High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low {Delta}{Delta}Cp of unfolding

Chie Motono, Tairo Oshima and Akihiko Yamagishi,1

Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan


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To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change {Delta}{Delta}G° of each unfolding process. Other thermodynamic parameters, enthalpy change {Delta}{Delta}H, entropy change {Delta}{Delta}S and heat capacity change {Delta}{Delta}Cp, were derived from the temperature dependence of {Delta}{Delta}G°. The main feature of the thermophilic enzyme was its lower dependence of {Delta}{Delta}G° on temperature resulting from a low {Delta}{Delta}Cp. The thermophilic IPMDH had a significantly lower {Delta}{Delta}Cp, 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low {Delta}{Delta}Cp of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area {Delta}{Delta}ASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature Ts. The {Delta}{Delta}G° maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature Tm is not necessarily accompanied by a higher {Delta}{Delta}G° maximum.

Keywords: heat capacity change/3-isopropylmalate dehydrogenase/protein unfolding/thermal stability/thermophilic protein


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In spite of numerous attempts to understand the extreme thermostability of proteins of thermophiles, it is not sufficiently clear what kind of energetic mechanisms are responsible for the thermal adaptation of the proteins. It is widely accepted that the stabilization of thermophilic proteins results from combination of weak non-covalent forces and protein–solvent interactions, which are also typical for mesophilic proteins.

Much research has been devoted to determining which forces are responsible for exceptional thermostability of the proteins isolated from thermophilic organisms. Recent structural studies of thermophilic and mesophilic protein pairs have revealed that there is no single structural feature that can account for the higher thermal stability of thermophilic proteins. There are many factors responsible for thermostability and every protein does not use the same strategy (Fujinaga et al., 1993Go; Russell et al., 1994Go; Korndörfer et al., 1995Go). Factors that have been shown to have an effect on the thermostability of proteins are salt links, hydrogen bonds (Shortle, 1992Go), hydrophobic interactions, internal packing, stabilization of helices (Serrano and Fersht, 1989Go), additional aromatic–aromatic interactions (Burley and Petsko, 1985Go) and a decrease in the main-chain flexibility (Leszczynski and Rose, 1986Go; Hering et al., 1992Go; Daggett and Levitt, 1993Go; Hardy et al., 1994Go). Recent studies have emphasized an increased number of salt bridges as the main stabilizing factors in hyperthermophilic enzymes from archea, but the mechanism is not common to all thermophiles (Britton et al., 1995Go; McCrary et al., 1996Go; Aguilar et al., 1997Go).

In this investigation, we compared the thermodynamic stability of two analogous 3-isopropylmalate dehydrogenases (IPMDH: EC 1.1.1.85): the thermophilic IPMDH from an extreme thermophile, Thermus thermophilus, and the mesophilic counterpart originating from Escherichia coli. IPMDH is a bifunctional enzyme involved in the leucine biosynthesis pathway. It catalyzes the dehydrogenation and concomitant decarboxylation of 3-isopropylmalate substrate, yielding 2-oxoisocaproate and carbon dioxide, using NAD+ as a cofactor. The enzyme from T.thermophilus is a functional dimer composed of two identical subunits, each with 345 amino acid residues (Yamada et al., 1990Go). The polypeptide chain of a subunit is folded into two domains with similar folding topologies based on parallel {alpha}/ß motifs (Imada et al., 1991Go).

IPMDH from T.thermophilus, which we will call Tt-IPMDH, is 51% identical with E.coli enzyme (Ec-IPMDH) in amino acid sequence (Kirino et al., 1994Go). The temperatures of the native environment of the organisms are 37 and 75–80°C for E.coli and T.thermophilus, respectively. The IPMDHs differ in their half denaturation temperature: 63 and 83°C for Ec-IPMDH and Tt-IPMDH, respectively. The two IPMDHs also differ in the level of enzyme activity, when measured at the same temperature (Wallon et al., 1996Go). The structural comparison of Tt-IPMDH with their mesophilic counterparts from E.coli and Salmonella typhimurium has shown that main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N- and C-termini and a larger number of proline residues (Wallon et al., 1997Go). It is still unclear, however, how these structural factors affect the thermostability or the unfolding of the IPMDH molecule.

We have previously reported equilibrium unfolding processes of Tt-IPMDH and Ec-IPMDH (Motono et al., 1999Go). Thermal unfolding and guanidine hydrochloride-induced unfolding of these IPMDHs were irreversible. We therefore searched for conditions for reversible urea-induced unfolding. We found that 90% of unfolded IPMDHs could be refolded in the presence of a small amount of Tween 20. We analyzed the unfolding processes of the IPMDHs at 300 K and obtained the free energy change upon unfolding, {Delta}G° (Motono et al., 1999Go). The unfolding process of Ec-IPMDH could be fitted to a three-state transition (see Equation 1Go in Materials and methods). The protein concentration dependence of unfolding curves revealed that dissociation of dimeric Ec-IPMDH occurred at the second transition and that the intermediate remained dimeric. The intermediate of Ec-IPMDH lost 50% of its secondary and tertiary structures and was enzymatically inactive. The unfolding curve of Tt-IPMDH at 300 K could be fitted to a two-state model from a native dimer to two unfolded monomers (see Equation 2Go in Materials and methods). The unfolding curves monitored by the changes in secondary structure (CD at 222 nm), tertiary structure (intrinsic fluorescence) and enzyme activity coincided well with each other, showing that the unfolding process was a two-state transition. There was a reproducible small shoulder in the unfolding curve in the presence of intermediate concentrations of urea. The unfolding curves of Tt-IPMDH could also be fitted to the three-state transition model (Equation 1Go), with a dimeric intermediate state corresponding to the small shoulder. In this study, however, the unfolding process of Tt-IPMDH is treated as the simpler model, the two-state transition model.

The main goal of this study was the thermodynamic characterization of the higher thermal stability of Tt-IPMDH. Here we report a comparison of the unfolding process of Tt-IPMDH and that of Ec-IPMDH in terms of thermodynamic parameters. A spectroscopic measurement of urea-induced unfolding was repeated at various temperatures where the unfolding of the IPMDHs is reversible, then the temperature dependence of {Delta}G°, i.e. protein stability curve (Becktel and Schellman, 1987Go), was obtained. Other thermodynamic parameters were estimated from these protein stability curves.


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Reagents and proteins

3-Isopropylmalate (IPM) was purchased from Wako Pure Chemicals (Osaka, Japan) and ultrapure urea from Nacalai Tesque (Kyoto, Japan). All other reagents were of analytical grade. Water was purified with a Milli-Q purification system (Millipore). Ec-IPMDH and Tt-IPMDH were overexpressed in E.coli BL21 and purified as described previously (Motono et al., 1999Go). The concentration of the Tt-IPMDH protein was measured spectroscopically using an extinction coefficient of 30 400 at 280 nm (Yamada et al., 1990Go). The concentration of the Ec-IPMDH was measured using a BCA Protein Assay Kit (Pierce). All the IPMDH concentrations in this paper have units of molarity (moles of monomer per liter). All samples and solutions were filtered through microporous filters (0.45 µm, Millipore) before use.

Protein unfolding and refolding monitored by intrinsic fluorescence

Fluorescence measurements were carried out with a Hitachi spectrofluorimeter as described previously (Motono et al., 1999Go). Briefly, Ec-IPMDH or Tt-IPMDH was incubated with urea in 50 mM potassium phosphate (pH 7.0), 1 mM DTT and 0.01% Tween 20 at each temperature for 24 or 48 h, respectively. The fluorescence intensity was monitored at 340 or 332 nm for Tt-IPMDH or Ec-IPMDH, respectively (excitation at 280 nm). All the measurements were repeated at least three times and corrected for the background signal of buffer containing the corresponding concentration of urea. To demonstrate the reversibility of the unfolding process, IPMDHs unfolded with 9 M urea were diluted 1:20 to various urea concentrations, incubated for a minimum of 24 h and then the activity and the fluorescence were measured.

Protein stability curve analysis

The urea unfolding titration at each temperature was fitted independently to the unfolding models (Equation 1 or 2GoGo), to obtain {Delta}G°. As reported previously (Motono et al., 1999Go), we employed a three-state model for Ec-IPMDH:

(1)
where {Delta}G1° and {Delta}G2° are the free energy changes related to the respective unfolding steps in the absence of urea. An overall free energy change, {Delta}Gt°, was assumed to be the sum of {Delta}G1° and {Delta}G2°. In this model, N2, I2 and D + D are a native dimer, a dimeric intermediate and unfolded monomers, respectively.

For the analysis of the unfolding data of Tt-IPMDH, a two-state model was used:

(2)

In the two-state model, a native dimer N2 unfolds directly to monomers D + D.

From multiple independent fits, the temperature dependence of {Delta}Gt° of the enzymes was obtained. The gas constant R = 8.314 J/mol.K and 1 cal = 4.186 J were used for calculations.

Privalov showed that {Delta}Cp of protein unfolding can be taken as a constant within experimental error for a given protein (Privalov, 1979Go). With this assumption, {Delta}G is given by a modified Gibbs–Helmholtz equation:

(3)
where T is temperature, Ts is the temperature at which {Delta}G is maximum, i.e. the entropy change {Delta}S = 0, {Delta}Hs is the change in enthalpy at Ts and is equal to the maximum of {Delta}G, {Delta}Gs, and {Delta}Cp is the change in heat capacity. Using Ts as a standard temperature, the entropy and enthalpy changes are described by

(4)


(5)

The temperature dependence of {Delta}Gt° was fitted to Equation 3Go to obtain {Delta}Hs, Ts and {Delta}Cp.

{Delta}ASA calculation

The change in solvent-accessible surface area upon unfolding of a protein, {Delta}ASA, was calculated to predict the {Delta}Cp value. The solvent-accessible surface area was calculated using the Homology module in InsightII (MSI) with a solvent probe radius of 1.4 Å and van der Waals radii given by Richards (Richards, 1977Go). X-ray crystallographic structures of Ec-IPMDH and Tt-IPMDH (Imada et al., 1991Go; Wallon et al., 1997Go) were used as native proteins. The unfolded proteins were modeled as extended ß-strands using the Biopolymer module in InsightII.


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Urea-induced unfolding profiles of Ec-IPMDH and Tt-IPMDH at various temperatures

Figure 1AGo shows urea-induced equilibrium unfolding curves of 0.82 µM Ec-IPMDH monitored by fluorescence emission over a range of temperature from 278 to 318 K. Figure 1BGo shows the result of the corresponding measurements of 0.82 µM Tt-IPMDH over a range of temperature from 277 to 333 K. Prior to the fluorescence measurement, Ec-IPMDH or Tt-IPMDH was incubated with urea at the temperature of interest for 20–24 or 44–48 h, respectively, to reach unfolding equilibrium. At this temperature range, both Ec-IPMDH and Tt-IPMDH unfolded reversibly with urea.



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Fig. 1. Urea-induced unfolding of Ec-IPMDH (A) and Tt-IPMDH (B) at various temperatures. (A) 0.82 µM Ec-IPMDH was incubated with urea at 318 K (•), 315.5 K ({circ}), 312.5 K ({blacksquare}), 310.5 K ({square}), 308 K ({blacklozenge}), 278 K ({lozenge}), 283 K ({blacktriangleup}), 303 K ({triangleup}), 286 K ({blacktriangledown}) or 293 K ({triangledown}). (B) 0.82 µM Tt-IPMDH was incubated with urea at 333 K (•, left), 329 K ({circ}), 326 K ({blacksquare}), 323 K ({square}), 277 K ({blacklozenge}), 318K ({lozenge}), 286 K ({blacktriangleup}), 293 K ({triangleup}), 313 K ({blacktriangledown}), 303 K ({triangledown}) or 308 K (•, right). Lines represent the fits to the three-state unfolding model (Equation 1Go) or to the two-state unfolding model (Equation 2Go) for panels A and B, respectively.

 
As shown in Figure 1BGo, the Tt-IPMDH unfolding curves shifted to a higher urea concentration as the temperature was increased from 277 to 308 K. In contrast, the midpoints of the unfolding curves shifted to a lower urea concentration as the temperature was increased from 308 to 333 K. The cooperative profile of the unfolding curves did not change much between 277 and 333 K.

The temperature dependence of the unfolding process of Ec-IPMDH was clearly different from that of Tt-IPMDH (Figure 1AGo). Each unfolding curve of Ec-IPMDH was biphasic, as reported in our previous work (Motono et al., 1999Go). The curve at 300 K was well fitted to a three-state transition model (Equation 1Go). The first phase of the Ec-IPMDH unfolding shifted to a higher urea concentration as the temperature was increased from 278 to 293 K and then shifted to a lower urea concentration as the temperature was increased from 293 to 318 K. The second phase appeared to be less sensitive to the temperature, but showed the same temperature dependence as did the unfolding curves of Tt-IPMDH.

Comparison of the temperature dependence of {Delta}G° of Ec-IPMDH and Tt-IPMDH

To estimate the free energy change {Delta}G° at each temperature, each unfolding curve of Ec-IPMDH (Figure 1AGo) or Tt-IPMDH (Figure 1BGo) was independently fitted to the three-state unfolding model (Equation 1Go) or the two-state model (Equation 2Go), respectively. The free energy change was plotted against temperature (Figure 2A and BGo). For Ec-IPMDH, {Delta}G1° and {Delta}G2° are the free energy changes in the absence of urea for the first and the second transitions, respectively. {Delta}Gt° indicates the overall free energy change of a whole dimer, which is the sum of {Delta}G1° and {Delta}G2° for Ec-IPMDH.



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Fig. 2. (A) Protein stability curves ({Delta}G° versus T) of Ec-IPMDH obtained from the data in Figure 1AGo on the basis of the three-state unfolding model. {Delta}G1° ({circ}) and {Delta}G2° ({square}) are the free energy changes of the first and the second transitions, respectively, in the absence of urea. {Delta}Gt° ({triangleup}) indicates the overall free energy change of the whole dimer, which is the sum of {Delta}G1° and {Delta}G2°. Lines represent the fits of {Delta}G1°, {Delta}G2° and {Delta}Gt° to the modified Gibbs–Helmholtz equation (Equation 3Go). (B) Protein stability curves of Tt-IPMDH obtained from the data in Figure 1BGo on the basis of the two-state unfolding model. A line represents the fit of {Delta}G° to the modified Gibbs–Helmholtz equation (Equation 3Go). Note that the second process in the three-state unfolding of Ec-IPMDH and the two-state unfolding of Tt-IPMDH are reverse second-order reactions.

 
By fitting the data of {Delta}G° to a modified Gibbs–Helmholtz equation (Equation 3Go), other thermodynamic parameters, {Delta}Cp, Ts and {Delta}Hs, were derived and are summarized in Table IGo. {Delta}Cp is the change in heat capacity. Because the curvature of the protein stability curve ({Delta}G vs T) is given by –{Delta}Cp/T, a smaller {Delta}Cp is responsible for a shallower stability curve. Ts is the temperature at which {Delta}G° is maximum, i.e. {Delta}S = 0. {Delta}Hs is the change in enthalpy at Ts and is equal to {Delta}Gs, the maximum of {Delta}G°. In Figure 2Go, the stability curves obtained from the fits are also indicated by broken lines for {Delta}G1° and {Delta}G2° and by continuous lines for {Delta}Gt°. {Delta}Gt° of Ec-IPMDH had a maximum of 32.7 kcal/mol at 298 K and {Delta}Cp was 20.7 kcal/mol.K. The {Delta}Gt° curve of Ec-IPMDH showed that the enzyme is stable between 267 and 328 K. The overall stability {Delta}Gt° of Tt-IPMDH showed a maximum (15.8 kcal/mol) at 304 K. {Delta}Cp upon overall unfolding was 1.73 kcal/mol.K. From the stability curve, Tt-IPMDH folds stably between 251 and 366 K.


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Table I. Thermodynamic parameters derived from the stability curves of Escherichia coli and Thermus thermophilus IPMDHs shown in Figure 2Go
 
The curve of {Delta}Gt° of Tt-IPMDH was flatter than that of Ec-IPMDH, which was due to its smaller {Delta}Cp. Accordingly, the {Delta}Gt° values of Tt-IPMDH remained positive at higher and lower temperatures, even though the maximum {Delta}Gt° of 15.8 kcal/mol was much smaller than that of Ec-IPMDH. In addition, Ts of Tt-IPMDH was 6 K higher than that of Ec-IPMDH, although the difference was smaller than the difference in the melting temperatures upon thermal unfolding, 20 K (Wallon et al., 1996Go).


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In our previous work, we analyzed the urea-induced unfolding of Ec-IPMDH and Tt-IPMDH at 300 K (Motono et al., 1999Go). Urea-induced unfolding of Ec-IPMDH and Tt-IPMDH was reversible and reached equilibrium in 24 and 48 h, respectively. Ec-IPMDH showed a biphasic unfolding process when monitored by CD at 222 nm or by intrinsic fluorescence and both curves coincided with each other. The unfolding data were fitted well by the three-state model (Equation 1Go). The intermediate state retained about half of the native CD signal and half of the native fluorescence intensity. The protein concentration dependence of the unfolding curve suggested that the intermediate retained a dimeric structure. The intermediate of Ec-IPMDH seemed to be a dimeric molecule with a partially unfolded structure.

The unfolding data of Tt-IPMDH could be fitted by both two- and three-state models equally well. However, the characteristics of the intermediate in the three-state model were considerably different from those of Ec-IPMDH. The intermediate retained most of the CD signal, fluorescence intensity and enzymatic activity of the native state enzyme. In this work, the two-state model was employed for the analysis of Tt-IPMDH.

We analyzed the temperature dependence of the urea-induced unfolding of both IPMDHs. The analysis revealed a higher temperature dependence of the unfolding curve of Ec-IPMDH. The higher temperature dependence was ascribed to a larger {Delta}Cp of Ec-IPMDH. Especially the first phase of the unfolding curve was responsible for the larger {Delta}Cp of the mesophilic enzyme. Although {Delta}Gt° maximum of Ec-IPMDH far surpassed that of Tt-IPMDH, {Delta}Gt° of Ec-IPMDH dropped to zero at a lower temperature than did Tt-IPMDH.

Mechanisms of higher stability of thermophilic proteins relative to mesophilic counterparts

Nojima et al. proposed three thermodynamic mechanisms to explain the higher denaturation temperature of thermophilic proteins relative to that of mesophilic counterparts (Nojima et al., 1977Go). In Figure 3Go, three possible curves for thermophilic proteins are compared with a stability curve for a mesophilic counterpart, which is represented by curve M. Curve A shows a `higher stability' model, as termed by Beadle et al. (Beadle et al., 1999Go): the overall increase in the free energy change of a thermophilic protein by shifting the curve upward results in a higher thermal denaturation temperature and a lower cold denaturation temperature. Curve B shows a `flattened' model: a lower {Delta}Cp of a thermophilic protein leads to a flattened stability curve, resulting in a higher thermal denaturation temperature and a lower cold denaturation temperature. Curve C shows a `shifted' model: the free energy profile of a thermophilic protein is shifted horizontally to a higher temperature, resulting in an increase in stability at higher temperature and a decrease in stability at lower temperature to enhance the possibility of cold denaturation. Such hypothetical mechanisms have been well documented also by other authors (McCrary et al., 1996Go; Beadle et al., 1999Go). Thus, the temperature dependence of the free energy of unfolding provides a suitable approach for characterizing thermophilic protein stability.



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Fig. 3. Three hypothetical models to explain the higher thermal stability of thermophilic proteins. Curve M depicts the dependence of {Delta}G on temperature for a typical mesophilic protein. Curve A shows an overall increase in {Delta}G of a thermophilic protein. In curve B, {Delta}G of a thermophilic protein is less dependent on temperature, resulting from a lower {Delta}Cp. This mechanism causes a higher Tm and a lower temperature of cold denaturation. Curve C results from a shift of the stability curve laterally to a higher temperature. All the curves were calculated based on the Gibbs–Helmholtz equation (Equation 6Go). The parameters {Delta}Hs, {Delta}Cp and Ts were 5 kcal/mol, 2.5 kcal/mol.K and 298 K for curve M; 13 kcal/mol, 2.5 kcal/mol.K and 298 K for curve A; 13 kcal/mol, 2.5 kcal/mol.K and 298 K for curve B; and 5 kcal/mol, 2.5 kcal/mol.K and 318 K for curve C, respectively.

 
As the first successful work, Nojima et al. studied the reversible thermal unfolding of phosphoglycerate kinases (PGK) from T.thermophilus and from yeast and compared the thermodynamic properties of the thermophilic and the mesophilic enzymes (Nojima et al., 1977Go). Stabilization of the thermophilic PGK was mainly explained by a small {Delta}Cp of unfolding: the flattened model (curve B). This mechanism has also been observed in Sac7d from a hyperthermophile, Sulfolobus acidocaldarius (McCrary et al., 1996Go), and in Sso7d from Sulfolobus solfataricus (Knapp et al., 1996Go). {Delta}Cp of 2.86 kcal/mol.K of trimeric adenylate kinase from a hyperthermophile, S.acidocaldarius, is also small for its protein size (Backmann et al., 1998Go). As clearly shown in Figure 2A and BGo, the high thermostability of Tt-IPMDH was primarily explained by the same mechanism as for T.thermophilus PGK. {Delta}Cp of Tt-IPMDH was significantly lower than that of Ec-IPMDH (Table IGo).

The mechanism represented in curve A has been reported for some hyperthermophilic proteins (Hiller et al., 1997Go; Grättinger et al., 1998Go). The higher stability model is also involved in stabilization of PGK from T.thermophilus to some extent (Nojima et al., 1977Go). Aspartate aminotransferase from S.solfataricus also achieves a higher Tm than its analogue from pig heart by a combination of a higher {Delta}G° and a smaller {Delta}Cp (Arnone et al., 1997Go). More recently, Beadle et al. clearly showed that higher thermal stability of the catalytic domain of cellulase E2 from Thermomonospora fusca is due to its larger {Delta}G° (Beadle et al., 1999Go). Our present results, however, demonstrated that an increased Tm is not necessarily accompanied by an increased thermodynamic stability {Delta}G°. {Delta}Gt° of Tt-IPMDH at its maximum (15.8 kcal/mol) was smaller than that of the mesophilic counterpart, 32.7 kcal/mol (Figure 2A and BGo). In spite of the smaller stability at its maximum, the lower {Delta}Cp of Tt-IPMDH led to a shallower stability curve, resulting in an enhanced Tm.

Tt-IPMDH has a lower {Delta}Cpthan the predicted value

{Delta}Cp of Ec-IPMDH was 20.7 kcal/mol.K. {Delta}Cp of Tt-IPMDH was much smaller, 1.73 kcal/mol.K. The {Delta}Cp associated with protein unfolding has been related primarily to the change in exposure of hydrophobic residues to water and to the change in the exposure of the polar backbone (Baldwin, 1986Go; Livingstone et al., 1991Go; Murphy and Freire, 1992Go). Myers et al. suggested that there is a linear correlation between the change in total accessible surface area upon unfolding, {Delta}ASA and {Delta}Cp (Myers et al., 1995Go). Data from 45 proteins indicate the following correlation:

(6)

{Delta}ASA values of both IPMDHs were calculated using a similar method to that of Myers et al., which is based on a uniform unfolded structure (extended ß-strand) (Myers et al., 1995Go). {Delta}ASA of Ec-IPMDH dimer was 78 014 Å2, giving a {Delta}Cp of 15.5 kcal/mol.K calculated from Equation 6Go . {Delta}ASA of Tt-IPMDH was 73 591 Å2, which predicted a {Delta}Cp of 14.6 kcal/mol.K. In Figure 4Go, the dependence of {Delta}Cp on {Delta}ASA of the 45 mesophilic proteins in the data set from Myers et al. (Myers et al., 1995Go) are shown with our experimental {Delta}Cp values for Ec-IPMDH and Tt-IPMDH. The linear correlation in Equation 6Go, which is shown in Figure 4Go, has been found only for small globular proteins ({Delta}ASA <35 000 Å2) that undergo a simple two-state unfolding mechanism. It has not been established that Equation 6Go holds for larger oligomeric proteins, thermophilic proteins or proteins that unfold in a multi-state manner. The experimental {Delta}Cp value for Ec-IPMDH was 5.2 kcal/mol.K higher than the value predicted using Equation 6Go, but did not deviated much from that relationship. Accordingly, the three-state unfolding probably does not have much effect on the {Delta}Cp between the native and the unfolded states. However, the measured {Delta}Cp upon the two-state denaturation of Tt-IPMDH, 1.73 kcal/mol.K, was much smaller than {Delta}Cp calculated from Equation 6Go, 14.6 kcal/mol.K, and clearly out of the correlation. A similar case has been reported for adenylate kinase from a hyperthermophile, S.acidocaldarius (Backmann et al., 1998Go). The measured {Delta}Cp of the adenylate kinase is 2.86 kcal/mol.K and is much smaller than the value of 12.4 kcal/mol.K predicted from Equation 6Go. This suggests that the correlation of {Delta}Cp and {Delta}ASA (Equation 6Go) should be viewed with caution concerning thermophilic proteins.



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Fig. 4. Dependence of {Delta}Cp on {Delta}ASA. The line represents the correlation (Equation 6Go), obtained by the fit to the data in Myers et al. (Myers et al., 1995Go) and those data are also shown by open circles ({circ}). {Delta}Cp of Ec-IPMDH ({blacktriangleup}) and that of Tt-IPMDH ({blacksquare}) were derived from the fit of the {Delta}G° data set in Figure 2Go to the Gibbs–Helmholtz equation (Equation 3Go). {Delta}Cp of adenylate kinase from Sulfolobus acidocaldarius (Backmann et al., 1998Go) is shown by a filled circle (•). All the data are for mesophilic proteins except for Tt-IPMDH and adenylate kinase from S.acidocaldarius.

 
Possible explanation for the small {Delta}Cpof Tt-IPMDH

{Delta}Cp of Tt-IPMDH was significantly lower than the predicted value. Sac7d from S.acidocaldarius achieves its thermostability also with the flattened model and its {Delta}Cp value remains lower than that of the mesophilic proteins with the same size, although {Delta}Cp of the mesophilic homologue was not estimated (McCrary et al., 1996Go). As for Sac7d, however, the experimental {Delta}Cp was comparable to {Delta}Cp calculated from the predicted {Delta}ASA upon unfolding. The significantly lower value of {Delta}Cp of Tt-IPMDH suggests that the unfolded state of Tt-IPMDH is different from that of Ec-IPMDH and different from a uniformly extended ß-strand which we used as a model of the unfolded state. Incomplete unfolding may be responsible for the relatively low {Delta}Cp. However, we did not find any evidence that unfolded Tt-IPMDH retained some secondary structure, judged from the CD measurements. It has been suggested that oligomeric proteins tend to have a lower {Delta}Cp value (Karantza et al., 1996Go; Backmann et al., 1998Go). However, the smaller {Delta}Cp of Tt-IPMDH is not due to its oligomeric structure. {Delta}Cp of the mesophilic counterpart, Ec-IPMDH, which is also dimeric and has a very similar structure to Tt-IPMDH, is comparable to the predicted {Delta}Cp. It is not likely that the difference in the unfolding mechanism affected the {Delta}Cp of Tt-IPMDH because Tt-IPMDH showed two-state unfolding in contrast to Ec-IPMDH. It should be noted that {Delta} (and {Delta}Cp) solely depends on the native and the unfolded states, irrespective of the unfolding mechanism, because of the fundamental characteristics of the thermodynamic parameters. Therefore, it is more likely that the smaller {Delta}Cp is a feature of at least some thermophilic proteins.

The relation between {Delta}ASA and {Delta}Cp has been explained by the hydrophobic hydration of the residues increased in the unfolded state. Accordingly, the significantly lower {Delta}Cp than that estimated from the predicted {Delta}ASA suggests that the model of the unfolded state does not represent the real unfolded state of Tt-IPMDH. The unfolded state may retain substantial interaction of hydrophobic residues. The interaction between hydrophobic residues in the denatured state of barnase has been reported by NMR and molecular dynamics simulation (Wong et al., 2000Go). The simulated denatured state of barnase contains residual structure in the form of dynamic, fluctuating secondary structure, as well as fluctuating tertiary contacts, including ion pairs.

Another possibility to perturb the unfolded states is electrostatic interactions. Recent studies have shown that favorable long-range electrostatic interactions among the charged groups of a protein surface is the strategy more often used to stabilize thermophilic proteins, rather than ion pairing (Karshikoff and Ladenstein, 1998Go; Grimsley et al., 1999Go; Loladze et al., 1999Go; Xiao and Honig, 1999Go; Perl et al., 2000Go; Spector et al., 2000Go). These results also suggest that electrostatic interactions stabilizing the folded proteins stabilize the unfolded state, so that the measured net contribution of the electrostatic interactions to protein stability is smaller than expected (Pace, 2000Go). The unfolded polypeptide chains may rearrange to compact conformations that improve electrostatic interactions.

Thermal stability of Tt-IPMDH is increased also by the shifted model

An additional reason for an increased Tm of Tt-IPMDH was a shift of the stability curve to higher temperature. The temperature at which {Delta}Gt° is maximum was 304 K for Tt-IPMDH, which was 6 K higher than that for Ec-IPMDH. The stability curve of the thermophilic enzyme is shifted horizontally to higher temperature, resulting in increased stability at higher temperature and an enhanced possibility of cold denaturation at lower temperature. At present, there is no report of a thermophilic protein that achieves higher thermostability solely with this mechanism. Hyperthermophilic rubredoxin, however, seems to achieve its higher Tm beyond 200°C partly with this shifted mechanism (Hiller et al., 1997Go).

In summary, from the quantitative comparison of the thermodynamic behavior of thermophilic IPMDH with that of its mesophilic counterpart, two thermodynamic mechanisms for protein stabilization were elucidated. The main stabilizing mechanism of the thermophilic enzyme was its lower dependence of {Delta}G° on temperature, resulting from its smaller {Delta}Cp. The observed {Delta}Cp of the thermophilic IPMDH was much smaller than the predicted value, which was calculated using its native structure and uniformly extended structure as an unfolded state. This discrepancy between the observed and predicted {Delta}Cp implies that the unfolded Tt-IPMDH retained some interactions among residues. Another stabilizing factor was a shift of the most stable temperature to higher temperature.


    Notes
 
1 To whom correspondence should be addressed.E-mail: yamagish{at}ls.toyaku.ac.jp Back


    Acknowledgments
 
This work was supported by the Proposal-Based R&D Program of the New Energy and Industrial Technology Development Organization (NEDO).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aguilar,C.F., Sanderson,I., Moracci,M., Ciaramella,M., Nucci,R., Rossi,M. and Pearl,L.H. (1997) J. Mol. Biol., 271, 789–802.[ISI][Medline]

Arnone,M.I., Birolo,L., Pascarella,S., Cubellis,M.V., Bossa,F., Sannia,G. and Marino,G. (1997) Protein Eng., 10, 237–248.[Abstract]

Backmann,J., Schäfer,G., Wyns,L. and Bönisch,H. (1998) J. Mol. Biol., 284, 817–833.[ISI][Medline]

Baldwin,R. (1986) Proc. Natl Acad. Sci. USA, 83, 8069–8072.[Abstract]

Beadle,B.M., Baase,W.A., Wilson,D.B., Gilkes,N.R. and Shoichet,B.K. (1999) Biochemistry, 38, 2750–2756.

Becktel,W.J. and Schellman,J.A. (1987) Biopolymers, 26, 1859–1877.[ISI][Medline]

Britton,K.L., Baker,P.J., Morges,K.M.M., Engel,P.C., Pasquo,A., Rice,D.W., Robb,F.T., Scandurra,R., Stillman,T. and Yip,K.S.P. (1995) Eur. J. Biochem., 229, 688–695.[Abstract]

Burley,S.K. and Petsko,G.A. (1985) Science, 229, 23–28.[ISI][Medline]

Daggett,V. and Levitt,M. (1993) J. Mol. Biol., 232, 600–619.[ISI][Medline]

Fujinaga,M., Berthet-Colominas,C., Yaremchuck,A.D., Tukalo,M.A. and Cusack,S. (1993) J. Mol. Biol., 234, 222–233.[ISI][Medline]

Grättinger,M., Dankersreiter,A., Schurig,H. and Jaenicke,R. (1998) J. Mol. Biol., 280, 525–533.[ISI][Medline]

Grimsley,G.R., Shaw,K.L., Fee,L.R., Alston,R.W., Huyghues-Despointes,B.M., Thurlkill,R.L., Scholtz,J.M. and Pace,C.N. (1999) Protein Sci., 8, 1843–1849.[Abstract]

Hardy,F., Vreind,G., van der Vinne,B., Frigerio,F., Grandi,G., Rnema,G. and Eijsink,V.G.H. (1994) Protein Eng., 7, 425–430.[Abstract]

Hering,T., Yutani,K., Inaka,K., Kuroki,R., Matsushima,M. and Kikuchi,M. (1992) Biochemistry, 31, 7077–7085.[ISI][Medline]

Hiller,R., Zhou,Z.H., Adams,M.W.W. and Englander,S.W. (1997) Proc. Natl Acad. Sci. USA, 94, 11329–11332.[Abstract/Free Full Text]

Imada,K., Sato,M., Tanaka,N., Katsube,Y., Matsuura,Y. and Oshima,T. (1991) J. Mol. Biol., 222, 725–738.[ISI][Medline]

Karantza,V., Freire,E. and Moudrianakis,E.N. (1996) Biochemistry, 35, 2037–2046.[ISI][Medline]

Karshikoff,A. and Ladenstein,R. (1998) Protein Eng., 11, 867–872.[Abstract]

Kirino,H., Aoki,M., Aoshima,M., Hayashi,Y., Ohba,M., Yamagishi,A., Wakagi,T. and Oshima,T. (1994) Eur. J. Biochem., 200, 275–281.

Knapp,S., Karshikoff,A., Berndt,K.D., Christova,P., Atanasov,B. and Ladenstein,R. (1996) J. Mol. Biol., 264, 1132–1144.[ISI][Medline]

Korndörfer,I., Steipe,B., Huber,R., Tomschy,A. and Jaenicke,R. (1995) J. Mol. Biol., 246, 511–521.[ISI][Medline]

Leszczynski,J. and Rose,G.D. (1986) Science, 234, 849–855.[ISI][Medline]

Livingstone,J., Spolar,R. and Record,T. (1991)Biochemistry, 30, 4237–4244.[ISI][Medline]

Loladze,V.V., Ibarra-Molero,B., Sanchez-Ruiz,J.M. and Makhatadze,G.I. (1999) Biochemistry, 38, 16419–16423.[ISI][Medline]

McCrary,B.S., Edmondson,S.P. and Shriver,J.W. (1996) J. Mol. Biol., 264, 784–805.[ISI][Medline]

Motono,C., Yamagishi,A. and Oshima,T. (1999) Biochemistry, 38, 1332–1337.[ISI][Medline]

Murphy,K.P. and Freire,E. (1992) Adv. Protein Chem., 43, 313–361.[ISI][Medline]

Myers,J., Pace,C.N. and Scholtz,J.M. (1995) Protein Sci., 4, 2138–2148.[Abstract/Free Full Text]

Nojima,H., Ikai,A., Oshima,T. and Noda,H. (1977) J. Mol. Biol., 116, 429–442.[ISI][Medline]

Pace,C.N. (2000) Nature Struct. Biol., 7, 345–346.[ISI][Medline]

Perl,D., Mueller,U., Heinemann,U. and Shimid,F.X. (2000) Nature Struct. Biol., 7, 380–383.[ISI][Medline]

Privalov,P.L. (1979) Adv. Protein Chem., 33, 167–241.[Medline]

Richards,F.M. (1977) Annu. Rev. Biophys. Bioeng., 6, 151–176.[ISI][Medline]

Russell,R.J.M., Hough,D.W., Danson,M.J. and Taylor,G.L. (1994) Structure, 2, 1157–1167.[ISI][Medline]

Serrano,L. and Fersht,A.R. (1989) Nature, 342, 296–299.[ISI][Medline]

Shortle,D. (1992) Q. Rev. Biophys., 25, 205–250.[ISI][Medline]

Spector,S. Wang,M., Carp,S.A., Robblee,J., Hendsch,Z.S., Fairman,R., Tidor,B. and Raleigh,D.P. (2000) Biochemistry, 39, 872–879.[ISI][Medline]

Wallon,G., Kirino-Kagawa,H., Yamagishi,A., Yamamoto,K., Lovett,S.T., Petsko,G.A. and Oshima,T. (1996) Biochim. Biophys. Acta, 1337, 105–112.[ISI]

Wallon,G., Kryger,G., Lovett,S.T., Oshima,T., Ringe,D. and Petsko,A. (1997) J. Mol. Biol., 266, 1016–1031.[ISI][Medline]

Wong,K.B., Clarke,J., Bond,C.J., Neira,J.L., Freund,S.M., Fersht,A.R. and Dagget,V. (2000) J. Mol. Biol., 296, 1257–1282.[ISI][Medline]

Xiao,A. and Honig,B. (1999) J. Mol. Biol., 289, 1435–1444.[ISI][Medline]

Yamada,T., Akutsu,N., Miyazaki,K., Kakinuma,K., Yoshida,M. and Oshima,T. (1990) J. Biochem.(Tokyo), 108, 449–456.[Abstract]

Received May 24, 2001; revised August 20, 2001; accepted August 25, 2001.