Without Salt, the ‘Thermophilic’ Protein Mth10b Is Just Mesophilic

Most proteins from thermophiles or hyperthermophiles are intrinsically thermostable. However, though Methanobacterium thermoautotrophicum ΔH is a thermophilic archaeon with an optimal growth temperature of 65°C, Mth10b, an atypical member the Sac10b protein family from M. thermoautotrophicum ΔH, seems not intrinsically thermostable. In this work, to clarify the molecular mechanism of Mth10b remaining stable under its physiological conditions, the thermodynamic properties of Mth10b were studied through equilibrium unfolding experiments performed at pH 7.0 monitored by circular dichroism (CD) spectra in detail. Our work demonstrated that Mth10b is not intrinsically thermostable and that due to the masking effect upon the large numbers of destabilizing electrostatic repulsions resulting from the extremely uneven distribution of charged residues over the surface of Mth10b, salt can contribute to the thermostability of Mth10b greatly. Considering that the intracellular salt concentration is high to 0.7 M, we concluded that salt is the key extrinsic factor to Mth10b remaining stable under its physiological conditions. In other word, without salt, ‘thermophilic’ protein Mth10b is just a mesophilic one.


Introduction
Organisms permeate almost everywhere on earth, including in harsh environments of extreme temperature, pH, salinity and pressure. Extremophiles are defined as organisms that flourish in habitats of extreme conditions. Among extremophiles, organisms adapted to high temperatures are called thermophiles, which grow optimally between 50 and 80uC, and hyperthermophiles, which grow optimally above 80uC.
The Sac10b protein family is generally regarded as a group of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Typical members of this family are small basic homodimeric proteins which bind to DNA without sequence specificity [28][29][30][31][32][33][34][35]. Recently, Mth10b, an atypical member of the Sac10b protein family was identified from M. thermoautotrophicum DH by our laboratory [36,37]. Though it is similar to typical Sac10b family proteins with respect to its primary, secondary, tertiary structure and in its preferred oligomeric forms, unlike typical Sac10b family proteins, Mth10b is an acidic one with potential isoelectric point of 4.56 and bind to neither DNA nor RNA in vitro [36,37]. When we try to purify recombinant Mth10b from Escherichia coli through maintaining cells lysate at high temperature, an interesting phenomenon was found: though M. thermoautotrophicum DH is a typical thermophilic archaeon that grows at temperatures in the range of 40-70uC, with an optimal temperature of 65uC [38], the recombinant Mth10b was precipitated absolutely with those unwanted proteins after the cells lysate was maintained at 60uC for 20 minutes [36]. This phenomenon suggests that Mth10b is not an intrinsically thermophlic protein and that there should be some extrinsic factors can help Mth10b remaining stable in vivo.
In order to clarify why Mth10b can maintain stable under its physiological conditions, in the present work, we studied the thermodynamic properties of Mth10b through denaturant-induced unfolding and heat-induced unfolding monitored by circular dichroism (CD) spectra in detail. Our results demonstrated that Mth10b is not intrinsically thermostable and that salt is the key factor to Mth10b remaining stable under the physiological conditions.

Materials
HEPES, GdnHCl, Urea, Tris, and isopropyl b-D-thiogalactoside (IPTG) were purchased from Sigma. The expression plasmid pET11a-mth10b containing the mth10b gene, and host strains E. coli DH5a and BL21 (DE3) were from our laboratory stocks. All chromatography apparatus and materials were purchased from GE Healthcare.

Protein preparation
The Mth10b protein was expressed in E. coli and purified to homogeneity as previously described [36]. Protein purity was higher than 95% as confirmed by 15% SDS-PAGE. The purified protein samples were dialyzed against 50 mM NH 4 HCO 3 then lyophilized and stored at 220uC.

Unfolding studies
Unfolding of Mth10b was studied by taking CD measurements, performed in basic running buffer (10 mM HEPES/pH 7.0) containing different concentrations of NaCl, with a Pistar-180 spectrometer with a Peltier temperature-controlled cell holder. The CD signal was monitored using a rectangular quartz cuvette with a path length of 1 mm.
For denaturant-induced unfolding, two most common chemical denaturants, GdnHCl and urea were employed respectively. The samples containing various concentrations of denaturant (GdnHCl or urea) were equilibrated at 25uC overnight and then measured by far-UV CD at 222 nm with an averaging time of 1 min. Reversibility of denaturant-induced unfolding was checked by diluting denatured protein in a high denaturant concentration into the buffer solution and comparing the CD spectrum with that of the native protein. CD spectra were scanned at 1 nm intervals from 200 (or 205) to 250 nm. Moreover, the urea stock solution was freshly prepared on the day of use.
For heat-induced unfolding, measurements were carried out in the presence of different concentrations of GdnHCl. Each sample was heated with a stepwise change of 2uC, and the far-UV CD signal at 222 nm was recorded, with a 2 min equilibration time, and a 1 min averaging time at each temperature point. Reversibility was checked by returning to the beginning temperature and comparing the CD spectrum with the premelt spectrum. CD spectra were scanned at 1 nm intervals from 205 to 250 nm.

Analysis of the denaturation data
Thermodynamic properties of Mth10b were calculated assuming a two-state denaturation process: N 2 K obs 2U. The observed equilibrium constant (K obs ) and the corresponding free energy change (DG) of unfolding at temperature T or denaturant concentration [D] were calculated according to: where P t is the total protein concentration in monomer units; R is the gas constant; T is the absolute temperature; y is the experimentally measured signal value at a given temperature (T) or given denaturant concentration ([D]); y N and y U are the intercepts; and m N and m U are the slopes of the native and unfolded baselines respectively. According to the linear free energy model [39][40][41], free energy changes (DG), enthalpy changes (DH) and entropy changes (DS) during protein unfolding are expected to vary linearly with denaturant concentration ([D]): Where For thermal unfolding, the free energy change (DG T ) of unfolding at temperature (T) can be represented as: where DH T and DS T are the enthalpy and entropy changes of unfolding at temperature (T), respectively. Assuming that the heat capacity change (DC p ) between the native and unfolded states of the system is relatively independent of temperature, the temperature dependences of DH T and DS T can be calculated according to: where DH m and DS m are the enthalpy and entropy changes of the protein at the transition midpoint, where T = T m . Substituting Equations 8 and 9 into Equation 7 gives the following named Gibbs-Helmholtz equation [41,42]: Within the transition range, where T ln(1{ can be simplified to the van't Hoff plot: The temperature of the transition midpoint (T m ), also named melting temperature, is a function of protein and denaturant concentration and can be calculated according to: Substituting Equations 4 and 5 into Equation 12 gives: Results

CD spectra characterization of Mth10b and unfolding reversibility
As far-UV CD spectrum of a protein conforms to its secondary structure, in this work, CD was employed to monitor unfolding of Mth10b. Far-UV CD spectra of Mth10b recorded in basic buffer containing different concentrations of GdnHCl are shown as traces in different colors in Figure 1a. The black trace shows typical CD spectrum of native Mth10b recorded at 25uC, agreeing well with that reported previously [36]. As shown in the figure, the CD signal of Mth10b reduced to about 50% in the presence of 2.4 M GdnHCl (the red trace); Mth10b lost about 70% CD signal in the presence of 4.0 M GdnHCl (the blue trace). The CD spectrum of denatured Mth10b was found no further change when the concentration of GdnHCl was increased to 6.0 M (the cyan trace). Residual CD signal suggested the existence of a compact denatured state of Mth10b. Moreover, the green trace represents a typical CD spectrum for 5-fold dilution of 6.0 M GdnHCl denatured protein, which is almost identical to that of native Mth10b, indicating that GdnHCl-induced unfolding of Mth10b is fully reversible. Similarly, urea-induced unfolding of Mth10b is also fully reversible and there also exist a similar compact denatured state of Mth10b (data not shown).
Due to Mth10b aggregated irreversibly at temperatures higher than 50uC, which is agreeing well with our previous report [36], heat-induced unfolding of Mth10b in basic running buffer could not be performed. However, we found that when the sample contains higher than 0.8 M GdnHCl, fully reversible thermal unfolding of Mth10b can be achieved successfully. Therefore, in this work, the thermal unfolding of Mth10b was carried out in the presence of different concentrations of GdnHCl. Figure 1b shows typical CD spectra of Mth10b in basic running buffer containing 1.2 M GdnHCl recorded at different temperatures. Compared with the spectrum recorded at 25uC (the black trace), when the temperature was increased to about 60uC, Mth10b lost about a quarter of CD signal (the red trace); when the temperature was increased to about 70uC, Mth10b lost about a half of CD signal (the blue trace); when the temperature was further increased to about 75uC, the spectrum displayed no obvious further change (the cyan trace). More residual signal than that existed in results of GdnHCl-induced unfolding performed at room temperature indicated that there is exist a different compact denatured state of Mth10b. This may be explained by that the two unfolding pathways are entirely different. The green trace in Figure 1b shows a typical CD spectrum after heat-induced unfolding at 75uC in the presence of 1.2 M GdnHCl, followed by re-equilibration at 25uC, which is almost identical to that of premelting Mth10b (the black trace).

Denaturant-induced unfolding
Denaturant induced unfolding of Mth10b was studied in basic running buffer at 25uC by far-UV CD monitored at 222 nm. In this work, urea and GdnHCl were chosen as the denaturant for unfolding, respectively. Figure   Moreover, the thermal transition curves also displayed good dependence on protein concentration (data not shown).

Heat-induced unfolding
The heat-induced unfolding profiles were analyzed by using the twostate denaturation model described in methods: the parameters DH m and DS m were obtained by fitting K obs to the van't Hoff plot. The obtained values of DH m and DS m appears decreased linearly with increasing GdnHCl concentration (Figure 4a and 4b

Discussion
Proteins from thermophilic and hyperthermophilic organisms are usually intrinsically thermostable. Though Mth10b is a protein from M. thermoautotrophicum DH, which is a thermophile with optimal growth temperature of 65uC, interestingly, Mth10b seems not intrinsically thermostable [36], suggesting that there are some intracellular extrinsic factors can help Mth10b remain stable under its physiological conditions. In this work, we set out to clarify the molecular mechanism of Mth10b remaining stable in vivo through equilibrium unfolding studies in detail.
GdnHCl and urea denaturation curves are generally employed to obtain an estimation of the conformational stability of proteins. And for most small proteins, as a denaturant, GdnHCl is found to be approximately 2.3 times as effective as urea [43]. However, results of denaturant-induced unfolding revealed that, unlike most proteins, the resistance of Mth10b to urea is much lower than to GdnHCl. The DG(H 2 O) obtained through urea-induced unfolding is just about a half of that obtained through GdnHCl-induced unfolding. Accordingly, thermal unfolding results showed that Mth10b can not remain stable at temperatures higher than 50uC in basic running buffer due to irreversible aggregation. But through heat-induced unfolding of Mth10b in the presence of GdnHCl combined with linear extrapolation method, we concluded that the melting temperature (T m ) of Mth10b in the absence of GdnHCl is close to 80uC. According to this T m value, Mth10b should remain stable at the growth temperature range of M. thermoautotrophicum DH (40-70uC) obviously. Then, what is the molecular basis leading to the huge divergence from different equilibrium unfolding experiments mentioned above.
It is well known that analysis of solvent denaturation curves can provide an estimate of the conformational stability of a protein and that GdnHCl and urea are two agents most commonly employed as protein denaturants [44,45]. However, due to the differences in the ionic character between urea and GdnHCl, analysis of urea and GdnHCl denaturation curves may provide different estimates of the conformational stability of a protein [46,47]. After investigated the effects of urea and GdnCl as denaturants of a synthetic coiled-coil peptide, containing variable numbers of interand intrahelical electrostatic interactions, Monera et al. suggested that the DG(H 2 O) obtained from urea-induced unfolding experiments reveals the sum effect of all kinds of interactions, while the DG(H 2 O) obtained from GdnHCl denaturation studies just represents the sum effect of all nonionic interactions due to the masking effect of GdnHCl on electrostatic interactions [47].
Recently, the crystal structure of Mth10b was solved by our laboratory (PDB code: 3TOE) [37]. Figure 6 shows the surface  electrostatic potential map of Mth10b. Though Mth10b is an acidic protein, the concave surface of the molecule is surprisingly dominated by positively-charged residues (Figure 6a). Accordingly, the electrostatic potential of the convex surface is extremely negative (Figure 6b). Considering that Mth10b has an extremely uneven charge distribution, which may be involved in the intracellular molecular recognition and interaction, we conclude that the dual character of GdnHCl may be the key factor resulting in the huge divergence in the conformational stability studies of Mth10b.
To verify this hypothesis, urea-induced unfolding studies of Mth10b were further performed in the presence of different concentrations of NaCl or GdnHCl. Results of NaCl dependent urea denaturation studies showed that the salt effect of NaCl contribute to the stability of Mth10b greatly. The salt effect of NaCl enhanced with the increasing concentration of NaCl and achieved saturation at the concentration higher than 0.5 M.
Accordingly, results of GdnHCl dependent urea denaturation studies showed that GdnHCl contribute to the stability of Mth10b at low concentrations as well as NaCl, but weaken it at high concentrations. This can be explained by the fact that GdnHCl is a salt as well as denaturant. At low concentrations, the salt property of GdnHCl is dominating. Gdn + and Cl 2 are presumed to shield the large numbers of electrostatic repulsions on the surface of Mth10b (Figure 6), thereby enhancing the stability of Mth10b. At high concentrations, the denaturant property of GdnHCl is dominating. Regardless of the types of electrostatic interactions present in the protein, the binding of the Gdn + ions to the proteins is presumed to predominate and to push the equilibrium toward the unfolded state [45,48]. Conversely, addition of NaCl has no obvious effect on GdnHCl-induced unfolding (data not shown).  These results agree well with our inference. We now return to explaining the huge divergence in the conformational stability studies of Mth10b. Due to nonionic character of urea, the DG(H 2 O) obtained from urea-induced unfolding reveals that the intrinsically conformational stability of Mth10b (34.5 kJ/mol) seems just like that of a typical mesophilic protein. For GdnHClinduced unfolding, as the unfolding transition occurred at GdnHCl concentration higher than 1 M and GdnHCl is more effective in masking electrostatic interactions [49], the DG(H 2 O) obtained from GdnHCl-induced unfolding is the conformational stability of Mth10b under saturated salt effect (63.0 kJ/mol). Similarly, as the thermal unfolding studies of Mth10b were performed in the presence of GdnHCl no less than 1 M, the obtained parameters (DH m (H 2 O), DS m (H 2 O) and T m ) through linear extrapolation are also under saturated salt effect. Therefore substituting the parameters obtained from thermal unfolding and the DG(H 2 O) at 25uC obtained from GdnHCl-induced unfolding into the Gibbs-Helmholtz equation, we got the heat capacity change (DC P ) upon unfolding of Mth10b with a value of 68.5 J?mol 21 ?K 21 ?residue 21 , which seems like the typical DC P value of a mesophilic protein (the average DC P value for mesophilis proteins is around 50 J?mol 21 ?K 21 ?residue 21 [50]). Then, substituting the DC P and those parameters obtained from thermal unfolding into the Gibbs-Helmholtz equation, the profile of temperature-dependent free energy change upon unfolding of Mth10b under saturated salt effect was obtained (Figure 7). According to the profile, under saturated salt effect Mth10b is very Taken together, our work demonstrated that Mth10b from thermophile is not intrinsically thermostable. As the charged residues distributed extremely uneven over the surface of Mth10b, the resulting electrostatic repulsions contribute to destabilizing the protein greatly. Due to the masking effect of soluble ions on electrostatic repulsions, salt contribute to the stability of Mth10b greatly. In other word, without salt, 'thermophilic' protein Mth10b is just a mesophilic one.