Stimulated Interaction between alpha  and beta  Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability*

Kyoko OgasaharaDagger , Masami Ishida§, and Katsuhide Yutani||

From the Dagger  Institute for Protein Research, Osaka University, Suita City, Osaka 565-0871, the § Tokyo University of Fisheries, Konan Minato-ku, Tokyo 108-8477, and the  Kwansei Gakuin University, Graduate School of Sciences, Gakuen 2-1 Sanda City, Hyogo 667-1337, Japan

Received for publication, October 24, 2002, and in revised form, January 6, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (alpha 2beta 2) composed of alpha  and beta 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the alpha  and beta  subunits in the hyperthermophile protein were of the order of 108 M-1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the alpha  subunit from the hyperthermophile was enhanced by 17 °C due to the formation of the alpha 2beta 2 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyperthermophilic proteins, which retain the folded conformation and maximally express their function near the boiling point of water, have been the target of extensive studies on protein stabilization, folding, structure, and evolutionary aspects over the past decade. Much work has been done to determine the three-dimensional structures of hyperthermophile proteins and to identify the structural determinants of the enhanced stability. A comparison of the structures of proteins from hyperthermophiles with their mesophilic counterparts has led to a better understanding of several features of the hyperthermophile proteins (1, 2, 19-22). One of these is that several hyperthermophile proteins have structures with a higher degree of oligomerization compared with the mesophilic homologues. Triose phosphate isomerase from hyperthermophiles is found to be tetrameric in contrast to the dimeric form from mesophilic sources (3-6). Hyperthermophilic phosphoribosylanthranilate isomerase is dimeric, but the proteins from mesophilic organisms are monomeric (7). Hyperthermophilic lactate dehydrogenase exists as tetrameric or octameric forms (8). Moreover, extra ion pairs or hydrophobic interactions have often been found in the subunit/subunit interface of proteins from hyperthermophiles (9-18). On the bases of these observations, a hypothesis has been proposed that the higher order oligomerization of subunits and strong subunit association are potentially important for enhanced stability of hyperthermophile proteins (19-22). However, there are few studies that characterize the strength of the subunit association in the hyperthermophile proteins and quantitatively elucidate the correlation between the subunit association and stability. Elucidating the subunit association feature in hyperthermophile proteins is an important subject for understanding the mechanism of anomalous stability and of protein-protein recognition itself in oligomeric proteins. Isothermal titration calorimetry is a powerful method for thermodynamically assessing protein-protein interactions, which are especially useful for measuring association parameters. There has been little application of isothermal titration calorimetry to characterize subunit association in hyperthermophile proteins.

We are now focusing our attention on the subunit association in tryptophan synthase from the hyperthermophile, Pyrococcus furiosus, in connection with thermal stability. Prokaryotic tryptophan synthase (EC 4.2.1.20) with the subunit composition alpha 2beta 2 is a multifunctional and allosteric enzyme. This alpha 2beta 2 complex has an alpha beta beta alpha arrangement (23) and can be isolated as the alpha  monomer and beta 2. The alpha  and beta 2 subunits catalyze inherent reactions (for reviews, see Refs. 24-28). When the alpha  and beta 2 subunits associate to form the alpha 2beta 2 complex, the enzymatic activity of each subunit is enhanced by 1 to 2 orders of magnitude (for reviews, see Refs. 24-28). The alpha /beta subunits interaction is important for the mutual activation of the each subunit in prokaryotic tryptophan synthase. We found that tryptophan synthase (PfTSase)1 from P. furiosus was also composed of alpha 2beta 2, and the enzymatic activities of the alpha  and beta 2 subunits separated in their active forms were stimulated by the formation of the alpha 2beta 2 complexes as well as the reported mesophilic prokaryotic bacterial tryptophan synthases (for reviews, see Refs. 24-28). The thermal stability of the alpha  subunit of PfTSase is remarkably higher than that from Escherichia coli (29). Tryptophan synthase from hyperthermophiles is an attractive model system for seeking correlation between subunit association and stability.

In this report, to elucidate the subunit interaction feature in PfTSase in connection with thermal stability and tryptophan synthase from E. coli (EcTSase), the subunit association and thermal stability were measured by isothermal titration calorimetry and differential scanning calorimetry, respectively. The results revealed that the binding between the alpha  and beta  subunits in PfTSase was strong compared with that in EcTSase, leading to the enhanced stability of the protein and the high temperature adaptation of the tryptophan synthase function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of alpha , beta 2, and alpha 2beta 2 from P. furiosus-- The alpha  subunit (Pfalpha ) from P. furiosus was expressed in the E. coli strain JM109/palpha 1974 (30) and purified as described previously (29). Each of the genes of trpB and trpBA from P. furiosus was transformed into the E. coli strain JM109 (30). E. coli, harboring each of the genes, was grown in 15 liters of Luria-Bertani medium supplemented with ampicillin at 100 mg/liter culture medium at 37 °C. The expressions of trpB and trpBA were induced by isopropyl-beta -D(-)-thiogalactopyranoside added at a concentration of 1 mM to the culture medium 1 h after starting the culture. After culturing for 20 h, the cells were harvested and suspended in 100 ml of 20 mM potassium phosphate buffer (pH 7.0) containing 0.02 mM PLP, 1 mM EDTA, and 5 mM DTT. After sonication and heat treatment of the homogenized solution for 10 min at 75 °C, cell debris and denatured E. coli proteins were removed by centrifugation at 15,000 rpm for 30 min at 4 °C.

For Pfbeta 2, the precipitate with ammonium sulfate at 60% saturation was dissolved in 50 ml of 25 mM potassium phosphate buffer (pH 7.0) containing 0.02 mM PLP, 5 mM EDTA, and 1 mM DTT and dialyzed against the same buffer overnight at 4 °C. The dialyzed sample was applied on a column (2.5 × 27 cm) of DEAE-Sephacel (Amersham Biosciences) and eluted with a linear gradient of 25 to 500 mM potassium phosphate buffer (pH 7.0) containing 5 mM EDTA and 1 mM DTT. The active fractions of the eluted solutions were concentrated and applied to a gel filtration column (Superdex TM200 26/60, Amersham Biosciences) and separated using 25 mM potassium phosphate buffer (pH 7.0) containing 5 mM EDTA and 1 mM DTT. The collected active fractions were finally purified by ion exchange chromatography (Q Sepharose 26/10, Amersham Biosciences) with a linear gradient of 25 to 200 mM potassium phosphate buffer (pH 7.0) containing 5 mM EDTA and 1 mM DTT.

For Pfalpha 2beta 2, the precipitate with ammonium sulfate at 60% saturation was dissolved in 50 ml of 10 mM potassium phosphate buffer (pH 7.0) containing 0.02 mM PLP, 5 mM EDTA, and 1 mM DTT and dialyzed against the same buffer overnight at 4 °C. The sample was separated on a column (2.5 × 27 cm) of DEAE-Sephacel (Amersham Biosciences) with a linear gradient of 10 to 500 mM potassium phosphate buffer (pH 7.0) containing 5 mM EDTA and 1 mM DTT. Next, the collected active fractions were separated by gel filtration (Superdex TM200 26/60, Amersham Biosciences) and finally purified by ion exchange chromatography (Q Sepharose 26/10, Amersham Biosciences) with a linear gradient of 10 to 300 mM potassium phosphate buffer (pH 7.0) containing 5 mM EDTA and 1 mM DTT. PLP at a concentration of 0.1 mM was added to the solutions of the purified Pfbeta 2 and Pfalpha 2beta 2.

The alpha  subunit from E. coli was purified as already described (31). The beta 2 subunit (32) from E. coli was purified as already described (33). All the purified proteins showed a single band on SDS-PAGE.

Protein Concentrations-- The protein concentrations were estimated from the absorbance of the protein solution at pH 7.0 using a cell with a light path length of 1 cm. The values of OD<UP><SUB>1 cm</SUB><SUP>1%</SUP></UP> were 6.92 for Pfalpha , 10.18 for Pfbeta 2 subunit, and 9.94 for Pfalpha 2beta 2. These values were determined based on protein assay by the Lowry method using bovine serum albumin as the standard protein. The concentrations of Ecalpha , Ecbeta 2, and Ecalpha 2beta 2 were determined using OD<UP><SUB>1 cm</SUB><SUP>1%</SUP></UP> values 4.4 (34), 6.5, and 6.0 (35), respectively.

Ultracentrifugation Analysis-- Ultracentrifugation analysis was carried out in a Beckman Optima model XL-A. Sedimentation equilibrium experiments were performed at 20 °C using an An-60 Ti rotor at a speed of 7,000-32,000 × g. Before taking the measurements, the protein solutions were dialyzed overnight against the desired buffer at 4 °C. The experiments at three different protein concentrations between 1.8 and 0.5 mg/ml were run in Beckman 4-sector cells. The partial specific volumes of 0.751 cm3/g for Pfalpha , 0.743 for Pfbeta 2, and 0.747 for Pfalpha 2beta 2 were calculated from the amino acid compositions (36). Analysis of the sedimentation equilibria was performed using the program XLAVEL (Beckman, version 2).

Isothermal Titration Calorimetry-- Isothermal titration calorimetry (ITC) was performed using an Omega Isothermal Titration Calorimeter (Microcal, Northampton, MA). Prior to the measurements, the solutions of the alpha  and beta 2 subunits were dialyzed against 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.1 mM DTT, and 0.02 mM PLP. The dialyzed samples were filtered through a 0.22-µm pore size membrane and then degassed in a vacuum. A 10-µl volume of the beta 2 subunit at a high concentration was injected into the 1.3155-ml sample cell containing the alpha  subunit with a 170-s equilibration period between injections. Integration of the thermogram and the binding isotherm were analyzed using the ITC data analysis module in ORIGIN software (Microcal Software, Northampton, MA).

Differential Scanning Calorimetry-- Differential scanning calorimetry (DSC) was carried out using differential scanning microcalorimeters, VP-DSC (Microcal) and Nano-DSC II model 6100 (Calorimetry Science Corp.) at a scan rate of 1 °C/min. Prior to the measurements, the protein solution was dialyzed against buffer described in the legend of Fig. 5. The dialyzed sample was filtered through a 0.22-µm pore size membrane and then degassed in a vacuum. The protein concentrations during the measurements were 0.2-1.4 mg/ml.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Confirmation of Association States of Recombinant beta 2 and alpha 2beta 2 from P. furiosus-- Pfalpha , which consists of 248 residues and has a molecular weight of 27,500, is found to exist in a monomer form in solution (29). Ultracentrifugation analysis was used to determine the association forms of the proteins translated by the trpB and trpBA gens from P. furiosus, which were expressed in E. coli. The apparent molecular weights (Mr app) at various pHs are shown in Fig. 1. The beta  chain is comprised of 388 residues and the calculated molecular weight is 42,500 (30). The Mr app of the recombinant beta  was 84,000-88,000 in the pH region above 4.7, indicating that the beta  chain exists in a dimeric form (Pfbeta 2). The Mr app of the recombinant complex of alpha  with beta  subunits was almost nearly equal to 2-fold (140,000) the calculated value for alpha beta around pH 7. These results show that tryptophan synthase from P. furiosus forms a complex of alpha 2beta 2 (Pfalpha 2beta 2) as observed for prokaryotic tryptophan synthases from mesophiles (24-28) and from the hyperthermophile (37, 38). The Mr app of the Pfbeta 2 decreased with decreasing pH below 4.0, resulting in dissociation to a monomer at pH 3.0. As shown in Fig. 1, the Mr app of Pfalpha 2beta 2 decreased with decreasing pH between pH 5 and 4, although that of Pfbeta 2 did not change.


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Fig. 1.   pH dependence of apparent molecular weight of Pfbeta 2 and Pfalpha 2beta 2. The buffers used were Gly-HCl, acetate, potassium phosphate, and Gly-KOH for the pH ranges of 2-3, 3.7-5.0, 5.0-7.0, and 8.0-9.0, respectively. The apparent molecular weights (Mr app) of Pfalpha 2beta 2 below pH 5 were estimated by fixing the molecular weight (27,500) of Pfalpha using the software analyzing association system (Beckman). Closed circles and open triangles are the values of Pfalpha 2beta 2 and Pfbeta 2, respectively.

Binding Titration of alpha  with beta 2 Subunits by Isothermal Titration Calorimetry-- To examine the inherent feature of the interaction between Pfalpha and Pfbeta 2 in comparison with EcTSase, an isothermal titration calorimetry (ITC) was used in the absence of any substrates or ligands, and the thermodynamic parameters of the binding of Pfalpha with Pfbeta 2 were estimated. The titration in this study was performed by injecting the beta 2 subunit into the alpha  subunit, in the calorimetry cell, at various temperatures and pH 7.0, because the solubility of Pfalpha was not sufficient for making a solution with a high concentration at pH 7.0. This was contrary to the injection used in our previous studies (33, 39). Fig. 2A displays the typical raw data for the calorimetric titration of the alpha  subunit with the beta 2 subunit at 40 °C. The binding of Pfalpha with Pfbeta 2 was exothermic. In Fig. 2B the titration curves are plotted as the sum of the heat released by each injection, normalized by the concentration of the alpha  subunit. The ITC titration curves for both PfTSase and EcTSase fitted well to a model of one set site (alpha  + beta  right-left-arrows alpha beta ) (Fig. 2B) and permitted the extraction of the enthalpy change (Delta H) upon formation of the complex, the association constant (K), and the stoichiometry (n) (40). The Gibbs energy change (Delta G) and the entropy change (Delta S) upon the subunit association can be evaluated using the following equation,


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Fig. 2.   Typical isothermal titration calorimetry for the association of an alpha  subunit with a beta 2 subunit at 40.0 °C and pH 7.0. The 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM DTT, 1.0 mM EDTA, and 0.02 mM PLP was used for the experiments. A, raw data of titration for the subunit association monitored by ITC. Heat effects were recorded as a function of time with successive 10-µl injections of the beta 2 subunit into the sample cell containing the alpha  subunit. 1, Pfbeta 2 (0.160 mM as a beta  monomer) was injected into Pfalpha (0.015 mM); 2, Ecbeta 2 (0.174 mM as a beta  monomer) was injected into Ecalpha (0.0189 mM). B, integration of the thermogram yielded a binding isotherm that fits a model of one set site (alpha  + beta  right-left-arrows alpha beta ) by using variables K, n, and Delta H. Each number, 1 and 2, corresponds to that in panel A, respectively. The solid lines represent the nonlinear regression of the data points according to the model. The parameters, association constant (K), stoichiometry (n) (beta /alpha ), and enthalpy change (Delta H) upon formation of the alpha 2beta 2 complex obtained were 1.6 × 108 M-1, 1.1, and -26.0 kJ/mol of alpha  subunit, respectively, for PfTSase and 3.6 × 106 M-1, 1.4, and -129.0 kJ/mol of alpha  subunit, respectively, for EcTSase.


&Dgr;G=<UP>−</UP>RT <UP>ln</UP> K=&Dgr;H−T&Dgr;S (Eq. 1)
where T and R are the absolute temperature and the gas constant, respectively. The thermodynamic parameters for the subunit association at various temperatures are listed in Table I.

                              
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Table I
Thermodynamic parameters of the association of the alpha  subunit with the beta  subunit in PfTSase, EcTSase, and hybrid complexes between subunits from PfTSase and EcTSase obtained by ITC measurements at pH 7.0 
Parameters obtained by ITC are represented per molar concentration of alpha  subunit.

The stoichiometry (molar ratio of beta /alpha ) of association between Pfalpha and Pfbeta was similar to unity and did not depend on temperature. The stoichiometry for EcTSase was 1.5. In a previous study in which Ecalpha is injected into the Ecbeta 2 solution, the stoichiometry is 1.4 (33, 39). The deviation from unity may be due to a decrease in the binding ability of the beta  subunit with PLP, because both Ecalpha and Ecbeta 2 showed a single band on SDS-PAGE (33). The K values were of the order of 108 M-1 in the temperature region of 40-60 °C for PfTSase and of the order of 106 M-1 in the temperature region of 20-40 °C for EcTSase (Table I and Fig. 3A). The K values of PfTSase were 2 orders higher than those of EcTSase. The negative values of Delta H for the interaction between Pfalpha and Pfbeta were smaller that those in EcTSase (Table I and Fig. 3B). In both cases of PfTSase and EcTSase, the Delta H values linearly correlated with temperature (Fig. 3B). The heat capacity change (Delta Cp) obtained from the slope of the linear correlation was estimated to be -1.96 and -5.56. kJ/K per mole of alpha  subunit for PfTSase and EcTSase, respectively. Fig. 4 shows the temperature dependences of Delta G and Delta S together with Delta H. In the case of PfTSase, the summation of small values of -Delta H and -TDelta S yielded the Gibbs energy (Delta G) for the subunit binding reaction. In contrast, for EcTSase, the large negative values of Delta H were compensated by using the large values of -TDelta S, resulting in a smaller negative Delta G. The subunit association in PfTSase was characterized by a large K, small negative Delta H, small negative Delta Cp, and small Delta S in comparison with EcTSase.


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Fig. 3.   Temperature dependences of the association constant and enthalpy change upon association of the alpha  subunit with the beta  subunit at pH 7.0. A and B display the temperature dependence of K and Delta H, respectively. Closed and open circles denote the values for PfTSase and EcTSase, respectively. In panel B, the linear lines show the linear correlation between Delta H and temperature. Delta Cp values obtained from the slope of a line were -1.96 and -5.56 kJ/K mol of the alpha  subunit for PfTSase and EcTSase, respectively.


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Fig. 4.   Temperature dependences of enthalpy change, entropy change, and Gibbs energy change upon association of the alpha  with beta  subunits at pH 7.0. A and B display the temperature dependence for PfTSase and EcTSase, respectively. Open triangles, Delta H; open circles, -TDelta S; closed circles, Delta G.

Thermal Stability of Subunits Alone and the Complex-- To explore the relationship between the K values and the stability of PfTSase, the thermal stability of each subunit and complex was measured by differential scanning calorimetry (DSC). The DSC measurement was carried out in the alkaline region, because the proteins became turbid by heating at neutral pH and they do not form a complex in the acidic region (Fig. 1). Fig. 5A shows the DSC curves for Pfalpha , Pfbeta 2, and Pfalpha 2beta 2 at pH 9.3-9.4. The Pfalpha exhibited a DSC curve with a single peak at 87.2 °C (curve a in Fig. 5A). For Pfbeta 2, a major peak appeared at 112.2 °C accompanied by a minor broad peak at 94.6 °C (curve b in Fig. 5A). It was confirmed that the major and minor peaks came from the holo-Pfbeta 2 and apo-Pfbeta 2 removing cofactor PLP, respectively. In the case of Pfalpha 2beta 2, separate two peaks appeared at 104.6 and 112.5 °C (curve c in Fig. 5A). The peak on the higher temperature can be assigned to that coming from Pfbeta 2, because the peak temperature (112.5 °C) was quite similar to that of Pfbeta 2 alone (112.2 °C). Therefore, the peak temperature at the lower temperature could be considered to arise from Pfalpha . Table II lists the Td values of individual subunits in the isolated and complex forms, where Td values represent the peak temperature of DSC curves. The Td value of Pfalpha alone was lower by 25 °C than that of Pfbeta 2. However, the Td value of Pfalpha was enhanced by 17.4 °C due to the complex formation. The Td value of Pfbeta 2 did not change due to the complex formation. On the other hand, the Td value of Ecalpha (53.0 °C) slightly increased by 1.7 °C due to the alpha 2beta 2 complex formation at pH 8.4 (curves a and c in Fig. 5B and Table II). The Td value of Ecbeta 2 (80.3 °C) at pH 8.2 did not change by complex formation (curves b and c in Fig. 5B and Table II). The stabilization of Pfalpha due to the complex formation might be correlated with a strong subunit association with a higher K value obtained by ITC. Remeta et al. (41) have reported that the DSC curve of Stalpha 2beta 2 at pH 8.0 showed two separate peaks at the denaturation temperatures of isolated Stalpha and Stbeta 2. Each of the Td values of Ecalpha and Ecbeta 2 was similar to those of the reported Stalpha and Stbeta 2. Td values of Pfalpha and Pfbeta 2 were drastically higher by 34.2 and 31.9 °C than those of Ecalpha and Ecbeta 2, respectively.


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Fig. 5.   DSC curves of the alpha , beta 2, and alpha 2beta 2. The DSC measurements were performed at a scan rate of 1 K/min using VP-DSC. The buffer conditions were 10 mM Gly-KOH with 1 mM EDTA and 0.02 mM PLP. pH indicates the values after the measurements. All of the sample solutions after the measurements were not turbid. Panels (A), (B) and (C) display the DSC curves for the PfTSase, EcTSase and hybrid complexes between subunits from PfTSase and EcTSase, respectively. In panel (A), a, Pfalpha (0.824 mg/ml) at pH 9.37; b, Pfbeta 2 (0.688 mg/ml) at pH 9.30; c, Pfalpha 2beta 2 (0.613 mg/ml) at pH 9.30. In panel (B), a, Ecalpha (1.679 mg/ml) at pH 8.40; b, Ecbeta 2 (1.207 mg/ml) at pH 8.21; c, Ecalpha 2beta 2 (0.999 mg/ml) at pH 8.40. In panels (A) and (B), Cp values for beta 2 and alpha 2beta 2 were normalized by the molar concentration of the beta  monomer and alpha beta dimer, respectively. In panel (C), a, complex of Pfalpha with Ecbeta 2 at pH 9.50; b, complex of Pfbeta 2 with Ecalpha at pH 9.49. Cp values for hybrid complexes were normalized by the optical density of 1.0 at 280 nm of the sample solutions. The hybrid complexes were prepared by mixing equivalent moles of the alpha  and beta  subunits from PfTSase and EcTSase.

                              
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Table II
Denaturation temperature (Td) of alpha  and beta 2 subunits in the isolated forms and in the alpha 2beta 2 complexes for both P. furiosus and E. coli proteins

ITC and DSC Measurements of Hybrid Complex between Pf Subunits and Ec Subunits-- To explore which of the alpha  and (or) beta  subunits corresponds to the strong association in PfTSase, the interaction between the Pf subunits and Ec subunits was examined by ITC. The ITC data at 40 °C demonstrated that the K values upon formation of the hetero complex between the Pf subunits and Ec subunits were lower than those of the homo complexes (Table I). The K value strongly decreased by 4 and 3 orders of magnitude for the Pfbeta 2-Ecalpha and Ecbeta 2-Pfalpha associations, respectively, compared with that for the Pfalpha -Pfbeta 2 association. These results suggest that the conformation of the subunit interface in PfTSase differs from that in EcTSase.

Fig. 5C shows the DSC curves of the complexes with hetero subunits. The peak positions for both Pfalpha 2Ecbeta 2 and Ecalpha 2Pfbeta 2 appeared at temperatures corresponding to each of the component subunits (Table II), indicating that the interaction between the subunits in the hybrid complexes did not contribute to enhancing the thermal stability of the subunits in contrast to the Pfalpha 2beta 2 (Fig. 5A and Table II).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Conformational Change upon the Subunit Association of PfTSase-- The K values of PfTSase in the range from 40 to 60 °C were of the order of 108 M-1 and 2 orders higher than those of EcTSase in the range from 20 to 40 °C (Table I and Fig. 3A). This means that the interaction between the alpha  and beta  subunits from the hyperthermophile is extremely strong. The protein-protein association with a K value of 8 orders or over has been reported in the hen egg white lysozyme, its antibody (42-45), barnase-barstar (46), and transthyretin-retinol binding protein (47) interactions, which are highly specific for biological significance. The association between the subunits in PfTSase is equivalent to such a highly specific interaction.

The negative value of Delta Cp of the subunit association for PfTSase (-1.96 kJ/K mol of alpha ) was less than half of that for EcTSase (-5.56 kJ/K mol of alpha ) (Table I and Fig. 3B). In the cases of TSases from mesophiles, Hiraga and Yutani (39) have reported that a heat capacity change (Delta Cpest) is estimated to be -1.05 kJ/K mol from the values of the water-accessible nonpolar (Delta Anp) and polar (Delta Ap) surface areas buried upon subunit association in the alpha /beta subunit interface in the crystal structure of the StTSase complex. The negative values of Delta Cp experimentally obtained upon the subunit association for EcTSase and StTSase (-7.29 and -6.83 kJ/K mol, respectively) are much larger than the estimated one (-1.05 kJ/mol) mentioned above. It has been evaluated that this difference comes from the folding of many residues coupled to the alpha /beta subunit association in EcTSase and StTSase (39), according to the method of Spolar and Record (48). Delta Anp and Delta Ap in the alpha /beta interface for PfTSase complex can not be estimated, because the structure of the PfTSase complex has not yet been determined. However, the number of residues of local folding coupled to the subunit association in PfTSase might be postulated to be slight, because of the smaller negative Delta Cp (-1.96 kJ/K mol of alpha ). For a rigid body association in which a specific site is recognized by a "lock and key" interaction, an experimental Delta Cp value might be similar to the Delta Cpest predicted from Delta Anp and Delta Ap resulting from burial of the pre-existing complementary surface (48). Negative Delta Cp values of association corresponding to the concept of "induced fit" are larger than the negative Delta Cpest predicted from Delta Anp and Delta Ap. In this case, the folding of many residues is coupled to the association and creates key parts of the protein-protein, protein-ligand, and protein-DNA interface (48). According to this criteria, the subunit association in PfTSase resembles a rigid body association. In contrast, the subunit association in EcTSase corresponds to an "induced fit" with large conformational changes.

The smaller negative Delta H of the subunit association for PfTSase relative to that for EcTSase (Table I and Fig. 3B) also indicates that the conformational changes upon the subunit association are much smaller in PfTSase than in EcTSase, because the negative value of the enthalpy change due to protein folding is high (49). In PfTSase, the small values of negative Delta H and positive Delta S yield the negative value of Delta G for driving the subunit association (Fig. 4A). The association constant between the hetero subunits from PfTSase and EcTSase at 40 °C were drastically decreased relative to those for PfTSase and EcTSase, resulting in the decreases in the negative Delta G (Table I). The negative Delta H for the hetero subunit associations also decreased. These results indicate that the alpha  and beta  subunits of PfTSase cannot strongly bind to each of subunits from EcTSase. The thermodynamic parameters of the subunit associations revealed that the binding between the alpha  and beta  subunits was much tighter in PfTSase than in EcTSase, and the conformational change coupled with the subunit association was low in PfTSase.

Structural Bases of Strong Subunit Association in PfTSase-- The structures of the Stalpha 2beta 2 complex form (23, 50-52) and an isolated Pfalpha monomer (29) have been determined by x-ray analysis. We tried to explore the cause responsible for the strong subunit association in PfTSase from comparison of the structures of the subunit interfaces in Pfalpha and Tsalpha . The crystal structure of Pfalpha alone (29) is the same topological pattern to that of Stalpha in the Stalpha 2beta 2 complex form (23). Pfalpha , Stalpha , and Ecalpha consist of 248, 268, and 268 residues, respectively. The sequence identities between Pfalpha and Stalpha and between Ecalpha and Stalpha are 31.5 and 85.1%, respectively.

We can find out the differences in the two structures as follows. The loops 2 and 6 in the Stalpha , which play an important role in the catalysis and allosteric communication between the active sites of the alpha  and beta  subunits, contact with Stbeta (23, 50-52). The B-factor averaged for the main-chain atoms of the loop 2 in Pfalpha is considerably lower than that in Stalpha (29), indicating that the loop 2 is less mobile in Pfalpha than in Stalpha . The loop 6 of Stalpha is highly mobile and 12 residues in the loop 6 have not been determined due to a weak electron density (23). In Pfalpha , only three residues are not determined, although Pfalpha does not form a complex with the beta  subunit, indicating that the number of mobile residues in Pfalpha is drastically reduced (29). The amino acid residues of the loop 6 in Pfalpha exchange by polar to nonpolar, acidic to basic, or less to more hydrophobic residues from those in StTSase, although the amino acid sequence of the loop 2 is highly conserved in both alpha  subunits (Fig. 6). In other regions of the subunit interface in StTSase, six hydrogen bonds are formed (53). The corresponding residues in PfTSase are presented in Fig. 6. The hydrogen bonding residues in StTSase are not conserved in both the alpha  and beta  subunits in PfTSase except for the Asnalpha 104-Glybeta 292 pair in StTSase. The remarkable deviations in the root mean square deviations of the Calpha atoms between Pfalpha and Stalpha are found in the loop 2 and in the residues of Val119, Phe120, and His121 in Pfalpha (29), which are the residues in Stalpha forming hydrogen bonds with the residues in Stbeta . From these observations, it seems that the conformations of the subunit interface in Pfalpha substantially differ from those in Stalpha , and the rigidly ordered conformations of the loops 2 and 6 in Pfalpha might contribute to creating the key part of the interface responsible for the strong subunit association in PfTSase.


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Fig. 6.   The amino acid sequences for Pfalpha , Ecalpha and Stalpha in the interfaces of the alpha  and beta  subunits. Bolds show residues that differed from Stalpha . Residues numbers of Pfalpha and Stalpha are shown.

Correlation between Subunit Association Constant and Protein Stability-- For dimeric phosphoribosylanthranilate isomerase (7) and tetrameric pyrrolidone carboxyl peptidases (54) from hyperthermophiles and dimeric 3-isopropylmalate dehydrogenase from thermophiles (55), the experimental data have shown that the subunit interaction is important to the increase in thermal stability in solution. For these proteins, however, the relationship between subunit association and stability has not been quantitatively evaluated. Schellman (56, 57) has developed an equation for the relationship between the binding constant of a ligand to a biopolymer and the melting temperature of a biopolymer as follows,


&Dgr;T=T−T<SUB>0</SUB>=[(TT<SUB>0</SUB>R)/&Dgr;H°] <UP>ln</UP>(1+KL)<SUP>&Dgr;n</SUP> (Eq. 2)
where To and T are the melting temperatures in the absence and presence of a ligand, respectively, L is the ligand concentration, Delta H° is the denaturation enthalpy in the absence of a ligand, Delta n is the difference in the number of bound molecules of a ligand in the unfolded and folded states, K is a binding constant, and R is the gas constant. This equation is applicable only when the enthalpy change of binding is negligible compared with the enthalpy change of the transition (57). Because the Delta H values of the subunit association for PfTSase were small, although the values for EcTSase were large (Table I), we used Equation 2 to verify whether the enhancement in the Td value of Pfalpha in the complex form (Fig. 5 and Table II) is due to the large K value of the subunit association. In the present case, a biopolymer and a ligand are the alpha  and beta  subunits, respectively. Table III shows the values of Delta T and Delta n calculated for PfTSase from Equation 2 using the K values obtained at various temperatures. The Delta n values were estimated to be 0.9-1.2 for PfTSase. The Delta n values obtained are rational as a number of beta  subunits bound to alpha  subunits in the PfTSase. This indicates that Equation 2 is applicable for the calculation of Delta T in PfTSase. The Delta T values for Pfalpha calculated from Equation 2, assuming a Delta n of 1.0, were 15.6-19.5 °C. These values were near the experimentally obtained ones (Tables I and II and Fig. 5). If the K value is assumed to be 1.0 × 106 M-1, Delta T was only 4.9 °C. This agrees with the fact that the enhancement in Td of Pfalpha is due to the alpha 2beta 2 complex formation originating from the strong subunit association with a K value of the order of 108 M-1. Because the conformational change coupled to the subunit association in PfTSase was low as judged from the thermodynamic parameters for the subunit association, it was proved that the increase in Td of Pfalpha due to the complex formation is not due to the subunit binding-induced conformational stabilization of the subunits but due to the shift in the equilibrium toward the native state, which is caused by the increase in the association constant. This reveals that the enhancement in the thermal stability of subunit resulting from subunit association can be quantitatively evaluated by the subunit association constant.

                              
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Table III
The estimations of increment in denaturation temperature (Delta T) of Pfalpha due to Pfalpha 2beta 2 complex formation using the association constant (K) at different temperatures
Delta n is the molar ratio of the association (Pfbeta per Pfalpha ). Delta T and Delta n were evaluated according to Equation 2. In Equation 2, T0 and T are the denaturation temperatures of Pfalpha alone (360.35 K) and of Pfalpha (377.75 K) in Pfalpha 2beta 2 complex form, respectively (Fig. 5). L is the concentration (8.72 × 10-6 M) of monomer Pfbeta under DSC measurements.

The Td values of the Pfbeta monomer in the acidic region remarkably decreased with lower pH levels,2 suggesting the importance of the beta -beta subunit interaction for the higher stability of Pfbeta 2 compared with Pfalpha . The Td (80.3 °C) value of Ecbeta 2 at pH 8.4 was higher than that of Ecalpha and comparable to that of Pfalpha at pH 9.4 (Fig. 5, A and B). The interaction between the beta  subunits from the mesophile also enhances the stability of this dimeric protein.

Conclusion-- The present study proved four significant aspects of the subunit association in the PfTSase compared with EcTSase: 1) Pfalpha and Pfbeta 2 tightly bind by the K value of the order of 108 M-1; 2) The negative values of Delta Cp and Delta H on the subunit association were low, indicating that the conformational change coupled to the subunit association is low; 3) The Td of Pfalpha was drastically enhanced by 17 °C in the alpha 2beta 2 complex form; 4) This increment could be quantitatively evaluated from the remarkably increased K value. It was found that the stimulated interaction between the subunits with the order of 108 M-1 or over of K values remarkably enhances the thermal stability of a protein without conformational changes.

    FOOTNOTES

* This work was supported in part by a grant-in-aid for special project research from the Ministry of Education, Science, and Culture of Japan to K. Y.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Kwansei Gakuin University, Graduate School of Sciences, Gakuen 2-1 Sanda City, Hyogo 667-1337, Japan, Tel.: 81-795-65-8482, Fax: 81-795-65-9077, E-mail: yutani@ksc.kwansei.ac.jp.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210893200

2 K. Ogasahara, M. Ishida, and K. Yutani, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TSase, tryptophan synthase; PfTSase, EcTSase and StTSase, tryptophan synthase from P. furiosus, E. coli and S. thyhimurium, respectively; Pfalpha , Pfbeta 2, and Pfalpha 2beta 2, alpha , beta 2 subunits and alpha 2beta 2 complex of PfTSase, respectively; Ecalpha , Ecbeta 2 and Ecalpha 2beta 2, alpha , beta 2 subunits and alpha 2beta 2 complex of EcTSase, respectively; Stalpha , Stbeta 2 and Stalpha 2beta 2, alpha , beta 2 subunits and alpha 2beta 2 complex of StTSase, respectively; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; PLP, pyridoxal 5'-phosphate; DTT, dithiothreitol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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