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

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
pET28a encoding artemin from Artemia urmiana was provided (3) and protein expression was 1 carried out in Escherichia coli BL21 (DE3) cells as previously described (9). Purification of the 2 His-tagged protein was carried out using Ni-NTA agarose column. Bound proteins were eluted 3 with a buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, and 250 mM imidazole, pH 8.0. 4 Aliquots of the eluted protein were taken, followed by dialysis against the phosphate buffer 5 overnight at 4°C. The protein concentrations were determined using Bradford's method and BSA 6 as standard (14).
control, the denatured lysozyme was refolded as described without artemin. In these Temperature-dependent structural changes in artemin 6 Intrinsic fluorescence was monitored at 330 nm with 280 nm excitation to probe changes in 7 tertiary structures of H-artemin ( Figure 1A). The results indicated that when the temperature 8 increased up to 30°C, the fluorescence intensity decreased sharply, and the lowest emission 9 intensity was recorded for the heated protein at 80°C ( Figure 1A). Besides, the wavelength of 10 maximum emission (λ max ) did not show any shift. 80°C. The larger size distribution of H-artemin can be explained with its ability to form dimeric 6 and oligomeric species as also indicated by SDS-PAGE ( Figure 2A). All results showed that the 7 conformational changes in artemin occur upon increasing temperature.  Oxidative-dependent structural changes in artemin 10 As depicted in Figure 4A    To evaluate the influence of the two stressors, i.e. heat and oxidant, on protein structure, artemin 9 was treated with 0-100 mM H 2 O 2 , followed by exposure to 50 and 70°C. Intrinsic fluorescence 10 measurements showed that under both temperature incubations, the intensity declined gradually 11 by increasing the oxidant concentrations from 0 to 100 mM ( Figure 6A). Besides, ANS 12 fluorescence indicated that the fluorescence intensity did not change considerably for HO-13 artemin incubated with 10-100 mM H 2 O 2 at elevated temperatures ( Figure 6B). This trend was 14 not similar to those observed for the individual oxidant treatments ( Figure 4B).      Denatured/reduced lysozyme was refolded by the dilution method and the kinetics of chaperone-11 assisted refolding was examined in the presence of 0.5 and 1 µg/mL artemin ( Figure 9). As 12 shown in Figure 9A, B, the heated artemin at 25 and 50°C was found efficient in suppressing 13 aggregation of the enzyme. In contrast, the 0.5 µg/mL chaperone incubated at elevated 14 temperatures, 60 to 80°C, accelerated the aggregation of lysozyme ( Figure 9A), and 1 µg/mL 15 artemin showed no effect on the refolding yield at similar conditions ( Figure 9B).

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Results showed that the oxidized chaperone (0.5 µg/mL) with 25 and 50 mM H 2 O 2 was efficient 18 in preventing the aggregation of lysozyme compared to the untreated control ( Figure 9C). In    Figure 10A, B, C) and temperatures (25, 4 50 and 70°C) ( Figure 10D, E, F). According to Figure 10A, the HO-artemin at 25 and 50°C 5 suppressed the aggregation of lysozyme, but at elevated temperature, 70°C, it could not 6 efficiently prevent the aggregation of lysozyme ( Figure 10A, B). These trends were similar to the 7 graphs obtained for H-artemin (Figure 9 A,B). In contrast, HO-artemin incubated with 100 mM 8 H 2 O 2 clearly exhibited an improved chaperone activity ( Figure 10C). 9 We also checked the effect of HO-artemin on refolding of lysozyme based on constant   Artemin is an abundant heat stable protein in Artemia encysted embryos and it was found that 19 high regulatory production of artemin under harsh environmental conditions is probably relevant 20 to stress resistance in this crustacean (2,16). Artemin demonstrated an ability in suppressing heat-induced aggregation of different protein substrates such as citrate synthase, carbonic 1 anhydrase, horseradish peroxidase and luciferase in vitro and in vivo, and also introduced as a 2 potent nucleic acid chaperone (6-9,11). The intrinsic conformational properties of artemin seem 3 to play a critical role in its biological activities. Our previous structural report documented that 4 artemin contains a high surface hydrophobicity compared to other molecular chaperones (9).     Accordingly, when such aromatic amino acids are located on the surface of the protein, the 10 fluorescence intensity of the residues is usually influenced in a higher degree by the quencher, 11 compared to the amino acids deeply embedded in the protein structure (23). Our intrinsic 12 fluorescence results showed that the fluorescence emission of the protein was significantly 13 reduced at elevated temperatures and the first transition observed at 50°C followed by the second 14 transition at 80°C ( Figure 1A). This is suggested that the induced conformational changes of 15 artemin resulted in the increased quenching probably due to the exposing internal aromatic 16 residues to solvent.

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The conformational changes of the protein was also investigated using ANS fluorescence. ANS 18 is widely used as a fluorescent hydrophobic probe for hydrophobic patches of proteins (22,24). It 19 is basically non-fluorescent in aqueous solutions but became highly fluorescent in non-polar 20 environment (22). Increases in temperature up to 60°C caused artemin to present more exposed probably due to the formation of a higher degree of oligomers. It is hypothesized that the highly 9 reactive thiol groups of cysteine residues may lead to protein oligomerization due to the excess 10 formation of disulfide bonds in O-artemin. Finally, the protein precipitation may occur as a result 11 of highly exposed hydrophobic regions and the formation of excess inter-protein disulfide 12 bridges (32) as showed by fluorescence ( Figure 4B) and SDS-PAGE analysis ( Figure 5). Totally, 13 these results indicate that upon oxidation, artemin undergoes dimerization and oligomerization. 15 In a final approach, we also examined the simultaneous effect of stressors on protein structure.  Figure 4B). Our suggestion is that the oxidant agent sequestered the exposed hydrophobic surfaces on the heated chaperone by stabilizing the 1 dimeric forms and preventing the protein from aggregation. Besides, we checked the tertiary 2 structural changes of HO-artemin using protein cross-linking analysis by SDS-PAGE (Figure 7).

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In agreement with the results obtained for O-artemin ( Figure 5) and H-artemin (Figure 2A), the 4 dimerization of HO-artemin was enhanced upon increasing the oxidant concentrations from 0-5 100 mM H 2 O 2 at both temperature treatments and the degree of dimerization was higher at 50°C 6 in comparison with 70°C (Figure 7).  indicated that the oligomerization of artemin as a consequence of self-association at elevated 12 temperatures. Our suggestion is that upon increasing temperature up to 60°C, artemin exposes 13 the maximum hydrophobic sites in order to stably bind the target folding intermediates. 14 Therefore, the presence of such binding substrates may stabilize artemin conformation at higher 15 temperatures and protect it from self-association/precipitation and this may result in the 16 reversible structural changes of artemin upon exposing to stress conditions. Enhanced peptide-17 substrate binding upon heat treatment was previously reported for other molecular chaperones 18 Hsp26 and gp96 (19,37). In the case of gp96, it was recognized that the heat-induced oligomers 19 retain peptide binding ability and it was suggested that these soluble aggregates could serve as a 20 reservoir, and be converted into activated chaperone molecules under certain circumstances (38).      exists mainly in rosette-like oligomeric forms. Upon heat shock, the exposed hydrophobic 4 packets of dimeric structures of the chaperone probably play a vital role in mediating the protein-5 chaperone interactions. At elevated temperatures, the highly exposed hydrophobic surfaces of the 6 chaperone lead to its self-assembly as aggregates. The oxidized artemin also forms stable dimers 7 through formation of disulfide bridges between the chaperone monomers and the protein 8 oligomerization/agglomeration occurs as a consequent of the exposure of a high degree of 9 hydrophobic surfaces and the formation of inter-protein disulfide bridges. Under both stress 10 conditions, the most active stable form of the chaperone is achieved through formation of the 11 stable dimers with an appropriate exposed hydrophobic sites. Through these mechanisms, 12 artemin oligomers dissociate into dimers and monomers upon heat and/or oxidative stresses, 13 which are able to bind non-native proteins, thus preventing their aggregation.
14 TABLES 1   The authors declare no competing financial interest. inter-subunit disulfide bond of artemin acts as a redox switch for its chaperone-like activity,