Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Abstract |
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Keywords: heat capacity change/3-isopropylmalate dehydrogenase/protein unfolding/thermal stability/thermophilic protein
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Introduction |
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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., 1993; Russell et al., 1994
; Korndörfer et al., 1995
). Factors that have been shown to have an effect on the thermostability of proteins are salt links, hydrogen bonds (Shortle, 1992
), hydrophobic interactions, internal packing, stabilization of helices (Serrano and Fersht, 1989
), additional aromaticaromatic interactions (Burley and Petsko, 1985
) and a decrease in the main-chain flexibility (Leszczynski and Rose, 1986
; Hering et al., 1992
; Daggett and Levitt, 1993
; Hardy et al., 1994
). 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., 1995
; McCrary et al., 1996
; Aguilar et al., 1997
).
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., 1990). The polypeptide chain of a subunit is folded into two domains with similar folding topologies based on parallel
/ß motifs (Imada et al., 1991
).
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., 1994). The temperatures of the native environment of the organisms are 37 and 7580°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., 1996
). 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., 1997
). 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., 1999). 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,
G° (Motono et al., 1999
). The unfolding process of Ec-IPMDH could be fitted to a three-state transition (see Equation 1
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 2
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 1
), 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 G°, i.e. protein stability curve (Becktel and Schellman, 1987
), was obtained. Other thermodynamic parameters were estimated from these protein stability curves.
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Materials and methods |
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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., 1999). The concentration of the Tt-IPMDH protein was measured spectroscopically using an extinction coefficient of 30 400 at 280 nm (Yamada et al., 1990
). 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., 1999). 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 2), to obtain
G°. As reported previously (Motono et al., 1999
), we employed a three-state model for Ec-IPMDH:
| (1) |
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 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 Cp of protein unfolding can be taken as a constant within experimental error for a given protein (Privalov, 1979
). With this assumption,
G is given by a modified GibbsHelmholtz equation:
![]() | (3) |
![]() | (4) |
![]() | (5) |
The temperature dependence of Gt° was fitted to Equation 3
to obtain
Hs, Ts and
Cp.
ASA calculation
The change in solvent-accessible surface area upon unfolding of a protein, ASA, was calculated to predict the
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, 1977
). X-ray crystallographic structures of Ec-IPMDH and Tt-IPMDH (Imada et al., 1991
; Wallon et al., 1997
) were used as native proteins. The unfolded proteins were modeled as extended ß-strands using the Biopolymer module in InsightII.
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Results |
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Figure 1A 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 1B
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 2024 or 4448 h, respectively, to reach unfolding equilibrium. At this temperature range, both Ec-IPMDH and Tt-IPMDH unfolded reversibly with urea.
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The temperature dependence of the unfolding process of Ec-IPMDH was clearly different from that of Tt-IPMDH (Figure 1A). Each unfolding curve of Ec-IPMDH was biphasic, as reported in our previous work (Motono et al., 1999
). The curve at 300 K was well fitted to a three-state transition model (Equation 1
). 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 G° of Ec-IPMDH and Tt-IPMDH
To estimate the free energy change G° at each temperature, each unfolding curve of Ec-IPMDH (Figure 1A
) or Tt-IPMDH (Figure 1B
) was independently fitted to the three-state unfolding model (Equation 1
) or the two-state model (Equation 2
), respectively. The free energy change was plotted against temperature (Figure 2A and B
). For Ec-IPMDH,
G1° and
G2° are the free energy changes in the absence of urea for the first and the second transitions, respectively.
Gt° indicates the overall free energy change of a whole dimer, which is the sum of
G1° and
G2° for Ec-IPMDH.
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Discussion |
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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 Cp of Ec-IPMDH. Especially the first phase of the unfolding curve was responsible for the larger
Cp of the mesophilic enzyme. Although
Gt° maximum of Ec-IPMDH far surpassed that of Tt-IPMDH,
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., 1977). In Figure 3
, 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., 1999
): 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
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., 1996
; Beadle et al., 1999
). Thus, the temperature dependence of the free energy of unfolding provides a suitable approach for characterizing thermophilic protein stability.
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The mechanism represented in curve A has been reported for some hyperthermophilic proteins (Hiller et al., 1997; Grättinger et al., 1998
). The higher stability model is also involved in stabilization of PGK from T.thermophilus to some extent (Nojima et al., 1977
). Aspartate aminotransferase from S.solfataricus also achieves a higher Tm than its analogue from pig heart by a combination of a higher
G° and a smaller
Cp (Arnone et al., 1997
). 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
G° (Beadle et al., 1999
). Our present results, however, demonstrated that an increased Tm is not necessarily accompanied by an increased thermodynamic stability
G°.
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 B
). In spite of the smaller stability at its maximum, the lower
Cp of Tt-IPMDH led to a shallower stability curve, resulting in an enhanced Tm.
Tt-IPMDH has a lower Cpthan the predicted value
Cp of Ec-IPMDH was 20.7 kcal/mol.K.
Cp of Tt-IPMDH was much smaller, 1.73 kcal/mol.K. The
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, 1986
; Livingstone et al., 1991
; Murphy and Freire, 1992
). Myers et al. suggested that there is a linear correlation between the change in total accessible surface area upon unfolding,
ASA and
Cp (Myers et al., 1995
). Data from 45 proteins indicate the following correlation:
![]() | (6) |
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., 1995
).
ASA of Ec-IPMDH dimer was 78 014 Å2, giving a
Cp of 15.5 kcal/mol.K calculated from Equation 6
.
ASA of Tt-IPMDH was 73 591 Å2, which predicted a
Cp of 14.6 kcal/mol.K. In Figure 4
, the dependence of
Cp on
ASA of the 45 mesophilic proteins in the data set from Myers et al. (Myers et al., 1995
) are shown with our experimental
Cp values for Ec-IPMDH and Tt-IPMDH. The linear correlation in Equation 6
, which is shown in Figure 4
, has been found only for small globular proteins (
ASA <35 000 Å2) that undergo a simple two-state unfolding mechanism. It has not been established that Equation 6
holds for larger oligomeric proteins, thermophilic proteins or proteins that unfold in a multi-state manner. The experimental
Cp value for Ec-IPMDH was 5.2 kcal/mol.K higher than the value predicted using Equation 6
, but did not deviated much from that relationship. Accordingly, the three-state unfolding probably does not have much effect on the
Cp between the native and the unfolded states. However, the measured
Cp upon the two-state denaturation of Tt-IPMDH, 1.73 kcal/mol.K, was much smaller than
Cp calculated from Equation 6
, 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., 1998
). The measured
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 6
. This suggests that the correlation of
Cp and
ASA (Equation 6
) should be viewed with caution concerning thermophilic proteins.
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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
Cp value remains lower than that of the mesophilic proteins with the same size, although
Cp of the mesophilic homologue was not estimated (McCrary et al., 1996
). As for Sac7d, however, the experimental
Cp was comparable to
Cp calculated from the predicted
ASA upon unfolding. The significantly lower value of
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
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
Cp value (Karantza et al., 1996
; Backmann et al., 1998
). However, the smaller
Cp of Tt-IPMDH is not due to its oligomeric structure.
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
Cp. It is not likely that the difference in the unfolding mechanism affected the
Cp of Tt-IPMDH because Tt-IPMDH showed two-state unfolding in contrast to Ec-IPMDH. It should be noted that
G° (and
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
Cp is a feature of at least some thermophilic proteins.
The relation between ASA and
Cp has been explained by the hydrophobic hydration of the residues increased in the unfolded state. Accordingly, the significantly lower
Cp than that estimated from the predicted
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., 2000
). 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, 1998; Grimsley et al., 1999
; Loladze et al., 1999
; Xiao and Honig, 1999
; Perl et al., 2000
; Spector et al., 2000
). 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, 2000
). 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 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., 1997
).
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 G° on temperature, resulting from its smaller
Cp. The observed
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
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.
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Notes |
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Acknowledgments |
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Received May 24, 2001; revised August 20, 2001; accepted August 25, 2001.