM: ↑
Dimerization	60–80°C: ↓	80–160 mM: ↓	50, 70°C; 10–100 mM: ↑
Reversibility	Irreversible	Irreversible	Irreversible
Chaperone Activity	25–50°C: Less to More	0–25 mM: Less to More	50–70°C; 25–100 mM:
Chaperone Activity	60–80°C: More to Less	50–100 mM: More to Less	Less to More

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Different mechanisms have been suggested for sHsps to protect protein substrates in different stresses [23]. Many sHsps are known to undergo temperature-dependent structural alterations, which modulate their chaperone activities [24]. These mechanisms may be mediated through conformational transitions of the dimeric and oligomeric forms of the chaperones to each other. For example, at elevated temperatures, dissociation of large oligomeric forms of sHsp26 into smaller, active species of the chaperone occurs [25]. Also temperature-dependent rearrangements in the tertiary structure of Hsp22 resulted in its improved chaperone activity [26]. The chaperoning potency of a redox-regulated chaperone, Hsp33, was mainly mediated by the heat effect and its monomer-dimer switch [27]. Besides, Hsp20.1 and Hsp14.1 oligomers dissociated to smaller oligomeric forms or even dimer/monomer species under acid stress [23]. The dissociation seems to be necessary for the exposure of additional hydrophobic sites on the surface of the protein molecule [28].

Heat-dependent structural transitions

At elevated temperatures, artemin has been expected to undergo heat-dependent structural transitions like other sHsps, leading to exposure of a large amount of hydrophobic residues on the protein surface. Since artemin contains seven Trp and five Tyr residues (S1 Fig), fluorescence studies could be applied to monitor its conformational changes. At present, there is no reliable information for the accurate spatial localization of these amino acids in artemin. It has been shown that Trp and Tyr fluorescence is usually quenched in a distance-dependent manner. Accordingly, when such aromatic amino acids are located on the surface of the protein, the fluorescence intensity of the residues is usually influenced in a higher degree by the quencher, compared to the amino acids deeply embedded in the protein structure [29]. As depicted in Fig 1A, the fluorescence emission of the protein was significantly reduced at elevated temperatures and the first transition was observed at 50°C, followed by the second transition at 80°C. It can be suggested that the induced conformational changes of artemin resulted in the increased quenching, probably due to the exposure of the buried aromatic residues to the solvent.

The conformational changes of the protein was also investigated using ANS fluorescence studies. ANS is widely used as a hydrophobic fluorescent probe for monitoring hydrophobic patches of proteins [28, 30]. It is a non-fluorescent probe in aqueous solutions, while it becomes highly fluorescent in non-polar environment [28]. Increasing temperature up to 60°C lead to higher exposure of artemin non-polar regions and binding ANS to these hydrophobic sites, where an enhanced fluorescence intensity and a weak blue shift in the λ_max (Fig 1B) were observed, similar to other reports [28, 31]. Such hydrophobic interactions in artemin likely play a critical role in acquiring its functional conformation and mediating the protein-chaperone interactions [30]. In contrast, no change was observed in fluorescence intensity, when the protein was incubated at lower temperatures, presumably reflecting negligible changes in the protein surface hydrophobicity, particularly the lack of ANS-binding sites. Due to the thermal inactivation of the excited state of the probe, the heat-treated proteins allowed to cool down at RT for 20 min followed by fluorescence measurements [30].

In a second approach, different monomer, dimer and oligomer forms of H-artemin were analyzed under thermal conditions using SDS-PAGE technique after addition of 0.5% glutaraldehyde as a fixative agent. Glutaraldehyde cross-linking is a commonly used method for determination of the subunit structure of oligomeric proteins [32]. The areas of the dimeric bands of the heated protein on SDS-PAGE were further checked using ImageJ software. SDS-PAGE and ImageJ analysis showed that the uncross-linked protein was mainly presented as monomers at 25°C (Fig 2A and 2B), while the protein was mostly observed in dimeric forms under cross-linking condition. The abundance of the 54 kDa protein band was slightly enhanced upon increasing temperatures from 25 to 50°C, whereas it became significantly weaker at 60°C and completely disappeared at 70 and 80°C, probably due to the formation of higher molecular weight oligomers and/ or aggregates via hydrophobic interactions that could not enter the gel (Fig 2A). Furthermore, we examined whether the heat-induced dimerization is mediated via disulfide bonds by addition of DTT as a reducing agent to the protein solutions before the heat treatment. Accordingly, the dimers were disappeared in the presence of DTT, confirming that the disulfide bond(s) are necessary for dimerization (Fig 2C and 2D). Size distribution analysis of H-artemin showed that the size of proteins was enhanced upon increasing temperatures, probably due to the formation of dimers, oligomers (Fig 3A and 3B) and protein oligomeric assembly and/or aggregates (Fig 3C and 3D) resulted from intermolecular hydrophobic interactions (Fig 1B).

Many previous reports revealed that N-terminus of sHsps is rich in hydrophobic residues and involved in binding client proteins due to the presence of these hydrophobic residues in this region, which is located on the inner surface of the oligomers [33]. Therefore, heat-induced oligomer dissociation may lead to the exposure of a large number of hydrophobic surfaces that are normally buried in the oligomer, including not only the N-terminus, but also the dimer–dimer and even the monomer–monomer interfaces [23]. As a result, the substrate-binding ability of the chaperones would be improved in a temperature-dependent manner to cope with the increasing number of the client substrates within cells [34]. Totally, our results revealed that at elevated temperatures, H-artemin presumably undergoes the structural changes, which are associated with a marked increase in the surface hydrophobicity and also degree of dimerization of the chaperone via disulfide bridges in order to improve the binding capacity of the chaperone to the client proteins in an unfolding intermediate state. As a suggestion, interaction of the protein substrates may subsequently prevent the self-association of the molecular chaperone with a highly exposed hydrophobic surface during the stress conditions.

Oxidative-dependent structural transitions

Since artemin contains a high content of cysteine residues with specific distributions, cysteines may be regarded as critical factors in regulating chaperone function [3, 15]. We have shown previously that oxidation/reduction of cysteine residues greatly influences chaperone potency of artemin [14]. Experimental results revealed that 9 out of 10 thiols are free in artemin monomers and there is one cysteine involved in inter-molecular disulfide bond formation. Moreover, our molecular modeling studies predicted that Cys22, with the highest accessible surface area, is likely the responsible residue for inter-subunit disulfide bond formation [14]. Accordingly, it was supposed that the formation of an inter-molecular disulfide bond between two monomers of artemin leads to its dimerization and switching between its less and more active forms. Here, we tried to provide detailed structural information on conformational changes of artemin during oxidative conditions. Intrinsic fluorescence measurements demonstrated that upon increasing hydrogen peroxide concentrations to 40 mM, artemin structure was still stable (Figs 4A and 5), when compared with many other oxidant susceptible proteins [35, 36]. However, higher concentrations of the oxidant (80–160 mM) led to a remarkable reduction in the fluorescence intensity of O-artemin with a slight red shift of λ_max (Fig 4A, insert), probably due to the changing in the microenvironment around the aromatic residues during oxidation. Trp and Tyr residues show high tendency to interact with negatively charged residues, cysteine and disulfide bonds in their spatial environments, where the interactions of sulfur atoms with their aromatic rings may lead to the fluorescence decay [37]. Cystines that are involved in disulfide bonds have a high propensity to interact with tryptophan residues [38]. Due to the micro-environment alteration of the fluorescent residues resulted from the breaking or forming of the disulfide bonds, the fluorescence properties of proteins could be changed [39]. Accordingly, variable degrees of fluorescence quenching of Trp and Tyr residues probably occurred by nearby formed disulfide and sulfhydryl groups in O-artemin (Fig 4A). Moreover, the quenching of Trp fluorescence upon exposing to the oxidant may also occur as a result of oxidation of the side chains of the aromatic residues and forming a range of aromatic products in oxidized proteins [40].

ANS binding indicated that increasing H₂O₂ content from 10 to 40 mM led to a decline in the fluorescence intensity (Fig 4B), as well as a shift in λ_max to longer wavelengths (Fig 4B, insert). Therefore, it seems that the surface hydrophobicity of O-artemin has been relatively decreased. It is suggested that oxidation of Cys residues and the subsequent disulfide bond formation resulted in oligomerization of artemin, where the hydrophobic patches were simultaneously buried deeply within the protein oligomer assemblies. Although, higher concentrations of the oxidant (80–160 mM) resulted in an increase in ANS binding, the increase was not as significant as that observed for H-artemin (Fig 1B). Similar trends were also recorded for the chaperone GroEL in the presence of H₂O₂ [41].

Glutaraldehyde cross-linking of the oxidized artemin in the presence of 0–160 mM H₂O₂ was carried out to further confirm the dimerization/oligomerization status of O-artemin. SDS-PAGE and ImageJ analysis demonstrated that the 54 kD dimeric bands were gradually sharpened along with increasing the oxidant content from 0 to 40 mM (Fig 5), and the bands were weakened at 100 mM H₂O₂, probably due to the formation of a higher degree of oligomers (Fig 5A and 5B). It is hypothesized that the highly reactive thiol groups of cysteine residues may lead to protein oligomerization due to the excess formation of disulfide bonds in O-artemin. Finally, the protein precipitation may occur as a result of highly exposed hydrophobic regions and the formation of excess inter-protein disulfide bridges [38], as showed by fluorescence (Fig 4B) and SDS-PAGE analysis (Fig 5). Taken together, the results indicate that upon oxidation, artemin undergoes dimerization and oligomerization.

Structural transitions of artemin under heat and oxidant treatments

Finally, to examine the simultaneous effect of stressors on protein structure, artemin was exposed to different concentrations of H₂O₂ (0–100 mM), followed by incubation at elevated temperatures (50, 70°C). The fluorescence measurements showed that the intensity of emission decreased upon increasing the oxidant content at both temperatures (Fig 6A). This result is in a good agreement with the fluorescence measurement of O-artemin (Fig 4A). Based on ANS binding studies, a decreasing trend for fluorescence intensity of HO-artemin was observed (Fig 6B) along with the increased oxidant concentrations (up to 100 mM H₂O₂). However, O-artemin showed an increased ANS binding upon treatment with 80–160 mM H₂O₂ (Fig 4B). Our suggestion is that the oxidant agent sequestered the exposed hydrophobic surfaces of the heated chaperone by stabilizing the dimeric forms, thereby preventing the protein from aggregation. Besides, we checked the tertiary structural changes of HO-artemin using SDS-PAGE analysis of the cross-linked protein (Fig 7A and 7B). In agreement with the results obtained for O-artemin (Fig 5) and H-artemin (Fig 2A), the dimerization of HO-artemin was enhanced upon increasing the oxidant concentrations from 0–100 mM H₂O₂ at both temperature treatments, and the degree of dimerization was higher at 50°C in comparison with 70°C (Fig 7).

The reversibility of the structural changes of artemin was investigated using fluorescence measurements (Fig 8). Both intrinsic (Fig 8A) and extrinsic (Fig 8B) fluorescence analyses confirmed the irreversible structural alteration of artemin under heat and oxidative conditions.

Heat/oxidative-dependent chaperone activity

To assess the potency of heated/oxidized artemin on the aggregation accompanying refolding of lysozyme, the refolding experiments were performed in the presence of 0.5 and 1 μg/mL artemin. Our results indicated that H-artemin incubated at 25 and 50°C extensively promoted the refolding of the enzyme (Fig 9A and 9B), however, at elevated temperatures (60–80°C), it accelerated the enzyme aggregation (Fig 9A). In addition, oxidation with lower concentrations of H₂O₂ (25, 50 mM) made the chaperone more active and subsequently promoted the refolding of the enzyme, but, the higher degree of artemin oxidation of (in the presence of 100 mM H₂O₂) did not change the lysozyme refolding yield (Fig 9C and 9D). Our suggestion is that the higher concentrations of the oxidant influenced the structure of artemin and a weak chaperone activity was observed as a result of oxidative modification of some amino acids of the protein, and alteration of the protein surface hydrophobicity [41], leading to self-association/agglomeration of the chaperone. Fluorescence (Fig 4) and protein cross-link (Fig 5) analysis confirmed this suggestion.

Finally, the chaperone activity of HO-artemin was investigated. The results revealed that the ability of HO-artemin in preventing the enzyme aggregation was significantly improved (Fig 10). Analysis of data showed that the oxidant did not strongly influence the heated chaperone (Fig 10A and 10B). A similar anti-aggregatory trend was found for H-artemin and HO-artemin, especially at 25 and 50 mM H₂O₂ (Figs 9B and 10A–10C). In contrast, HO-artemin exhibited a higher efficacy in suppressing the enzyme aggregation (Fig 10D–10F) when compared to O-artemin (50 and 100 mM H₂O₂) (Fig 9D). Our observations revealed that although the presence of both heat and oxidant stressors is required to achieve the most active form of the chaperone, heat has a stronger impact on the structure and function of artemin than the oxidizing agent.

Here, we reported that the mechanism of activation of artemin mainly relies on protein dimerization, which is strongly modulated through heat-induced conformational changes of the protein. Besides, such dimer and/or oligomer structures can be stabilized through the formation of inter-subunit disulfide bridge(s) [42] in the dimer form and switch a less active to more active form of the protein. Subsequently, stable dimerization of artemin monomers leads to the accumulation of highly active artemin species. It has been indicated that disulfide bonds improve the thermal stability of proteins with an activity in the oxidizing extracellular environment [38]. One explanation is that disulfide bonds reduce the conformational freedom and entropy of the protein in its unfolded state, and consequently destabilize this state with respect to the folded state [43]. Similar mechanisms have been proposed for other chaperones, Hsp33 and Hsp27, which control their dimerization by forming an intermolecular disulfide bond [27, 44]. Our observations suggested that there may be different mechanisms for artemin to protect client proteins in different stresses, which are basically mediated through protein’s dimerization (Scheme 1). The proposed mechanisms presumably play a vital role in conserving the Artemia cysts’s tolerance against severe environmental stresses.

Scheme 1. Model of chaperone function of artemin

Under physiological condition, artemin exists mainly in rosette-like oligomeric forms. Upon heat shock, the exposed hydrophobic patches of dimeric structures of the chaperone probably play a vital role in mediating the protein-chaperone interactions. At elevated temperatures, the highly exposed hydrophobic surfaces of the chaperone lead to its self-assembly as aggregates. The oxidized artemin also forms stable dimers through formation of disulfide bridges between the chaperone monomers and the protein oligomerization/agglomeration occurs as a consequent of the exposure of a high degree of hydrophobic surfaces and the formation of inter-protein disulfide bridges. Under both stress conditions, the most active stable form of the chaperone is achieved through formation of the stable dimers with an appropriate exposed hydrophobic sites. Through these mechanisms, artemin oligomers dissociate into dimers and monomers upon heat and/or oxidative stresses, which are able to bind non-native proteins, thus preventing their aggregation.

Our finding confirmed that artemin appears to exist in different conformational forms including monomer, dimer and oligomer as a function of heat and oxidant. Both hydrophobicity and dimerization are important to achieve the chaperone in a fully active form (S1 Graphical abstract). At elevated temperatures and higher degree of oxidation, both hydrophobicity and dimerization increased. In contrast, under both stress conditions, hydrophobicity did not change at the higher oxidant concentrations, while the dimerization was enhanced. Such dimerization strongly relied on disulfide bond formations between artemin monomers. The protein cross-link experiments indicated the oligomerization of artemin as a consequence of self-association at elevated temperatures. Our suggestion is that upon increasing temperature up to 60°C, artemin exposes the maximum hydrophobic sites in order to stably bind the target folding intermediates. Therefore, the presence of such binding substrates may stabilize artemin conformation at higher temperatures and protect it from self-association/precipitation and this may result in the reversible structural changes of artemin upon exposing to stress conditions. Enhanced peptide-substrate binding upon heat treatment was previously reported for other molecular chaperones Hsp26 and gp96 [25, 45]. In the case of gp96, it was recognized that the heat-induced oligomers retain peptide binding ability and it was suggested that these soluble aggregates could serve as a reservoir, and be converted into activated chaperone molecules under certain circumstances [46]. Our aggregation accompanying refolding experiments also showed the chaperone potency was considerably influenced by the temperature rather than the oxidant. Also, the function of artemin was improved at the lower oxidant concentrations probably due to proper dimerization of the chaperone through disulfide bond formation as it was detected by cross-link analysis. Despite the weak chaperone potency of oxidized artemin with high concentrations of H₂O₂, simultaneous incubation of artemin at elevated temperatures with higher oxidant concentrations significantly triggered the activation of chaperone. As the fluorescence studies showed, the presence of the oxidant resulted in a lower ANS binding through sequestering the exposed hydrophobic patches on the heated artemin probably to inhibit its aggregation. It is suggested that oxidation presumably acts by stabilizing the dimer structures of artemin through formation of disulfide bridges between the protein monomers and strengthens its stability and chaperoning potency.

Urmia Lake is one of the most hypersaline lakes and the largest natural Artemia habitats in the world located in the northwestern region of Iran [47–49]. Lake water salinity used to fluctuate between 140 and 220 g/L before 1999, but the salinity of lake increased to 340 g/L during recent years [2] possibly due to drought and increased in agricultural water consumption [50]. The lake itself is also exposed to harsh and continental climate, with winter temperature reaching -20°C and summer temperature of up to 40°C [50]. One of the other adaptations, is the ability of Artemia cysts to tolerate anoxia for periods of years, while fully hydrated and at physiological temperatures [51]. Accordingly, salinity, heat and anoxia are the serious challenges that Artemia cysts face during their lifetime. Large quantities of artemin, which accumulate during encystation of Artemia embryos, and not degraded in cysts during such extreme stresses can partly justify the tolerance of Artemia cysts [52]. High salinity can induce the formation of reactive oxygen species (ROS) within cells, and its over accumulation results in oxidative damage of membrane lipids, proteins and nucleic acids [53]. Artemin is a redox regulated chaperone and it probably acts as a reducing reservoir in the cytoplasm of the brine shrimp Artemia to protect cells from oxidative damage. In addition, activation of artemin under stress conditions can protect Artemia embryos against heat, salinity and oxidative stress conditions.

Conclusion

Our data suggest that the heat-induced dimerization of artemin is the most critical factor for its activation. It seems that in vivo cytosolic artemin may exist in a monomer–oligomer equilibrium in the cytoplasm of the cysts, which play a critical role in maintaining the cysts under such extreme conditions. Environmental stresses and/or intracellular portion of proteins may shift the equilibrium towards the active dimer forms. Moreover, the stabilization of artemin by disulfide bonds, has the potential to increase stress resistance, by protecting both RNA and protein substrates, in oviparously developing Artemia embryos. Further studies can be also performed in future for illustrating the substrate-chaperone interactions in stress conditions.

Supporting information

S1 Fig. Amino acid sequence of artemin from A. urmiana (GenBank accession no: EU380315.1).

(DOCX)

Click here for additional data file.^{(12.8KB, docx)}

S1 File

(PDF)

Click here for additional data file.^{(1.6MB, pdf)}

S1 Graphical abstract

(TIF)

Click here for additional data file.^{(157.8KB, tif)}

S1 Raw images

(PDF)

Click here for additional data file.^{(590.5KB, pdf)}

S1 Scheme

(TIF)

Click here for additional data file.^{(205.9KB, tif)}

Acknowledgments

The authors appreciate Tarbiat Modares University for the instrumental and technical supporting the work.

Data Availability

We confirm that the data provided in our Supporting Information as the "minimal data set", reach the conclusions drawn in the manuscript with related metadata and methods, and any additional data required to replicate the reported study findings. In addition, we also provided the original uncropped and unadjusted gel images as “S1_raw_images” file in the revision. We would like to provide contact information of three involved authours for ensuring data access. The email contacts include: Zeinab Takalloo (First Author): z.takalu@yahoo.com S. Shirin Shahangian (Professor Assistant & Project Advisor): shahangian@guilan.ac.ir Reza H. Sajedi (Professor of Biochemistry & Project Supervisor): sajedi_r@modares.ac.ir.

Funding Statement

MBGG (Mah Behin Gene Gostaran Company) provided some instruments for authors, and contributed in analysis of spectroscopic data.

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PLoS One. doi: 10.1371/journal.pone.0242206.r001

Decision Letter 0

Reza Yousefi

18 Jun 2020

PONE-D-20-06479

Stress-Dependent Conformational Changes of Artemin: Eﬀects of Heat and Oxidant

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Reviewer #1: Sajed et al. present results on the effect of heat and H2O2 on artemin, a protein from Artemia involved in survival of neurons. Despite the large number of experimental tools, there is a lack of experimental and interpretative rigor in the techniques used. For these reasons, some of which are detailed below, the conclusions reported in the text are not supported by the experimental results. In addition to these observations, the novelty of the work is not clear. What is really the applied outcome of the work? The current work has added additional information to artemin knowledge or not.

In conclusion, it is not possible to recommend acceptance of the manuscript.

Majors:

-What is the quality/state of the produced protein? Is the protein folded (include circular dichroism spectra)? Is the protein aggregated (include gel filtration information)? Is there a SDS-PAGE of the preparation that can be evaluated? The second (Fig 2A) and seventh (Fig. 2B) lanes are likely to refer to the produced protein. It seems to show the presence of monomer, dimer and a very large aggregate. Is it possible to separate those species by gel filtration chromatography? In this case, are the species states reversible? Why are the bands at very high concentration SDS-resistant?

-Have the authors considered aggregation/precipitation as a function of temperature-induced unfolding-refolding, and also after H2O2 treatment? See for instance: 1)Fig2A: samples at 70 and 80C have much less protein than the other lanes (authors need to use a software tool such as ImageJ to evaluate protein quantity in each lane). 2)Fig.2B: same ´protein missing problem´; samples DTT+GA- and DTT-GA+ have much less proteins than the others. 3)Figs 5 and 7 have the same problem. Same ´protein missing problem´ in many lanes. 4)Fig.3. Distributions ate 70 and 80C are so thin that it may indicate protein precipitated that was not recovered. This is a very important issue because it may explain the decrease in fluorescence in Figs1A and 4A (protein precipitated that was not recovered).

-Fluorescence. 1)An amino acid sequence should be added to help quantify the amount of Tyr and Trp. Authors state that Fig1A refers to ´Trp fluorescence emission´ but Tyr residues, if present, also contribute to emitted fluorescence. 2)The intensity of Trp fluorescence is temperature-dependent. Any study of heat-induced unfolding followed by Trp fluorescence has to present a curve control using N-acetyl-tryptophanamide (NATA) at the same buffer (there are plenty of book chapter and reviews about the topic). The authors need to comment on that. 3)Figs. 4A and 6A: Trp fluorescence is quenched by hydrogen peroxide. Is there a control to the experiments? 5)What is the rational for the bis-ANS concentration used? Is there a bis-ANS titration experiment to be evaluated?

-SDS-PAGE figures are of low quality and have little information about protein quantity. See that even the loaded marker has a band of very high molecular mass (Fig. 7 is the worse). Additionally, authors need to use a software tool such as ImageJ to evaluate protein quantity in each lane.

-Chaperone activity. Data seem to have some potential but it measures no refolding, maybe it probes protection against aggregation. To probe refolding, authors need to show that lysozyme regain its folded state (CD, NMR, enzymology, other). But even protection against aggregation seem to be minor, 20% or less.

Minors:

-Protein concentration should be given in micromolar to facilitate understanding of the amount of additives used.

-Material and Methods description lack some information, as for instance the temperature of some experiments.

Reviewer #2: This study examined the effects of temperature and an oxidizing agent (hydrogen peroxide) on the structure and chaperoning capacity of the brine shrimp protein artemin. This molecular chaperone has received a great deal of study in the past, and the present work is thus built on a large foundation of information. The principal contribution of these new data is the demonstration of how changes in oligomerization of artemin are driven by temperature and oxidative state, and how these structural changes lead to influences on chaperone potential. Activation of artemin relies chiefly on temperature-induced alterations in conformation that favor dimerization of the protein. Disulfide bridges between artemin monomers are shown to play important roles in the dimerization process. The work involves a large and diverse set of experiments, but the conclusions are nicely summarized in the two “schemes” the authors provide.

The experimental work appears to have been done carefully and the interpretations of the findings are well-supported by the results. The writing is generally clear, but there are dozens of errors in syntax and grammar that need to be corrected.

My main criticism of the paper is that the authors make no effort to link these in vitro findings to the biology of the organism itself. For example, they provide no information on the temperatures that the cysts of the shrimp experience under natural field conditions. Thus, it is not possible to determine whether the experimental conditions used with the isolated protein resemble the thermal conditions found in nature. I am concerned that some of the effects on the protein that were manifested under extremes of stress, notably some of the highest experimental temperatures, would never occur in the actual organism. In summary, whereas the in vitro biochemistry here is of high quality and yields interesting results, there is no way of determining whether these results, notably the stress-driven changes in dimerization state, actually occur in nature. The authors should try to link their in vitro findings to in vivo phenomena.

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PLoS One. 2020 Nov 16;15(11):e0242206. doi: 10.1371/journal.pone.0242206.r002

Author response to Decision Letter 0

16 Sep 2020

Dear Editors-in-Chief,

We are grateful for your consideration of this manuscript (Manuscript ID: PONE-D-20-06479), and we also very much appreciate your suggestions, which have been very helpful in improving the manuscript. We also thank the reviewers for their careful reading of our text. All the comments we received on this study have been taken into account in improving the quality of the article, and we present our reply to each of them separately. All corrections and modifications are highlighted in red in the revised version of the manuscript.

We hope that these changes to the manuscript will facilitate the decision to publish this study in your journal. We have made a considerable effort to take into account the interesting suggestions proposed by the reviewers. In any case, we are open to consideration of any further comment on our answers.

Sincerely,

Reza H Sajedi, PhD

Professor of Biochemistry

Department of Biochemistry,

Faculty of Biological Sciences,

Tarbiat Modares University, Tehran, Iran

e.mail: sajedi_r@modares.ac.ir

Comments from reviewers:

Reviewer 1:

Sajedi et al. present results on the effect of heat and H2O2 on artemin, a protein from Artemia involved in survival of neurons. Despite the large number of experimental tools, there is a lack of experimental and interpretative rigor in the techniques used. For these reasons, some of which are detailed below, the conclusions reported in the text are not supported by the experimental results. In addition to these observations, the novelty of the work is not clear. What is really the applied outcome of the work? The current work has added additional information to artemin knowledge or not. In conclusion, it is not possible to recommend acceptance of the manuscript.

ANSWER: Thank you very much for sharing us your voluble comments and concerns. As the first point, it is needed to mention that artemin is a thermostable protein in Artemia embryos (the name of the protein is similar to a member of the glial cell-line derived neurotrophic factor family, expressing in numerous human mammary carcinoma cell lines). Artemin acts as a highly efficient molecular chaperone in the encysted Artemia embryos (brine shrimps) against extreme environmental stress conditions. We previously worked on the chaperoning potency of this protein and confirmed that artemin can act as an efficient molecular chaperone in vitro and in vivo (References 5, 8, 11-13, 15 in the manuscript). Moreover, we used recombinant artemin for different purposes. For example, enhancing the soluble production of some aggregation-prone proteins in bacterial cells such as aequorin (Khosrowabadi et al., 2018), luciferase (Takalloo et al., 2016), endostatin (non-published data), and also increasing the thermostability of an industrial enzyme (xylanase) (non-published yet). Moreover, the anti-amyloidogenic effect of artemin on α-synuclein protein (Marvastizadeh et al., 2020) and blocking fibrillization of tau protein by artemin (Khosravi et al., 2017) were further confirmed. More recently, artemin gene was transferred into plant cells and expressed, in order to produce the stress-tolerant transgenic plants and the first generation of the plants showed a higher resistance against desiccation and high temperatures (data not published yet).

Despite such successes, there were no enough details on the activation mechanisms or structural modifications of this chaperone. In addition, the structural changes of artemin under oxidative stress has not been investigated to date. Accordingly, in the present study, we tried to provide some basis on the induced conformational changes of artemin upon exposing to stress conditions and the mechanisms involved in interactions with the protein substrates. This is the first report in providing details on the conformational changes of artemin’s subunits (in particular during oxidative stress). We believe that these information would help us not only to justify the protective function of artemin in Artemia cysts or other transgenic organisms, but also to effectively select more target protein substrates for further in vitro and in vivo studies.

According to the respectful reviewer’s comments, we have expanded details in the introduction and discussion to more clarify the aim and novelty of the present work for the reviewer as the other readers (Page 4; Lines 6-11, 14-16, 20-22, Page 18; Lines 13-16). In addition, we added some related explanations (as also asked by the respectful reviewer 2) in the conclusion (Page 28; Lines 19-22, Page 29; Lines 1-18, 22, 23, Page 30; Line 1).

Major comments:

1. What is the quality/state of the produced protein? Is the protein folded (include circular dichroism spectra)? Is the protein aggregated (include gel filtration information)? Is there a SDS-PAGE of the preparation that can be evaluated? The second (Fig 2A) and seventh (Fig. 2B) lanes are likely to refer to the produced protein. It seems to show the presence of monomer, dimer and a very large aggregate. Is it possible to separate those species by gel filtration chromatography? In this case, are the species states reversible? Why are the bands at very high concentration SDS-resistant?

ANSWER: Regarding the state of the protein, artemin (at 25 ºC) has a normal three dimensional (3D) structure, as also confirmed by fluorescence spectroscopy (Fig. 1A, Fig. 4A). We also checked the secondary structure of purified artemin by CD spectroscopy (shown in below). Secondary structure analysis by K2D2 online software predicted an α-helical content of approximately 47.89%, followed by random coil (41.91%), and β-sheet (10.28%), as also showed in our previous works.

Moreover, regarding the form of artemin in normal conditions, please notice that, like other small heat shock proteins (such as Hsp26, Hsp20.1 and Hsp14.1), artemin probably exists in a monomer–oligomer equilibrium in the cells (Scheme 1, Page 29; Lines 19-20). Overall, dynamic oligomerization and the ability to switch between oligomeric states appear to be crucial for the function of a variety of known chaperones, but in very different ways. Previous SEM analysis also revealed the Rosset like structure of artemin composing hexameric structure of artemin in the Artemia (franciscana) cysts (Graaf et al., 1990; Chen et al., 2007). It is suggested that environmental stresses and/or intracellular portion of proteins may shift the equilibrium towards the active dimer forms through forming disulfide bridges between the monomers. Our cross-link analysis by SDS-PAGE revealed that the ratio of dimeric states of the chaperone was changed upon exposing to stress conditions (Fig. 2 A, Fig. 5, Fig. 7).

Analysis of purified artemin by SDS-PAGE (in below) under reducing and non-reducing conditions was performed, which revealed monomeric forms (under reducing), and dimeric/other oligomeric structures (under non-reducing conditions).

Purified artemin fractions in non-reduced (1) and reduced (2-11) SDS-PAGE.

In addition, performing gel filtration needs a high concentration of purified protein. Gel filtration can dilute the protein concentration 10 times. Thus, if our protein concentration is not high enough, the monitor can not detect all oligomeric forms of the protein after gel filtration. Accordingly, we preferred to use cross-link analysis followed by SDS-PAGE. In comparison, using SDS-PAGE followed by staining with nitrate silver enabled us to use lower concentrations of proteins and also detect various forms of protein even at very low concentrations (~5 ng).

Regarding the reversibility of the protein states, our suggestion is that under normal (or non-stress) conditions, some transitions may occur between different protein states. However, in stress conditions, we checked the reversibility of artemin modifications after exposing to stress conditions (Fig. 8), and the results of fluorescence spectroscopy indicated that the structural changes were irreversible.

About the final question, it is needed to mention that the protein samples were treated with 0.5% glutaraldehyde as a fixative agent and then loaded onto the 10% SDS-PAGE. The fixed protein subunits by glutaraldehyde prevented the subunits dissociation upon exposing to SDS or other agents during SDS-PAGE analysis.

2. Have the authors considered aggregation/precipitation as a function of temperature-induced unfolding-refolding, and also after H2O2 treatment? See for instance: 1)Fig2A: samples at 70 and 80C have much less protein than the other lanes (authors need to use a software tool such as ImageJ to evaluate protein quantity in each lane). 2)Fig.2B: same ´protein missing problem´; samples DTT+GA- and DTT-GA+ have much less proteins than the others. 3)Figs 5 and 7 have the same problem. Same ´protein missing problem´ in many lanes. 4)Fig.3. Distributions at 70 and 80C are so thin that it may indicate protein precipitated that was not recovered. This is a very important issue because it may explain the decrease in fluorescence in Figs1A and 4A (protein precipitated that was not recovered).

ANSWER: We completely agree with the respectful reviewer’s comment. We also believe that exposing the chaperone to elevated temperatures (≥ 70 ºC), as well as higher concentrations of H2O2 (≥ 80 mM) probably lead to forming higher molecular weights of protein assembles and probably protein aggregates as we also mentioned in our manuscript before (Page 15; lines 11-13, Page 15; lines 17-20, Page 18; lines 5-7; Page 19; lines 14-18 in previous version of the manuscript). As the dear reviewer also mentioned, these aggregates or precipitates could also result in decrease in the fluorescence intensity as was observed for heated artemin at elevated temperatures or oxidized protein with higher concentrations of the oxidant (Fig. 1A, Fig. 3A). We referred these suggestions in the previous version of the manuscript, but we also proposed that the presence of protein substrates may stabilize artemin conformations at higher temperatures or oxidant contents and protect it from self-association/precipitation and this may even induce the reversibility of structural changes of artemin upon exposing to stress conditions, however in the present study we did not test this hypothesis (Page 16; lines 8-10, Page 21; lines 7-11 in previous version of the manuscript).

Besides, we analyzed the percent of dimeric forms of the treated proteins (as the active state of the chaperone) using ImageJ software and added related data in the manuscript (Page 7; Lines 16-18, Page 10; Line 8-9, 17-18, Page 11; Lines 2-3, Page 13; Lines 1, 6-7, Page 14; Lines 10-11, 14-15, Page 21; Lines 1-2, Page 23; Line 22, Figures 2B, D, 5B, 7B).

3. Fluorescence. 1) An amino acid sequence should be added to help quantify the amount of Tyr and Trp. Authors state that Fig1A refers to ´Trp fluorescence emission´ but Tyr residues, if present, also contribute to emitted fluorescence. 2)The intensity of Trp fluorescence is temperature-dependent. Any study of heat-induced unfolding followed by Trp fluorescence has to present a curve control using N-acetyl-tryptophanamide (NATA) at the same buffer (there are plenty of book chapter and reviews about the topic). The authors need to comment on that. 3)Figs. 4A and 6A: Trp fluorescence is quenched by hydrogen peroxide. Is there a control to the experiments? 5)What is the rational for the bis-ANS concentration used? Is there a bis-ANS titration experiment to be evaluated?

ANSWER: 1) According to the respectful reviewer’s comment, the amino acid sequences of the protein was added to the manuscript (Page 19; Line 17, Page 31; F1 S1).

Regarding the second comment, although tyrosine (Tyr) has a quantum yield similar to Trp, the indole group of Trp is considered the dominant source of UV absorbance at ∼280 nm and emission at ∼350 nm in proteins (Teale et al., 1957). Furthermore, in native proteins, Tyr emission is often quenched, presumably by its interaction with the peptide chain or via energy transfer to Trp (Lakowicz et al., 2006). In comparison, the spectroscopic properties of Trp are complex, in particular the high sensitivity to the (local) environment and its (at least) two different fluorescence lifetimes (∼0.5 and ∼3.1 ns) (Amar et al., 2014). Accordingly, the fluorescence of the protein upon excitation at 280 nm generally referred as the Trp fluorescence. But as the reviewers suggested, we corrected the “Trp fluorescence” phrases to avoid any misunderstanding (Page 9; Line 18, Page 12; Line 13).

2) About using a curve control using NATA; the quantum yield of fluorescence of the Trp residues generally diminished as the temperature increased, also with no change in the shape of their emission spectra (Gally and Edelman, 1962). Besides, NATA is often used as a model for the Trp residues in proteins and peptides. It is used to quantify the number of Trp residues changed in proteins. However, here we tried to compare the conformational changes between different treated proteins, and not to quantify the number of exposed Trp residues. There are also many reliable studies, in which the comparison has been performed in the similar manner as we used (Wang et al., 2002; Gosslau et al., 2001; Waseem Akhtar et al., 2004; Choi et al., 2013; Shearstone and Baneyx, 1999; Dahl eta al., 2015; Jung et al., 2015; Fan et al., 2006 and so on).

3, 4) Yes, we are agreed with the respectful reviewer that the quenching of Trp and Tyr fluorescence upon exposing to the oxidant may also occur as a result of oxidation of the aromatic residues. However, in this regard, the accessibility of the fluorophore to the solvent and thus to the quencher depends on its position within the protein. Trp is quite sensitive to its local environment. Then, buried Trp residues should have a lower accessibility to the solvent that those present at the surface (J. R. Albani, Elsevier Science, 2011). Accordingly, the residues on the surface of the protein will be influenced to a greater degree by the oxidant, while the buried Trp residues are not easily accessible to the solvent. There are also many reports in which the fluorescence intensity of a protein has been measured in the similar manner as we used (Wang et al., 2002; Khoshaman et al., 2015; Gosslau et al., 2001, and so on). As control, we added H2O2 at different concentrations to the protein solutions and recorded the fluorescence intensity after 2 minutes, and the differences between the control (0 mM H2O2) and the samples with higher oxidant contents were insignificant. Accordingly, as another probable reason, the decrease in the fluorescence intensity of the oxidized protein may be as a result of the formed disulﬁde bonds in the oxidized protein. The prone contacts between cystines and the aromatic rings lead to the ﬂuorescence decay. We also added the earlier possibility into the manuscript for more clarifications (Page 23; Lines 8-10).

5) ANS generally is used in a broad range of 10-100 µM, but we chose the optimum concentration (30 µM) based on a dozen of studies reported previously (Das et al., 1995; Wang et al., 2002; Gosslau et al., 2001; Jung et al., 2015).

4. SDS-PAGE figures are of low quality and have little information about protein quantity. See that even the loaded marker has a band of very high molecular mass (Fig. 7 is the worse). Additionally, authors need to use a software tool such as ImageJ to evaluate protein quantity in each lane.

ANSWER: The figures were provided in a very high quality version (in TIFF format with dpi: 300). The quality probably decreased during converting TIFF to PDF by the journal. We have provided some of the figure (in DOX version) for more clarifications (Please check the below figures for example).

Figure 2

Figure 7

Moreover, as the dear reviewer suggested, we evaluated the quantity of dimeric forms (as the active state of the chaperone) in each lane by ImageJ software (Page 7; Lines 16-18, Page 10; Line 8-9, 17-18, Page 11; Lines 2-3, Page 13; Lines 1, 6-7, Page 14; Lines 10-11, 14-15, Page 21; Lines 1-2, Page 23; Line 22, Figures 2B, D, 5B, 7B).

5. Chaperone activity. Data seem to have some potential but it measures no refolding, maybe it probes protection against aggregation. To probe refolding, authors need to show that lysozyme regain its folded state (CD, NMR, enzymology, other). But even protection against aggregation seem to be minor, 20% or less.

ANSWER: Thank you for this point. Please notice that it is not possible to determine the CD or NMR spectra of a protein when we want to examine the interactions between two or more proteins. In the case of molecular chaperones, refolding experiments are among the most popular and efficient methods used for evaluating the potential of molecular chaperones as also used for many other chaperones (Wang et al., 2010; Dong et al., 2002; Choi et al., 2013; Rozema et al., 1996; Wang et al., 2010; Dong et al., 2004; Dong et al., 2001; Gull et al., 2017; Takalloo et al., 2019; Antonio-Pérez et al., 2012; Niknaddaf, 2018). But regarding the refolding yield, depending on type of the target protein substrate and the protein substrate: chaperone ratio, results may be different. Finally, please remind that the main purpose of performing refolding experiments was not increasing the refolded yield of lysozyme, but simply compare the chaperonin potency of different treated artemin (heated, oxidized, and heated oxidized artemin).

Minor comments:

1. Protein concentration should be given in micromolar to facilitate understanding of the amount of additives used.

ANSWER: As the respected reviewer suggested, we replaced all protein concentrations in mg/mL with µM (Page 6; Line 19, Page 7; Lines 3, 7, 10, 13, 20, Page 8; Lines 4, 19, Page 9; Line 19, Page 10; Lines 2, 18, 22, Page 12; Lines 12, 16, Page 13; Line 7, Page 14; Lines 2, 7, 15, Page 15; Lines 9, 13, Page 16; Line 13).

2. Material and Methods description lack some information, as for instance the temperature of some experiments.

ANSWER: We added more details regarding the experimental preparation to the manuscript (Page 6; Lines 1-10, 14, 15, Page 7; Lines 1, 15).

Apart from the changes (requested by respected reviewers), some other alterations have been made and highlighted in red in the revised manuscript, e.g.: Page 2; Lines 2, 6, 10, 12, 18, Page 3; Lines 8, 9, 11, 18, 21, 23, Page 4; Lines 4, 5, 12, 13, 17, Page 7; Line 4, 6, 11, 12, 19, 21, Page 8; Lines 2, 6, 11, 17, 20, Page 9; Lines 1, 2, 20, Page 10; Lines 3, 10, 19, Page 11; Lines 7, 8, 18, Page 12; Line 6, Page 14; Lines 4, 9, 17, Page 15; Lines 3, 21, Page 16; Lines 4, 5, 6, Page 17; Lines 1, 2, 10, 12, Page 18; Lines 3, 5, 7, 8, 9, 12, Page 19; Lines 2, 3, 5, 6, 7, 8, 9, 1, 11, 15, 16, 17, 18, 19, Page 20; Lines 3, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, Page 21; Lines 4, 6, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 22, 23, Page 22; Lines 5, 8, 10, 12, 13, 14, 15, 16, 20, 22, 23, Page 23; Lines 1, 2, 3, 4, 5, 6, 14, 15, 17, 18, 20, 21, 22, 23, Page 24; Lines 6, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, Page 25; Lines 2, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 22, Page 26; Line 2, 4, 8, Page 28; Line 6, Page 29; Line 19.

Due to the alterations (requested by respected reviewers) 15 new references (Ref 1, 2, 16, 17, 18, 20, 21, 41, 47-53) were added to the manuscript.

Again, we appreciate all of your insightful comments. We worked hard to be responsive to them. Thank you for taking the time and energy to help us improve the paper.

-Reviewer 2

This study examined the effects of temperature and an oxidizing agent (hydrogen peroxide) on the structure and chaperoning capacity of the brine shrimp protein artemin. This molecular chaperone has received a great deal of study in the past, and the present work is thus built on a large foundation of information. The principal contribution of these new data is the demonstration of how changes in oligomerization of artemin are driven by temperature and oxidative state, and how these structural changes lead to influences on chaperone potential. Activation of artemin relies chiefly on temperature-induced alterations in conformation that favor dimerization of the protein. Disulfide bridges between artemin monomers are shown to play important roles in the dimerization process. The work involves a large and diverse set of experiments, but the conclusions are nicely summarized in the two “schemes” the authors provide.

ANSWER: The constructive comments by the respectful reviewer are really appreciated. Accordingly, we have carefully revised the conclusion and other sections of the manuscript and added some related descriptions regarding the biology, lifetime and the living environments of the organism and tried to explore the relationship between our data and Artemia biology (Page 3; Lines 2-6, Page 28; Lines 19-22, Page 29; Lines 1-18, 22-23, Page 30; Line 1).

For more clarification, it is needed to mention that our main purpose was not solely to link the chaperoning function of artemin to its conformational changes during stress condition in Artemia cysts as its origin. To date, we have used recombinant artemin for different purposes. For example, enhancing the soluble production of some aggregation-prone proteins in bacterial cells such as aequorin (Khosrowabadi et al., 2018), luceiferase (Takalloo et al., 2016), endostatin (non-published data), and also increasing the thermostability of an industrial enzyme (xylanase) (non-published yet). Moreover, the anti-amyloidogenic effect of artemin on α-synuclein protein (Marvastizadeh et al., 2020) and blocking fibrillization of tau protein by artemin (Khosravi et al., 2017) were further confirmed. More recently, artemin gene was transferred into plant cells and expressed, in order to produce the stress-tolerant transgenic plants and the first generation of the plants showed a higher resistance against desiccation and high temperatures (data not published yet).

Accordingly, to avoid any misunderstanding, we have expanded details in the introduction and discussion to more clarify the aim and novelty of the present work for the reviewer as the other readers (Page 4; Lines 6-11, 14-16, 20-22, Page 18; Lines 13-16).

Besides, we also re-read the whole manuscript and corrected the grammatical errors and typing mistakes as much as possibl, e.g.: Page 2; Lines 2, 6, 10, 12, 18, Page 3; Lines 8, 9, 11, 18, 21, 23, Page 4; Lines 4, 5, 12, 13, 17, Page 7; Line 4, 6, 11, 12, 19, 21, Page 8; Lines 2, 6, 11, 17, 20, Page 9; Lines 1, 2, 20, Page 10; Lines 3, 10, 19, Page 11; Lines 7, 8, 18, Page 12; Line 6, Page 14; Lines 4, 9, 17, Page 15; Lines 3, 21, Page 16; Lines 4, 5, 6, Page 17; Lines 1, 2, 10, 12, Page 18; Lines 3, 5, 7, 8, 9, 12, Page 19; Lines 2, 3, 5, 6, 7, 8, 9, 1, 11, 15, 16, 17, 18, 19, Page 20; Lines 3, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, Page 21; Lines 4, 6, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 22, 23, Page 22; Lines 5, 8, 10, 12, 13, 14, 15, 16, 20, 22, 23, Page 23; Lines 1, 2, 3, 4, 5, 6, 14, 15, 17, 18, 20, 21, 22, 23, Page 24; Lines 6, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, Page 25; Lines 2, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 22, Page 26; Line 2, 4, 8, Page 28; Line 6, Page 29; Line 19.

Due to the alterations (requested by respected reviewers) 15 new references (Ref 1, 2, 16, 17, 18, 20, 21, 41, 47-53) were added to the manuscript.

Again, we appreciate you taking the time to offer us your comments and insights related to the paper.

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PLoS One. doi: 10.1371/journal.pone.0242206.r003

Decision Letter 1

Reza Yousefi

19 Oct 2020

PONE-D-20-06479R1

Stress-dependent conformational changes of artemin: eﬀects of heat and oxidant

PLOS ONE

Dear Dr. Sajedi,

==============================

Your article will be accepted if the following changes are applied correctly.

1. The numbers identifying the affiliations of authors are not used correctly.

2. The language of this manuscript still needs significant improvement. Below please find some examples of typo grammatical errors which must be fixed.

Abstract section, lines 3-6, the sentence needs amending.

Pg.4, line 2: moderates should be replaced with modulates.

Pg.6, line 5: Microtubule must be corrected as micro tube.

3. Length of the final conclusion needs to be greatly shortened and additional and repetitive explanations must to be avoided.

4. While drawing the GA and Scheme 1, if more than one color is used, it will probably be easier for readers to understand the messages.

.==============================

Please submit your revised manuscript by Dec 03 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Reza Yousefi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Your article will be accepted if the following changes are applied correctly.

1) The numbers identifying the affiliations of authors are not used correctly.

2) The language of this manuscript still needs significant improvement. Below please find some examples of typo grammatical errors which must be fixed.

Abstract section, lines 3-6, the sentence needs amending.

Pg.4, line 2: moderates should be replaced with modulates.

Pg.6, line 5: Microtubule must be corrected as micro tube.

3) Length of the final conclusion needs to be greatly shortened and additional and repetitive explanations must to be avoided.

4) While drawing the GA and Scheme 1, if more than one color is used, it will probably be easier for readers to understand their messages.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

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Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

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Reviewer #3: No comment. In my opinion, all of the points raised in the first revision were addressed by the authors. Accordingly, the manuscript is acceptable for publication in PLOS ONE.

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Reviewer #3: Yes: Mohsen Asghari

PLoS One. 2020 Nov 16;15(11):e0242206. doi: 10.1371/journal.pone.0242206.r004

Author response to Decision Letter 1

25 Oct 2020

Dear Editors-in-Chief,

We are grateful for your consideration of this manuscript (Manuscript ID: PONE-D-20-06479). All the comments we received on this study have been taken into account in improving the quality of the article, and we present our reply to each of them separately. All corrections and modifications are highlighted in red in the revised version of the manuscript.

1. The numbers identifying the affiliations of authors were checked and corrected (Page 1).

2. We re-read the whole manuscript and corrected the grammatical errors and typing mistakes as much as possible, e.g.: Page 2; Lines 4, 5, 6, 7, 10, 14, Page 3; Line 11, Page 4; Lines 1, 3, 20, 21, Page 6; Lines 5, 6, 8, Page 9; Lines 9, 10, Page 10; Lines 1, 9, 16, Page 11; Lines 3, 4, 6, 21, Page 12; Line 11, Page 16; Lines 10, 16, 17, Page 17; Lines 1, 3, 14, 19, 20, Page 18; Lines 9, 10, 13, 14, Page 19; Lines , 18, 20, Page 20; Lines 3, 4, 14, Page 21; Lines 5, 15, 18, 20, 22, 23, Page 22; Lines 1, 3, 6, 9, 14, 21, Page 23; Lines 1, 9, 10, Page 24; Lines 17, 18, 22, Page 25; Line 18, Page 26; Line 9, Page 27; Lines 5, 19, 20, 23.

3. Regarding the conclusion section, as the respectful reviewer recommended us to add some related information about the physiology of Artemia cysts in the conclusion section, and accordingly the conclusion was too lengthy. As the dear editor suggested, we removed the first part of the conclusion and added that to the discussion (Page 28; Lines 3-18), and shortened conclusion as much as possible (Page 28; Lines 21-22, Page 29; Lines 1-6).

4. We corrected the scheme 1 and graphical abstract as the respectful reviewer suggested.

Again, we appreciate you taking the time to offer us your comments and insights related to the paper. We hope that these changes to the manuscript will facilitate the decision to publish this study in your journal. In any case, we are open to consideration of any further comment on our manuscript.

Sincerely,

Reza H Sajedi, PhD

Professor of Biochemistry

Department of Biochemistry,

Faculty of Biological Sciences,

Tarbiat Modares University, Tehran, Iran

E.mail: sajedi_r@modares.ac.ir

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PLoS One. doi: 10.1371/journal.pone.0242206.r005

Decision Letter 2

Reza Yousefi

29 Oct 2020

Stress-dependent conformational changes of artemin: eﬀects of heat and oxidant

PONE-D-20-06479R2

Dear Professor Sajedi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Reza Yousefi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0242206.r006

Acceptance letter

Reza Yousefi

6 Nov 2020

PONE-D-20-06479R2

Stress-dependent conformational changes of artemin: eﬀects of heat and oxidant

Dear Dr. Sajedi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Amino acid sequence of artemin from A. urmiana (GenBank accession no: EU380315.1).

(DOCX)

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S1 File

(PDF)

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S1 Graphical abstract

(TIF)

Click here for additional data file.^{(157.8KB, tif)}

S1 Raw images

(PDF)

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S1 Scheme

(TIF)

Click here for additional data file.^{(205.9KB, tif)}

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Data Availability Statement

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PERMALINK

Stress-dependent conformational changes of artemin: Effects of heat and oxidant

Zeinab Takalloo

Zahra Afshar Ardakani

Bahman Maroufi

S Shirin Shahangian

Reza H Sajedi

Roles

Abstract

Introduction

Materials and methods

Chemicals

Expression and purification of recombinant artemin

Protein treatments

Heated artemin (H-artemin)

Oxidized artemin (O-artemin)

Heated oxidized artemin (HO-artemin)

Conformational analysis

Intrinsic fluorescence measurements

ANS fluorescence measurements

Protein cross-link analysis by SDS-PAGE

DLS analysis

Chaperone-like activity assay

Preparation of denatured reduced lysozyme

Aggregation accompanying refolding

Results

Temperature-dependent structural changes in artemin

Fig 1.

Fig 2. Protein cross-link analysis of heated artemin by non-reducing SDS-PAGE.

Fig 3. DLS analysis of heat-treated artemin.

Oxidative-dependent structural changes in artemin

Fig 4.

Fig 5.

Structural changes of artemin under heat and oxidative conditions

Fig 6.

Fig 7.

Structural changes of artemin under stress conditions are irreversible

Fig 8. Structural changes of artemin under different stress conditions are irreversible.

Chaperone-like activity of artemin under heat and oxidative stress

H-artemin

Fig 9.

O-artemin

HO-artemin

Fig 10. Effects of heated-oxidized artemin on aggregation accompanying refolding of lysozyme.

Discussion

Table 1. A summarize of the structural and functional changes of artemin upon heat and H2O2 treatments.

Heat-dependent structural transitions

Oxidative-dependent structural transitions

Structural transitions of artemin under heat and oxidant treatments

Heat/oxidative-dependent chaperone activity

Scheme 1. Model of chaperone function of artemin

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Reza Yousefi

Roles

Author response to Decision Letter 0

Decision Letter 1

Reza Yousefi

Roles

Author response to Decision Letter 1

Decision Letter 2

Reza Yousefi

Roles

Acceptance letter

Reza Yousefi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Table 1. A summarize of the structural and functional changes of artemin upon heat and H₂O₂ treatments.