Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Valerio Consalvi1,2, Roberta Chiaraluce1, Laura Giangiacomo1, Roberto Scandurra1, Petya Christova3, Andrej Karshikoff4, Stefan Knapp5 and Rudolf Ladenstein4

1 Dipartimento di Scienze Biochimiche `A. Rossi Fanelli', Università `La Sapienza', Piazzale A. Moro 5, 00185 Rome, Italy, 3 Institute of Organic Chemistry, Biophysical Chemistry Laboratory, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, 4 Department of Biosciences, NOVUM Karolinska Institutet, 14157 Huddinge, Sweden and 5 Department of Structural Chemistry, Discovery Research Oncology, Pharmacia and Upjohn, 20014 Nerviano (MI), Italy


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Domain II (residues 189–338, Mr = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.coli using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90–8.00 with melting temperatures between 75.1 and 68.0°C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70°C of 70.2 ± 4.0 kcal/mol and a {Delta}Cp value of 1.4 ± 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 ± 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 ± 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.maritima glutamate dehydrogenase is a thermostable protein with a {Delta}Gmax of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T.maritima domain II glutamate dehydrogenase.

Keywords: domain/glutamate dehydrogenase/hyperthermophiles/protein/thermodynamic stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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In recent years, considerable efforts have been made to understand the mechanisms that determine the extraordinary stability of proteins isolated from hyperthermophilic organisms. To date, most data concerning this topic have been derived from comparisons of the three-dimensional structures of thermostable proteins with their mesophilic counterparts. On the basis of these comparisons, it has been demonstrated that thermostability of a protein is a property that requires only minute structural changes that lead to an optimization of stabilizing hydrophobic and/or hydrophilic interactions (Spassov et al., 1995Go; Jaenicke and Bohm, 1998Go). In particular, it has been recognized that an increased number of salt bridges, often organized in networks, plays a predominant role in the stabilization of proteins from hyperthermophiles (Ladenstein and Antranikian 1998Go; Yip et al., 1998Go). However, comparative studies suffer from the fact that structures of proteins from different mesophilic and thermophilic organisms show not only modifications necessary to increase the thermal stability of the investigated protein, but also species-specific variations. These limitations suggest that such studies should be accompanied by thermodynamic measurements of protein stability and site-directed mutagenesis approaches to verify the mechanisms proposed by the comparison. Studies of the thermodynamic stability of proteins from hyperthermophiles have been reported for only a few selected examples (Jaenicke et al., 1996Go; Knapp et al., 1996Go; Pfeil et al., 1997Go; Wassenberg et al., 1997Go, 1999Go; Motono et al., 1999Go; Zaiss and Jaenicke, 1999Go), despite the large amount of structural information and the availability of a number of natural and recombinant hyperthermophilic proteins. The complex folding pathways, the often irreversible folding/unfolding transitions and the formation of intermediate(s) during refolding are the major obstacles encountered in a thorough quantitative thermodynamic analysis of large oligomeric proteins, as in the case of the hexameric hyperthermophilic glutamate dehydrogenases (GDH) (Consalvi et al., 1996Go) which have been used to study the determinants of thermal stability (Lebbink et al., 1998Go, 1999Go).

One approach to overcoming the problems encountered in the study of the thermodynamic stability of hyperthermophilic GDH is to study the stability of domains of this enzyme which form in principle independent folding units that maintain the native structure. This strategy gives an insight into the intrinsic stability of the studied domain, which might be additionally stabilized in the entire protein by domain interactions and subunit interactions within the oligomer. This concept has already been proven in a study on D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima, where it was demonstrated that the chemical stability of the isolated coenzyme-binding domain is comparable to that of the native enzyme (Jecht et al., 1994Go; Jaenicke et al., 1996Go).

On these premises we decided to study domain II of T.maritima GDH that was expressed independently from the rest of the protein in Escherichia coli. This domain forms an autonomous and stable folding unit and is soluble as a monomer in solution. Circular dichroism (CD) spectroscopy reveals that the expressed domain contains the anticipated secondary structure elements previously observed in the crystal structure (Knapp et al., 1997Go) of the hexameric enzyme. In the pH range studied, the GDH domain unfolds reversibly and therefore offers a suitable model system to acquire thermodynamic data for the unfolding of a part of an extremely thermostable enzyme. The studied protein domain is thermally and chemically stable and the reversibility of the thermal and chemical transitions allowed a detailed thermodynamic analysis of unfolding equilibria.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
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Chemicals and buffers

Guanidinium chloride (GdmCl), urea, dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) were obtained from Fluka (Buchs, Switzerland). All the experiments were performed in the presence of DTT and EDTA to prevent the oxidation of the single Cys residue in the domain. Thermal denaturation experiments were performed in 20 mM sodium phosphate, pH 5.9, 6.3, 7.0, 7.6 and 8.0, containing 100 µM DTT and 100 µM EDTA. GdmCl and urea unfolding/refolding experiments and analytical ultracentrifugation experiments were carried out in 20 mM sodium phosphate, pH 7.0, containing 100 µM DTT and 100 µM EDTA. Buffer solutions were filtered (0.22 µm) and carefully degassed. All buffers and solutions were prepared with ultra-high-quality water (ELGA UHQ).

Protein expression and purification

DNA coding for the domain II of T.maritima GDH was amplified by PCR and cloned into a pET15b expression vector (Novagen). The recombinant protein domain was expressed in the host cells BL21 (DE3). Expression was induced for 3 h at an optical density (OD600) of 0.6 using 1 mM IPTG. Cells were harvested by centrifugation and lysed in a French press in a buffer containing 30 mM Tris–HCl, pH 7.4, 50 mM NaCl and 2 mM DTT. The lysate was incubated at 76°C for 20 min and the precipitated E.coli proteins were separated from the thermostable soluble domain by centrifugation. The supernatant was concentrated and applied in 2 ml fractions to an 80x1 cm i.d. Superose 12 column (Pharmacia, Uppsala, Sweden) equilibrated with a buffer containing 30 mM Tris–HCl, pH 7.4, 1 mM DTT and 100 mM NaCl. Fractions containing the protein were pooled and applied to a Mono Q column (Pharmacia) and eluted with a step gradient. The recombinant protein eluted at a salt concentration of 150 mM and was pure after this purification step. The protein was stored at 4°C until it was used for calorimetric or spectroscopic experiments.

Spectroscopic techniques

Intrinsic fluorescence emission measurements were carried out with a Perkin-Elmer LS50B spectrofluorimeter using a 1 cm pathlength quartz cuvette. Fluorescence emission spectra were recorded between 280 and 400 nm (1 nm sampling interval) at 20°C with the excitation wavelength set at 274 nm. The protein concentration was 0.15 mg/ml.

CD spectra were recorded on a Jasco J-720 spectropolarimeter. Far-UV (190–250 nm) and near-UV (250–310 nm) CD measurements were performed at 20°C in 0.1 and 1.0 cm pathlength quartz cuvettes, sealed with a PTFE stopper, at an enzyme concentration of 0.20 and 3.60 mg/ml, respectively. The results were expressed as mean residue ellipticity ([]) assuming a mean residue weight of 110 per amino acid residue. Spectra were accumulated four times. All values were corrected for solvent contributions.

Analytical ultracentrifugation

All experiments were conducted at 20°C on a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics. The protein concentration was in the range 1–5 mg/ml. Sedimentation velocity experiments were performed at 40 000 r.p.m. Data were collected at 280 nm at a spacing of 0.005 cm with three averages in the continuous scan mode and were analyzed with the program DCDT (W.Stafford, Boston Biomedical Research Institute) (Stafford, 1992Go). Sedimentation coefficients were corrected to s20,w using standard procedures. Sedimentation equilibrium experiments were performed at 25 000 and 32 000 r.p.m. Data were collected at 280 nm at a spacing of 0.001 cm with 10 averages in the step scan mode. Equilibrium was checked by comparing scans up to 24 h. Data sets were edited with REEDIT (J.Lary, National Analytical Ultracentrifugation Center, Storrs, CT) and fitted with NONLIN (PC version provided by E.Braswell, National Analytical Ultracentrifugation Center, Storrs, CT) (Johnson et al., 1981Go). Data from different speeds were combined for global fitting. Fits to a single species give a Z-average molecular weight. For fits to a monomer–dimer association scheme, the monomer molecular weight was fixed at the value determined from the amino acid sequence. In all experiments the buffer used was 20 mM sodium phosphate, pH 7.0, containing 100 µM DTT and 100 µM EDTA.

Thermal denaturation

For thermal scans, the protein samples (0.2 mg/ml) at different pH values between 5.9 and 8.0 were heated from 10 to 95°C and subsequently cooled to 10°C with a heating/cooling rate ranging from 0.75 to 1.50 °C/min controlled by a Jasco programmable Peltier element. A scan rate of 1 °C/min was chosen in consideration of the observed independence of thermal transitions of the heating/cooling rate. Far-UV CD spectra were recorded every 5–2.5°C and the dichroic activity at 222 nm was continuously monitored every 0.5°C with a 4 s averaging time. All the spectra were corrected for solvent contribution at increasing temperature for all the different pH values examined.

Reversible thermal denaturation was analyzed by fitting baseline and transition region data to a two-state model (Santoro and Bolen, 1988Go) according to the following equation:


where y is the observed signal, yN and yD are the native and denatured baseline intercepts, mN and mD are the native and denatured baseline slopes, T is the temperature, {Delta}Hvh is the van't Hoff enthalpy, R is the gas constant and Tm is the temperature of the transition midpoint. The heat capacity change upon thermal denaturation, {Delta}Cp, corresponding to


was estimated from the slope of the {Delta}Hvh versus Tm plot, assuming that {Delta}Hvh and {Delta}Cp do not vary significantly with pH (Privalov and Khechinashvili, 1974Go; Becktel and Schellman, 1987Go). The temperature dependence of the free energy of unfolding, {Delta}G(T), was calculated at any temperature from a modified Gibbs–Helmholtz equation:


Unfolding/refolding equilibria in the presence of GdmCl and urea

For equilibrium transition studies the protein (final concentration 0.15 mg/ml) was incubated at 20°C at increasing concentrations of GdmCl (0–4.3 M) or urea (0–7.2 M) in 20 mM sodium phosphate, pH 7.0, containing 100 µM DTT and 100 µM EDTA. Far-UV CD spectra were recorded after 24 h at 20°C. To probe the reversibility of the unfolding process, the protein was unfolded at 20°C in 4.5 M GdmCl or in 7.6 M urea at 1.5 mg/ml protein concentration in 20 mM sodium phosphate, pH 7.0, containing 1 mM DTT and 1 mM EDTA. After 24 h, the refolding transition was started by 10-fold dilution of the unfolding mixture at 20°C into solutions of the same buffer used for the unfolding containing decreasing denaturant concentrations. The final enzyme concentration was 0.15 mg/ml. After 24 h, far-UV CD spectra were recorded at 20°C. GdmCl and urea-induced unfolding equilibria were analyzed by fitting baseline and transition region data to a two-state linear extrapolation model (Santoro and Bolen, 1988Go) according to the following equation:


where {Delta}Gunfolding is the free energy change for unfolding for a given denaturant concentration, {Delta}GH2O is the free energy change for unfolding in the absence of denaturant, m is a slope term which quantitates the change in {Delta}Gunfolding per unit concentration of denaturant, R is the gas constant, T is the temperature and Kunfolding is the equilibrium constant for unfolding. The model expresses the signal as a function of denaturant concentration:


where yi is the observed signal, yN and yD are the baseline intercepts corresponding to native and denatured protein, respectively, mN and mD are the corresponding baseline slopes, [X]i is the denaturant concentration after the ith addition, {Delta}GH2O is the extrapolated free energy of unfolding in the absence of denaturant, m is the slope of a {Delta}Gunfolding versus [X] plot, R is the gas constant and T is the temperature. [X]0.5 is the denaturant concentration at the midpoint of the transition and, according to Equation 5Go, is calculated as


Differential scanning calorimetry (DSC)

The protein was extensively dialyzed against the buffer used in the calorimetric experiments. Data were measured on a MicroCal MCS calorimeter controlled by the MCS observer program (MicroCal). Samples were routinely degassed for 5 min before they were used for a calorimetric analysis. Buffer baselines were collected under identical conditions and were subtracted from the corresponding data of the protein samples. The scanning rate during the calorimetric measurement was 1°C/min. A second (re-heating) scan was always performed after the DSC experiment in order to assess the reversibility of the unfolding transition. Calorimetric enthalpies ({Delta}Hcal) were calculated by integration of the measured heat absorption peak after subtraction of the buffer baseline. Calorimetric data were calculated using software provided by the manufacturer.


    Results
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 Abstract
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 Materials and methods
 Results
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Purification and spectral properties of domain II

Cloning of GDH domain II into a pET15b expression vector yielded an efficient expression system. Large amounts (10 mg/l) of the pure protein were obtained after heat treatment of the lysate which takes advantage of the thermal stability of this domain, followed by a few additional purification steps. Analysis of the far-UV CD data (Figure 1AGo) using the method of Yang (Yang et al., 1986Go) resulted in a secondary structure determination of 45% {alpha}-helical and 27% ß-sheet, respectively. In the three-dimensional crystal structure of the hexamer this domain has a similar secondary structure composition (Knapp et al., 1997Go). The near-UV CD spectrum (data not shown) is characterized by three negative contributions at 275, 269 and 262 nm that can be ascribed to tyrosine and phenylalanine residues. No CD signal was observed above 275 nm in accordance with the lack of tryptophan residues (Knapp et al., 1997Go). The absence of tryptophan was also evident in the fluorescence spectrum (Figure 1BGo) which showed an emission maximum at 306 nm, typical for tyrosine residues (Schmid, 1989Go).



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Fig 1. Spectroscopic analysis of GDH domain II. Spectra were recorded in 20 mM phosphate, pH 7.0, in the presence of 100 µM DTT and 100 µM EDTA at 20°C. (A) Far-UV CD spectrum recorded in a 0.1 cm pathlength quartz cuvette at 0.20 mg/ml protein concentration. (B) Fluorescence emission spectrum (274 nm excitation wavelength) recorded at 0.15 mg/ml in a 1 cm pathlength quartz cuvette.

 
Analytical ultracentrifugation

Sedimentation velocity experiments yielded s20,w = 1.54 ± 0.05 consistent with the molecular weight of the monomer assuming a spherical shape for the protein. In addition, equilibrium sedimentation experiments yielded a value of Mz = 17 000 ± 1000, which has to be compared with 16 222, the value obtained from the sequence (Wyman and Ingalls, 1943Go). This comparison indicated that the protein is entirely monomeric under the conditions used in the experiments, as confirmed by the small value of the association constant [K1,2 = (1.3 ± 0.2)x10–2 ml/mg] obtained by fitting the data to a monomer–dimer equilibrium.

Thermal unfolding

In the investigated pH range (5.9–8.0), the far-UV CD spectrum of the native protein was found to be identical with that of the heat-denatured sample after cooling. Monitoring a second heat-denaturation cycle by far-UV CD spectroscopy resulted in an identical thermal transition profile (Figure 2AGo). In addition, gel filtration chromatography of the protein refolded after heat denaturation revealed that during thermal unfolding the protein did not aggregate or polymerize (data not shown). Complete reversibility was observed only in the pH range 5.9–8.0. At pH values >8 and <5.9, the unfolding transition was irreversible, thus limiting the pH window in which a thermodynamic analysis of the process could be performed. Complete reversibility of the thermal unfolding transition was also observed at the higher protein concentration used in DSC experiments in the pH interval 6.0–8.0 (Figure 2BGo, Table IGo). The presence of two data points with a lower degree of reversibility and the minor differences observed between calorimetric and van't Hoff enthalpies suggest the limitation of the system.




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Fig. 2. Reversible thermal denaturation of GDH domain II. (A) The molar ellipticity measured at 222 nm was monitored continuously in the temperature range from 10 to 95°C (•) and during a reverse scan from 95 to 10°C ({circ}).The protein concentration was 0.2 mg/ml and the pH was 6.3. Data were measured at 0.5°C intervals. The solid line represents a non-linear regression analysis (Equation 1Go). Reversibility points ({circ}) were not included in the non-linear regression analysis. The inset shows the far-UV CD spectra measured at 5°C interval during the heating cycle (10–95°C). (B) Calorimetric scan recorded at pH 6.5 (solid line) and a second scan of the same sample after cooling (dotted line). The reversibility of this experiment, calculated by comparing the area under the heat absorption peak, was 92%.

 

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Table I. Thermal unfolding parameters of GDH domain II determined by differential scanning calorimetrya
 
An isodichroic point observed in the far-UV CD spectra at 205 nm (Figure 2AGo, inset) supports the two-state nature of the unfolding transition. This assumption received additional support by DSC because the measured data (Figure 3AGo) fit perfectly to a theoretical two-state model (Figure 3BGo). Finally, the two-state nature of the unfolding transition was supported by good agreement between calorimetric enthalpies and van't Hoff enthalpies (Table IGo). The thermodynamic unfolding parameters calculated by non-linear regression analysis (Equation 1Go) of the temperature-dependent changes of the molar ellipticity at 222 nm are reported in Table IIGo. The {Delta}Hvh values obtained from the spectroscopically monitored thermal unfolding transitions depend linearly on Tm. From this temperature dependence a heat capacity difference upon unfolding ({Delta}Cp) of 1.4 ± 0.3 kcal/mol K was determined by linear regression analysis.




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Fig. 3. DSC data for GDH domain II thermal unfolding. (A) Raw calorimetric data after subtraction of the buffer baseline. Shown is a calorimetric scan at pH 6.5 ({circ}) and the corresponding sigmoidal baseline. (B) Same scan after subtraction of the sigmoidal baseline and fitting of the data to a two-state model (solid line).

 

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Table II. Thermal denaturation parameters for GDH domain II calculated from far-UV CD transitiona
 
Thermodynamic data for the thermal unfolding transition determined by far-UV CD spectroscopy were compared with directly measured calorimetric data. Calorimetrically measured enthalpies ({Delta}Hcal) and melting temperatures (Tm) (Table IGo) are comparable to the data derived from spectroscopic measurements (Table IIGo). The thermal unfolding of GDH domain II is characterized by an enthalpy change of 74.5 kcal/mol at the maximum melting temperature of 72.8°C. In the investigated pH range, a stability maximum was observed at pH 5.9. Using the measured data and Equation 3Go (Becktel and Schellman, 1987Go), the maximum stability of this protein was observed at 33°C and pH 5.9, where the native state of the protein is stabilized by a free energy change ({Delta}G) of 3.7 kcal/mol.

Unfolding/refolding equilibria in the presence of GdmCl and urea

Incubation of domain II in the presence of increasing urea and GdmCl concentrations in 20 mM phosphate buffer, pH 7.0, containing 100 µM EDTA and 100 µM DTT for 24 h at 20°C resulted in progressive changes in the far-UV CD spectra. The thermodynamic unfolding parameters of the transition curves (Figure 4Go) were calculated using the spectral changes at 222 nm. Extrapolation to zero denaturant concentration resulted in a free energy difference ({Delta}GH2O) of 6.11 ± 0.18 kcal/mol for GdmCl and 4.24 ± 0.13 kcal/mol for urea unfolding, respectively. The corresponding m values for GdmCl and urea denaturation were 3.36 ± 0.10 and 1.46 ± 0.04 kcal/mol M, respectively; the midpoint corresponding to the denaturant concentration at which 50% of the protein was in the unfolded state was determined to be 1.83 ± 0.05 M (GdmCl) and 2.96 ± 0.09 M (urea).



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Fig. 4. GdmCl- and urea-induced unfolding/refolding equilibria of GDH domain II. Far-UV CD spectra were recorded after 24 h incubation at 20°C at the indicated GdmCl (•) or urea ({blacksquare}) concentrations, at a protein concentration of 0.15 mg/ml. The solid lines result from a non-linear regression analysis using Equation 5Go. Reversibility points ({circ}, {square}) were not included in the non-linear regression analysis.

 

    Discussion
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 Materials and methods
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Domain II of T.maritima GDH expressed in E.coli is a small thermostable protein with the same relative composition of secondary structure elements as that reported for the domain in the context of the hexameric structure (Knapp et al., 1997Go). It is monomeric in solution and does not show any tendency to associate. In GDH domain II the aromatic residues appear to be locked in tertiary contacts, despite the lack of the proper interdomain and intersubunit contacts present in the native hexamer.

The equilibrium unfolding process of T.maritima domain II GDH monitored by far-UV CD spectroscopy and DSC was monophasic. The simplest model that accounts for the experimental observations is


in which the protein is assumed to exist in the native (N) or in the denatured (D) state. Therefore, unfolding data were evaluated assuming a two-state transition as suggested by the presence of an isodichroic point at 205 nm. In addition, the measured DSC data fit perfectly to a theoretical two-state model. The measured calorimetric enthalpies ({Delta}Hcal) are in good agreement with the van't Hoff enthalpies ({Delta}Hvh) and the minor differences observed are not unprecedented in proteins whose unfolding transitions are consistent with the classical two-state model (Pace et al., 1999Go; Waldner et al., 1999Go). These results strongly support a cooperative two-state unfolding of this protein (Privalov and Khechinashvili, 1974Go; Privalov, 1979Go).

Thermal unfolding was highly reversible in the investigated pH range (5.9–8.0). However, irreversible unfolding was observed at higher or lower pH values. The energetics of domain II thermal unfolding studied by far-UV CD and DSC are comparable and the differences are within the range of accuracy of the data. The heat capacity difference between the native and the thermally unfolded state ({Delta}Cp) was determined from the temperature dependence of {Delta}H. In principle, {Delta}Cp can be also determined directly from the individual DSC measurements. However, when comparing different calorimetric measurements the directly measured heat capacity differences scattered significantly. Such a discrepancy between the {Delta}Cp obtained calorimetrically and that assessed from the {Delta}H versus Tm plots has been discussed earlier and can be explained by inaccuracies in the baseline determination especially for the baseline of the denatured state (Liu and Sturtevant, 1996Go). {Delta}H, determined by the integral over the whole curve, is less prone to such inaccuracies and leads to more accurate values. The {Delta}Cp obtained from the temperature dependence of the enthalpies measured by CD spectroscopy corresponded to 1.4 kcal/mol K. The determination of {Delta}Cp was particularly difficult for this protein because the melting temperature interval for which unfolding enthalpies could be determined was very small. The reason for this limited Tm window is that Tm does not depend strongly on pH in the region where a reversible unfolding was observed, thus explaining the relatively large inaccuracy of the {Delta}Cp determination. However, it can still be concluded that the heat capacity change upon unfolding is surprisingly small. As a consequence, the {Delta}Cp measured per amino acid residue is only 9.7 cal/mol K. Specific heat capacity differences measured for small mesophilic proteins usually range between 10 and 18 cal/mol residue K (Myers et al., 1995Go). Also, the estimation of {Delta}Cp based on the domain II amino acid composition would be 2.1 kcal/mol K (14 cal/mol residue K) (Murphy and Gill, 1991Go). The stability of a protein [{Delta}G(T)] is determined by Tm, {Delta}H and {Delta}Cp or, equivalently, by {Delta}Hm, {Delta}Sm and {Delta}Cp with the slope given by {Delta}Sm and the curvature by {Delta}Cp/T (Becktel and Schellmann, 1987). In order to achieve thermostability, the stability curves of proteins from hyperthermophilic organisms may be either shifted upwards, to higher Tm, or flattened, or several of these solutions may be combined (Beadle et al., 1999Go; Dams and Jaenicke, 1999Go). In recent studies it was suggested that the stability curves of at least some of these proteins are flattened rather than shifted upwards or to higher Tm (Jaenicke, 1991Go; Knapp et al., 1996Go). The small heat capacity change upon unfolding observed in the case of GDH domain II supports this view. Indeed, the specific {Delta}Cp calculated per amino acid residue corresponds very well with the value obtained for Sso7d (9.7 cal/mol residue K), a small, extremely thermostable protein from the hyperthermophile Sulfolobus solfataricus (Knapp et al., 1996Go). A definite explanation for the small {Delta}Cp values measured for some hyperthermostable proteins is still lacking, even if a possible hypothesis may be referred to an incomplete exposure of hydrophobic residues to the solvent upon thermal unfolding.

The thermodynamic analysis of the reversible two-state chemical denaturation induced by urea and GdmCl indicated once more that domain II of T.maritima GDH is a single cooperative unit. The transition midpoints for chemical denaturations are those expected for a stable protein but lower than the values found for natural and recombinant proteins of hyperthermophilic sources (Jaenicke et al, 1996Go). Similar considerations hold also for the melting points. Interestingly, domains excised from several monomeric or oligomeric proteins from T.maritima usually display thermal and chemical stabilities similar to or higher than those of the intact protein as well as melting points higher than the optimal growth temperature of the microorganism (Jecht et al., 1994Go; Wassenberg et al., 1997Go; Zaiss and Jaenicke, 1999Go). However, in the case of GDH domain II the stabilization induced by interdomain interaction and/or by subunit assembly appears to be significant, since the intrinsic stability of this domain is much lower than that reported for the hexamer (Lebbink et al., 1998Go, 1999Go). The association to an oligomer as a prerequisite for stabilization may be an important property of hexameric GDHs. In line with this consideration, it has been reported that the recombinant monomer of mesophilic GDH from Closteidium symbiosum (Millevoi et al., 1998Go) and the refolding intermediate(s) of Pyrococcus furiosus GDH (Consalvi et al., 1996Go) are less resistant towards denaturation than the native hexameric enzymes. The large difference in stability derived from urea and GdmCl denaturation studies points to the different preferential stabilization of the denatured states exerted by the two denaturants (Fersht, 1999Go). A higher accessibility of the protein to the solvent upon unfolding by GdmCl may be suggested by the higher m value measured in the presence of this denaturant (Myers et al., 1995Go).

One important factor discussed in the recent literature is the contribution of ion pairs and ion pair networks to protein stability (Lebbink et al., 1998Go, 1999Go; Vetriani et al., 1998Go; Frankenberg, 1999Go). In particular, for glutamate dehydrogenases this mechanism seems to play an important role since the number of ion pairs and the length of ion pair networks correlate well with an increase in thermal stability (Yip et al., 1998Go). The largest ion pair network that has been reported in the crystal structure of T.maritima glutamate dehydrogenase spans the cleft between the two domains of the six subunits involving residues R190, E186, K193, E231, E371, R367 and K375 (Knapp et al., 1997Go). Independent expression of domain II leads to the disruption of the network. Since the ion pair network is the predominant interaction between the two GDH domains, it is reasonable to assume that most of the stabilization of domain II in the intact subunit can be accounted for by the stabilizing contribution of these ion pairs. Experiments that aim to clarify the extent to which this network contributes to the interdomain stabilization of T.maritima glutamate dehydrogenase are in progress in our laboratories.


    Notes
 
2 To whom correspondence should be addressed. E-mail: pasquo{at}caspur.it Back


    Acknowledgments
 
We are grateful to Professor Emilia Chiancone and to Dr Joyce Lebbink for helpful discussions and critical comments on the manuscript. This work was supported by the European Commission (Biotech Generic Project `Extremophiles as Cell Factories', contract ERBBIO4CT960488), by the CNR Target Project Biotechnology, by MURST and partially by the Academic Exchange Program of the Royal Swedish Academy of Sciences.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received December 14, 1999; revised April 19, 2000; accepted May 18, 2000.