Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability
2003; Elsevier BV; Volume: 278; Issue: 11 Linguagem: Inglês
10.1074/jbc.m210893200
ISSN1083-351X
AutoresKyoko Ogasahara, Masami Ishida, Katsuhide Yutani,
Tópico(s)Tryptophan and brain disorders
ResumoTryptophan synthase from hyperthermophile,Pyrococcus furiosus, was found to be a tetrameric form (α2β2) composed of α and β2subunits. 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 α and β subunits in the hyperthermophile protein were of the order of 108m−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 α subunit from the hyperthermophile was enhanced by 17 °C due to the formation of the α2β2 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. Tryptophan synthase from hyperthermophile,Pyrococcus furiosus, was found to be a tetrameric form (α2β2) composed of α and β2subunits. 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 α and β subunits in the hyperthermophile protein were of the order of 108m−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 α subunit from the hyperthermophile was enhanced by 17 °C due to the formation of the α2β2 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. 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 (1Jaenicke R. Bohm G. Methods Enzymol. 2001; 334: 458-469Google Scholar, 2Petsko G.A. Methods Enzymol. 2001; 334: 469-478Google Scholar, 19Jaenicke R. Schurig H. Beaucamp N. Ostendorp R. Adv. Protein Chem. 1996; 48: 181-269Google Scholar, 20Jaenicke R. Bohm G. Curr. Opin. Struct. Biol. 1998; 8: 738-748Google Scholar, 21Backmann J. Schafer G. Methods Enzymol. 2001; 334: 328-342Google Scholar, 22Rees D. Methods Enzymol. 2001; 334: 423-437Google Scholar). 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 (3Kohlhoff M. Dahm A. Hensel R. FEBS Lett. 1996; 383: 245-250Google Scholar, 4Beaucamp N. Hofmann A. Kellerer B. Jaenicke R. Protein Sci. 1997; 6: 2159-2165Google Scholar, 5Bell G.S. Russell R.J.M. Kohlhoff M. Hensel R. Danson M.J. Hough D.W. Taylor G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1419-1421Google Scholar, 6Walden H. Bell G.S. Russell R.J.M. Siebers B. Hensel R. Taylor G.L. J. Mol. Biol. 2001; 306: 745-757Google Scholar). Hyperthermophilic phosphoribosylanthranilate isomerase is dimeric, but the proteins from mesophilic organisms are monomeric (7Thoma R. Hennig M. Sterner R. Kirchner K. Structure. 2000; 8: 256-276Google Scholar). Hyperthermophilic lactate dehydrogenase exists as tetrameric or octameric forms (8Dames T. Ostendorp R. Ott M. Rutkat K. Jaenicke R. Eur. J. Biochem. 1996; 240: 274-279Google Scholar). Moreover, extra ion pairs or hydrophobic interactions have often been found in the subunit/subunit interface of proteins from hyperthermophiles (9Yip K.S.P. Stillman T.J. Britton K.L. Artymiuk P.J. Baker P.J. Sedelnikova S.E. Engel P.C. Pasquo A. Chiaraluce R. Consalvi V. Scandurra R. Rice D.W. Structure. 1995; 3: 1147-1158Google Scholar, 10Lim J.-H. Yu Y.G. Han Y.S. Cho S. Ahn B.-Y. Kim S.-H. Cho Y. J. Mol. Chem. 1997; 270: 259-274Google Scholar, 11Russell R.J.M. Ferguson J.M.C. Hough D.W. Danson M.J. Taylor G.L. Biochemistry. 1997; 36: 9983-9994Google Scholar, 12Auerbach G. Ostendorp R. Prade L. Korndorfer I. Dams T. Huber R. Jaenicke R. Structure. 1998; 6: 769-781Google Scholar, 13Villeret V. Clantin B. Tricot C. Legrain C. Roovers M. Stalon V. Glansdorff N. Van Beeumen J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2801-2806Google Scholar, 14Maes D. Zeelen J.P. Thanki N. Beaucamp N. Alvarez M. Thi M.H.D. Backmann J. Martial J.A. Wyns L. Jaenicke R. Wierenga R.K. Proteins Struct. Funct. Genet. 1999; 37: 441-453Google Scholar, 15Arnott M.A. Michael R.A. Thompson C.R. Hough D.W. Danson M.J. J. Mol. Biol. 2000; 304: 657-668Google Scholar, 16Tanaka H. Chinami M. Mizushima T. Ogasahara K. Ota M. Tsukihara T. Yutani K. J. Biochem. (Tokyo). 2001; 130: 107-118Google Scholar, 17Zhang X. Meining W. Fischer M. Bacher A. Ladenstein R. J. Mol. Biol. 2002; 306: 1099-1114Google Scholar, 18Ishikawa K. Matsui I. Payan F. Cambillau C. Ishida H. Kawarabayasi Y. Kikuchi H. Roussei A. Structure. 2002; 10: 877-886Google Scholar). 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 (19Jaenicke R. Schurig H. Beaucamp N. Ostendorp R. Adv. Protein Chem. 1996; 48: 181-269Google Scholar, 20Jaenicke R. Bohm G. Curr. Opin. Struct. Biol. 1998; 8: 738-748Google Scholar, 21Backmann J. Schafer G. Methods Enzymol. 2001; 334: 328-342Google Scholar, 22Rees D. Methods Enzymol. 2001; 334: 423-437Google Scholar). 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 α2β2 is a multifunctional and allosteric enzyme. This α2β2 complex has an αββα arrangement (23Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Google Scholar) and can be isolated as the α monomer and β2. The α and β2 subunits catalyze inherent reactions (for reviews, see Refs. 24Yanofsky C. Crawford I.P. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1972: 1-31Google Scholar, 25Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 49: 127-186Google Scholar, 26Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Google Scholar, 27Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172Google Scholar, 28Pan P. Woehl E. Dunn M.F. Trends Biochem. Sci. 1997; 22: 22-27Google Scholar). When the α and β2 subunits associate to form the α2β2 complex, the enzymatic activity of each subunit is enhanced by 1 to 2 orders of magnitude (for reviews, see Refs. 24Yanofsky C. Crawford I.P. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1972: 1-31Google Scholar, 25Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 49: 127-186Google Scholar, 26Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Google Scholar, 27Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172Google Scholar, 28Pan P. Woehl E. Dunn M.F. Trends Biochem. Sci. 1997; 22: 22-27Google Scholar). The α/β subunits interaction is important for the mutual activation of the each subunit in prokaryotic tryptophan synthase. We found that tryptophan synthase (PfTSase) 1The abbreviations used are: TSase, tryptophan synthase; PfTSase, EcTSase andStTSase, tryptophan synthase from P. furiosus,E. coli and S. thyhimurium, respectively; Pfα, Pfβ2, andPfα2β2, α, β2subunits and α2β2 complex ofPfTSase, respectively; Ecα, Ecβ2 andEcα2β2, α, β2subunits and α2β2 complex ofEcTSase, respectively; Stα, Stβ2 andStα2β2, α, β2subunits and α2β2 complex ofStTSase, respectively; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; PLP, pyridoxal 5′-phosphate; DTT, dithiothreitol 1The abbreviations used are: TSase, tryptophan synthase; PfTSase, EcTSase andStTSase, tryptophan synthase from P. furiosus,E. coli and S. thyhimurium, respectively; Pfα, Pfβ2, andPfα2β2, α, β2subunits and α2β2 complex ofPfTSase, respectively; Ecα, Ecβ2 andEcα2β2, α, β2subunits and α2β2 complex ofEcTSase, respectively; Stα, Stβ2 andStα2β2, α, β2subunits and α2β2 complex ofStTSase, respectively; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; PLP, pyridoxal 5′-phosphate; DTT, dithiothreitol fromP. furiosus was also composed of α2β2, and the enzymatic activities of the α and β2 subunits separated in their active forms were stimulated by the formation of the α2β2complexes as well as the reported mesophilic prokaryotic bacterial tryptophan synthases (for reviews, see Refs. 24Yanofsky C. Crawford I.P. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1972: 1-31Google Scholar, 25Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 49: 127-186Google Scholar, 26Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Google Scholar, 27Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172Google Scholar, 28Pan P. Woehl E. Dunn M.F. Trends Biochem. Sci. 1997; 22: 22-27Google Scholar). The thermal stability of the α subunit of PfTSase is remarkably higher than that from Escherichia coli (29Yamagata Y. Ogasahara K. Hioki Y. Lee S.J. Nakagawa A. Nakamura H. Ishida M. Kuramitsu S. Yutani K. J. Biol. Chem. 2001; 276: 11062-11071Google Scholar). 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 inPfTSase 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 α and β subunits inPfTSase was strong compared with that inEcTSase, leading to the enhanced stability of the protein and the high temperature adaptation of the tryptophan synthase function. The α subunit (Pfα) from P. furiosus was expressed in theE. coli strain JM109/pα1974 (30Ishida M. Oshima T. Yutani K. FEMS Microbiol. Lett. 2002; 216: 179-183Google Scholar) and purified as described previously (29Yamagata Y. Ogasahara K. Hioki Y. Lee S.J. Nakagawa A. Nakamura H. Ishida M. Kuramitsu S. Yutani K. J. Biol. Chem. 2001; 276: 11062-11071Google Scholar). Each of the genes of trpB andtrpBA from P. furiosus was transformed into theE. coli strain JM109 (30Ishida M. Oshima T. Yutani K. FEMS Microbiol. Lett. 2002; 216: 179-183Google Scholar). 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-β-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 Pfβ2, the precipitate with ammonium sulfate at 60% saturation was dissolved in 50 ml of 25 mmpotassium 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 mmpotassium 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, AmershamBiosciences) with a linear gradient of 25 to 200 mmpotassium phosphate buffer (pH 7.0) containing 5 mm EDTA and 1 mm DTT. For Pfα2β2, 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 (AmershamBiosciences) with a linear gradient of 10 to 500 mmpotassium 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, AmershamBiosciences) 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 purifiedPfβ2 andPfα2β2. The α subunit from E. coli was purified as already described (31Yutani K. Ogasahara K. Tsujita T. Kanemoto K. Matsumoto M. Tanaka S. Miyashita T. Matsushiro A. Sugino Y. Miles E.W. J. Biol. Chem. 1987; 262: 13429-13433Google Scholar). The β2 subunit (32Zhao G.-P. Somerville R.L. J. Biol. Chem. 1992; 267: 526-541Google Scholar) from E. coli was purified as already described (33Ogasahara K. Hiraga K. Ito W. Miles E.W. Yutani K. J. Biol. Chem. 1992; 267: 5222-5228Google Scholar). All the purified proteins showed a single band on SDS-PAGE. 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 1cm1% were 6.92 for Pfα, 10.18 forPfβ2 subunit, and 9.94 forPfα2β2. These values were determined based on protein assay by the Lowry method using bovine serum albumin as the standard protein. The concentrations ofEcα, Ecβ2, andEcα2β2 were determined using OD 1cm1% values 4.4 (34Ogasahara K. Yutani K. Suzuki M. Sugino Y. Nakanishi M. Tsuboi M. J. Biochem. (Tokyo). 1980; 88: 1733-1738Google Scholar), 6.5, and 6.0 (35Hathaway G.M. Crawford I.P. Biochemistry. 1970; 9: 1801-1808Google Scholar), respectively. 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 Pfα, 0.743 for Pfβ2, and 0.747 for Pfα2β2 were calculated from the amino acid compositions (36Durchschlag H. Hinz H.-J. Thermodynamic Data for Biochemistry and Biotechnology. Springer-Verlag, Berlin, Germany1986: 45-128Google Scholar). Analysis of the sedimentation equilibria was performed using the program XLAVEL (Beckman, version 2). Isothermal titration calorimetry (ITC) was performed using an Omega Isothermal Titration Calorimeter (Microcal, Northampton, MA). Prior to the measurements, the solutions of the α and β2 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 β2 subunit at a high concentration was injected into the 1.3155-ml sample cell containing the α 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 (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. Pfα, which consists of 248 residues and has a molecular weight of 27,500, is found to exist in a monomer form in solution (29Yamagata Y. Ogasahara K. Hioki Y. Lee S.J. Nakagawa A. Nakamura H. Ishida M. Kuramitsu S. Yutani K. J. Biol. Chem. 2001; 276: 11062-11071Google Scholar). Ultracentrifugation analysis was used to determine the association forms of the proteins translated by thetrpB and trpBA gens from P. furiosus, which were expressed in E. coli. The apparent molecular weights (M r app) at various pHs are shown in Fig. 1. The β chain is comprised of 388 residues and the calculated molecular weight is 42,500 (30Ishida M. Oshima T. Yutani K. FEMS Microbiol. Lett. 2002; 216: 179-183Google Scholar). TheM r app of the recombinant β was 84,000–88,000 in the pH region above 4.7, indicating that the β chain exists in a dimeric form (Pfβ2). TheM r app of the recombinant complex of α with β subunits was almost nearly equal to 2-fold (140,000) the calculated value for αβ around pH 7. These results show that tryptophan synthase from P. furiosus forms a complex of α2β2(Pfα2β2) as observed for prokaryotic tryptophan synthases from mesophiles (24Yanofsky C. Crawford I.P. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1972: 1-31Google Scholar, 25Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 49: 127-186Google Scholar, 26Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Google Scholar, 27Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172Google Scholar, 28Pan P. Woehl E. Dunn M.F. Trends Biochem. Sci. 1997; 22: 22-27Google Scholar) and from the hyperthermophile (37Tang X.-F. Ezaki S. Atomi H. Imanaka T. Eur. J. Biochem. 2000; 267: 6369-6377Google Scholar, 38Hettwer S. Sterner R. J. Biol. Chem. 2002; 277: 8194-8201Google Scholar). The M r app of thePfβ2 decreased with decreasing pH below 4.0, resulting in dissociation to a monomer at pH 3.0. As shown in Fig. 1, the M r app ofPfα2β2 decreased with decreasing pH between pH 5 and 4, although that of Pfβ2did not change. To examine the inherent feature of the interaction between Pfα andPfβ2 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 Pfα with Pfβ2 were estimated. The titration in this study was performed by injecting the β2 subunit into the α subunit, in the calorimetry cell, at various temperatures and pH 7.0, because the solubility ofPfα 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 (33Ogasahara K. Hiraga K. Ito W. Miles E.W. Yutani K. J. Biol. Chem. 1992; 267: 5222-5228Google Scholar, 39Hiraga K. Yutani K. Eur. J. Biochem. 1996; 240: 63-70Google Scholar). Fig.2 A displays the typical raw data for the calorimetric titration of the α subunit with the β2 subunit at 40 °C. The binding of Pfα with Pfβ2 was exothermic. In Fig.2 B the titration curves are plotted as the sum of the heat released by each injection, normalized by the concentration of the α subunit. The ITC titration curves for both PfTSase andEcTSase fitted well to a model of one set site (α + β ⇄ αβ) (Fig. 2 B) and permitted the extraction of the enthalpy change (ΔH) upon formation of the complex, the association constant (K), and the stoichiometry (n) (40Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Google Scholar). The Gibbs energy change (ΔG) and the entropy change (ΔS) upon the subunit association can be evaluated using the following equation, ΔG=−RT lnK=ΔH−TΔSEquation 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.Table IThermodynamic parameters of the association of the α subunit with the β subunit in PfTSase, EcTSase, and hybrid complexes between subunits from PfTSase and EcTSase obtained by ITC measurements at pH 7.0Temperature (°C)n(β/α)KΔHΔGΔSΔCp108m−1kJ/molJ/kmolkJ/kmolPfα/β association351.20.7−9.4−46.3119.6−1.96401.11.6−26.0−49.274.043.11.22.7−27.2−51.075.4451.23.1−33.2−51.758.3471.13.9−38.4−52.644.5501.14.0−43.9−53.228.0551.16.5−53.4−55.45.9601.29.5−58.7−57.3−4.3Ecα/β association211.50.0293−15.4−36.471.7−5.56251.40.0286−33.5−36.911.4301.50.0599−56.1−39.3−55.5351.50.0400−87.4−38.9−157.1371.50.0422−101.4−39.4−200.0371.40.0340−104.4−38.8−211.5381.50.0322−105.4−38.8−213.940.071.40.0356−129.0−39.3−286.3Hybrid α/β associationPfα/Ecβ2401.10.00470−24.0−34.032.0Ecα/Pfβ2400.70.00061−2.9−28.782.0Parameters obtained by ITC are represented per molar concentration of α subunit. Open table in a new tab Parameters obtained by ITC are represented per molar concentration of α subunit. The stoichiometry (molar ratio of β/α) of association betweenPfα and Pfβ was similar to unity and did not depend on temperature. The stoichiometry for EcTSase was 1.5. In a previous study in which Ecα is injected into theEcβ2 solution, the stoichiometry is 1.4 (33Ogasahara K. Hiraga K. Ito W. Miles E.W. Yutani K. J. Biol. Chem. 1992; 267: 5222-5228Google Scholar,39Hiraga K. Yutani K. Eur. J. Biochem. 1996; 240: 63-70Google Scholar). The deviation from unity may be due to a decrease in the binding ability of the β subunit with PLP, because both Ecα andEcβ2 showed a single band on SDS-PAGE (33Ogasahara K. Hiraga K. Ito W. Miles E.W. Yutani K. J. Biol. Chem. 1992; 267: 5222-5228Google Scholar). The K values were of the order of 108m−1 in the temperature region of 40–60 °C for PfTSase and of the order of 106m−1 in the temperature region of 20–40 °C for EcTSase (Table I and Fig.3 A). The K values of PfTSase were 2 orders higher than those ofEcTSase. The negative values of ΔH for the interaction between Pfα and Pfβ were smaller that those in EcTSase (Table I and Fig. 3 B). In both cases of PfTSase and EcTSase, the ΔH values linearly correlated with temperature (Fig.3 B). The heat capacity change (ΔCp) obtained from the slope of the linear correlation was estimated to be −1.96 and −5.56. kJ/K per mole of α subunit for PfTSase andEcTSase, respectively. Fig. 4shows the temperature dependences of ΔG and ΔS together with ΔH. In the case ofPfTSase, the summation of small values of −ΔHand −TΔS yielded the Gibbs energy (ΔG) for the subunit binding reaction. In contrast, forEcTSase, the large negative values of ΔH were compensated by using the large values of −TΔS, resulting in a smaller negative ΔG. The subunit association in PfTSase was characterized by a largeK, small negative ΔH, small negative ΔCp, and small ΔS in comparison withEcTSase.Figure 4Temperature dependences of enthalpy change, entropy change, and Gibbs energy change upon association of the α with β subunits at pH 7.0. A and B display the temperature dependence for PfTSase and EcTSase, respectively.Open triangles, ΔH; open circles, −TΔS; closed circles, ΔG.View Large Image Figure ViewerDownload (PPT) 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.5 A shows the DSC curves forPfα, Pfβ2, andPfα2β2 at pH 9.3–9.4. ThePfα exhibited a DSC curve with a single peak at 87.2 °C (curve a in Fig. 5 A). ForPfβ2, a major peak appeared at 112.2 °C accompanied by a minor broad peak at 94.6 °C (curve b in Fig. 5 A). It was confirmed that the major and minor peaks came from the holo-Pfβ2 and apo-Pfβ2 removing cofactor PLP, respectively. In the case of Pfα2β2, separate two peaks appeared at 104.6 and 112.5 °C (curve c in Fig.5 A). The peak on the higher temperature can be assigned to that coming from Pfβ2, because the peak temperature (112.5 °C) was quite similar to that ofPfβ2 alone (112.2 °C). Therefore, the peak temperature at the lower temperature could be considered to arise fromPfα. Table II lists the T d values of individual subunits in the isolated and complex forms, where T d values represent the peak temperature of DSC curves. TheT d value of Pfα alone was lower by 25 °C than that of Pfβ2. However, theT d value of Pfα was enhanced by 17.4 °C due to the complex formation. The T dvalue of Pfβ2 did not change due to the complex formation. On the other hand, the T dvalue of Ecα (53.0 °C) slightly increased by 1.7 °C due to the α2β2 complex formation at pH 8.4 (curves a and c in Fig. 5 B and TableII). The T d value ofEcβ2 (80.3 °C) at pH 8.2 did not change by complex formation (curves b and c in Fig.5 B and Table II). The stabilization of Pfα due to the complex formation might be correlated with a strong subunit association with a higher K value obtained by ITC. Remetaet al. (41Remeta D.P. Miles E.W. Ginsburg A. Pure Appl. Chem. 1995; 67: 1859-1866Google Scholar) have reported that the DSC curve ofStα2β2 at pH 8.0 showed two separate peaks at the denaturation temperatures of isolatedStα and Stβ2. Each of theT d values of Ecα andEcβ2 was similar to those of the reportedStα and Stβ2.T d values of Pfα andPfβ2 were drastically higher by 34.2 and 31.9 °C than those of Ecα andEcβ2, respectively.Table IIDenaturation temperature (Td) of α and β2 subunits in the isolated forms and in the α2β2 complexes for both P. furiosus and E. coli proteinsSubunitsT d(°C)ΔT d2-cThe differences in theT d values of individual subunits in the isolated and homo complex forms.(°C)Isolated form2-aFrom panels A andB in Fig. 5.Homo complex form2-aFrom panels A andB in Fig. 5.Hybrid complex form2-bFrom panel C in Fig. 5for the hybrid complexes between the Pf subunits andEc subunits.Pfα87.2 (pH 9.37)104.6 (pH 9.30)86.4 (pH 9.50)17.4Pfβ2112.2 (pH 9.30)112.5 (pH 9.30)111.6 (pH 9.49)0.3Ecα53.0 (pH 8.40)54.7 (pH 8.40)53.5 (pH 9.49)1.7Ecβ280.3 (pH 8.21)79.9 (pH 8.40)71.5 (pH 9.50)−0.4The T d values represent the peak temperature of the DSC profiles measured at scan rate of 1 °C/min.2-a From panels A andB in Fig. 5.2-b From panel C in Fig. 5for the hybrid complexes between the Pf subunits andEc subunits.2-c The differences in theT d values of individual subunits in the isolated and homo complex forms. Open table in a new tab The T d values represent the peak temperature of the DSC profiles measured at scan rate of 1 °C/min. To explore which of the α and (or) β subunits corresponds to the strong association in PfTSase, the interaction between the Pf subunits and Ecsubunits 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 Kvalue strongly decreased by 4 and 3 orders of magnitude for thePfβ2-Ecα andEcβ2-Pfα associations, respectively, compared with that for thePfα-Pfβ2 association. These results suggest that the conformation of the subunit interface inPfTSase differs from that in EcTSase. Fig. 5 C shows the DSC curves of the complexes with hetero subunits. The peak positions for bothPfα2 Ecβ2 andEcα2 Pfβ2 appeared at temperatures corresponding to each of the component subunits (TableII), i
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