1

Stimulated Interaction between αααα and ββββ Subunits of Tryptophan Synthase from

Hyperthermophile Enhances Its Thermal Stability.**

Kyoko Ogasahara1, Masami Ishida2, and Katsuhide Yutani3*

1Institute for Protein Research, Osaka University, Suita City, Osaka 565-0871, Japan,

2Tokyo University of Fisheries, Konan Minato-ku, Tokyo 108-8477, Japan, 3Kwansei

Gakuin University, Graduate School of Sciences, Gakuen 2-1 Sanda City, Hyogo

667-1337, Japan

Running Title: Subunit Interaction and Stability of Hyperthermophile Tryptophan

Synthase

Keywords: tryptophan synthase; hyperthermophile, subunit interaction; calorimetry;

DSC; ITC; thermal stability; subunit association constant.

__________________________________________________________________

**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.

*Correspondence to: Katsuhide Yutani, 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

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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1Abbreviations: TSase, tryptophan synthase; PfTSase, EcTSase and StTSase, tryptophan

synthase from P. furiosus, E. coli and S. thyhimurium, respectively; Pfα, Pfβ2, and

Pfα2β2, α, β2 subunits and α2β2 complex of PfTSase, respectively; Ecα, Ecβ2 and

Ecα2β2, α, β2 subunits and α2β2 complex of EcTSase, respectively; Stα, Stβ2 and Stα2β2,

α, β2 subunits and α2β2 complex of StTSase, respectively; ITC, isothermal titration

calorimetry; DSC, differential scanning calorimetry; PLP, pyridoxal 5'-phosphate; DTT,

dithiothreitol.

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SUMMARY

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found

to be a tetrameric form (α2β2) composed of α and β2-subunits. In order 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 E. coli. The association constants

between the α and β 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 α subunit from the

hyperthermophile was enhanced by 17 oC 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 to that of

the counterpart from E. coli.

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INTRODUCTION

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. A lot of efforts 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 thrown light upon several

features of the hyperthermophile proteins (for reviews, see refs 1, 2). One of them is

that several hyperthermophile proteins have structures with a higher degree of

oligomerization compared to the mesophilic homologues. Triose phosphate isomerase

(TIM) 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 (9-17). 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 (18-20). However, there are few studies

to characterize the strength of the subunit association in the hyperthermophile proteins

and also to 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

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is a powerful method to thermodynamically assess 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 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

multi-functional and allosteric enzyme. The α2β2 complex with an αββα arrangement

(21) can be isolated as the α monomer and β2. The α and β2 subunits catalyze inherent

reactions (for reviews, see refs 22-26). 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 22-26). 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) from Pyrococcus 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 α2β2 complexes as well as the reported

mesophilic prokaryotic bacterial tryptophan synthases (for reviews, see refs 22-26). The

thermal stability of the α subunit of PfTSase is remarkably higher than that from E. coli

(27). Tryptophan synthase from hyperthermophiles is an attractive model system for

seeking correlation between subunit association and stability.

In this study, in order to elucidate the subunit interaction feature in PfTSase in

connection of thermal stability compared with tryptophan synthase from Escherichia

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 in PfTSase was strong

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compared to that in EcTSase, leading to the enhanced stability of the protein and the

high temperature adaptation of the tryptophan synthase function.

EXPERIMENTAL PROCEDURS

Expression and Purification of αααα, ββββ2 and αααα2ββββ2 from P. furiosus-----The

α subunit (Pfα) from P. furiosus was expressed in the Echerichia coli strain

JM109/pα1974 (28) and purified as described (27). Each of the genes of trpB and

trpBA from P. furiosus was transformed into the E. coli strain JM109 (28). E. coli

harboring each of the genes was grown in 15 L of Luria-Bertani medium supplemented

with ampicillin at 100 mg per 1 L of culture medium at 37 oC. The expressions of trpB

and trpBA were induced by isopropyl-β-D(-)-thiogalactopyranoside added at a

concentration of 1 mM to the culture medium one hour after starting the culture. After

culturing for 20 hrs, 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

oC, cell debris and denatured E. coli proteins were removed by centrifugation at 15,000

rpm for 30 min at 4 oC.

For Pfβ2, the precipitate with ammonium sulfate at 40 % 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 oC. The

dialyzed sample was applied on a column (2.5x27 cm) of DEAE-Sephacel (Amersham

Pharmacia Biotech) and eluted with a linear gradient of 25 to 200 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 Pharmacia Biotech) and separated using 25 mM

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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 (SP

Sepharose 26/16, Amersham Pharmacia Biotech) with a linear gradient of 25 to 100

mM potassium 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

oC. The sample was separated on a column (2.5x27 cm) of DEAE-Sephacel (Amersham

Pharmacia Biotech) 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 Pharmacia

Biotech) and finally purified by ion exchange chromatography (SP Sepharose 26/16,

Amersham Pharmacia Biotech) 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 Pfβ2 and Pfα2β2.

The α subunit from E. coli was purified as already described (30). The β2 subunit

(31) from E. coli was purified as already described (32).

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 OD1%1cm were 6.92 for Pfα, 1.18 for Pfβ2 subunit, and 9.94 for Pfα2β2.

These values were determined based on protein assay by the Lowry method using

bovine serum albumin as the standard protein. The concentrations of Ecα, Ecβ2, and

Ecα2β2 were determined using OD1%1cm of 4.4 (33), 6.5 and 6.0 (34), respectively.

Ultracentrifugation Analysis---Ultracentrifugation analysis was carried out in a

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Beckman Optima model XL-A. Sedimentation equilibrium experiments were performed

at 20 oC using an An-60 Ti rotor at a speed of 7000-32,000 g. Before taking the

measurements, the protein solutions were dialyzed overnight against the desired buffer

at 4 oC. 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 (35). 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 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 ten-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-sec 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 micro-calorimeters, VP-DSC

(MicroCal, Northanpton) and Nano-DSC II model 6100 (Calorimetry Science Corp.) at

scan rate of 1 K/min. Prior to the measurements, the protein solution was dialyzed

against a buffer. 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 to 1.4 mg /ml.

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RESULTS

Confirmation of Association States of Recombinant ββββ2 and αααα2ββββ2 from P.

furiosus-----Pfα, which consists of 248 residues and has a molecular weight of 27.5 k,

is found to exist in a monomer form in solution (27). 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

(MWapp) at various pHs are shown in Fig. 1. The β chain is comprised of 388 residues

and the calculated molecular mass is 42.5 k (29). The MWapp of the recombinant β was

84 to 88 k in the pH region above 4.7, indicating that the β chain exists in a dimeric

form (Pfβ2). The MWapp of the recombinant complex of α with β subunits was almost

nearly equal to two-fold (140 k) the calculated value for αβ around pH 7. These results

show that tryptophan synthase from P. furiosus forms a complex of α2β2 (Pf(α2β2) as

observed for prokaryotic tryptophan synthases from mesophiles (23-25) and also from

the hyperthermophile (36, 37). The MWapp of the Pfβ2 decreased with decreasing pH

below 4.0, resulting in dissociation to a monomer at pH 3.0. As shown in Fig. 1, the

MWapp of Pfα2β2 decreased with decreasing pH between pH 5 and 4, although that of

Pfβ2 did not change.

Binding Titration of αααα with ββββ2 subunits by Isothermal Titration

Calorimetry-----To examine the inherent feature of the interaction between Pfα and

Pfβ2 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

injection of the β2 subunit into the α subunit in the calorimetry cell at various

temperatures and pH 7.0, because the solubility of Pfα was not sufficient for making a

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solution with a high concentration at pH 7.0. This was contrary in injection to our

previous studies (32, 38). Figure 2A displays the typical raw data for the calorimetric

titration of the α subunit with the β2 subunit at 40 oC. The binding of Pfα with Pfβ2 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 α subunit. The ITC titration

curves for both PfTSase and EcTSase fitted well to a model of one set site (α + β <->

αβ) (Fig. 2B) and permitted the extraction of the enthalpy change (∆H) upon formation

of the complex, the association constant (K), and the stoichiometry (n). (40). The Gibbs

energy change (∆G) and the entropy change (∆S) upon the subunit association can be

evaluated using the following equation,

∆G = - RT ln K = ∆H - T ∆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 1.

The stoichiometry (molar ratio of β/α) of association between Pfα 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 the Ecβ2 solution, the

stoichiometry is 1.4 (32, 38). The deviation from unity may be due to a decrease in the

binding ability of the β subunit with PLP, because both Ecα and Ecβ2 showed a single

band on SDS-PAGE (34). The K values were of the order of 108 M-1 in the temperature

region of 40 to 60 oC for PfTSase and of the order of 106 M-1 in the temperature region

of 20 to 40 oC for EcTSase (Table 1 and Fig. 3A). The K values of PfTSase were 2

orders higher than those of EcTSase. The negative values of ∆H for the interaction

between Pfα and Pfβ were smaller that those in EcTSase (Table 1 and Fig. 3B). In the

both cases of PfTSase and EcTSase, the ∆H values linearly correlated with temperature

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(Fig. 3B). The heat capacity change (∆Cp) obtained from the slope of the linear

correlation was estimated to be -1.96 and -5.56. kJ/K per mol of α subunit for PfTSase

and EcTSase, respectively. Figure 4 shows the temperature dependences of ∆G and ∆S

together with ∆H. In the case of PfTSase, the summation of small values of -∆H and

-T∆S yielded the Gibbs energy (∆G) for the subunit binding reaction. In contrast, for

EcTSase, the large negative values of ∆H were compensated by the large values of -T∆S,

resulting in a smaller negative ∆G. The subunit association in PfTSase was

characterized by a large K, small negative ∆H, small negative ∆Cp, and small ∆S in

comparison with EcTSase.

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).

Figure 5A shows the DSC curves for Pfα, Pfβ2 and Pfα2β2 at pH 9.3-9.4. The Pfα

exhibited a DSC curve with a single peak at 87.2 oC (curve a in Fig 5A). For Pfβ2, a

major peak appeared at 112.2 oC accompanied by a minor broad peak at 94.6 oC (curve

b in Fig 5A). 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 oC (curve c in Fig. 5A). The peak on the higher

temperature can be assigned to that coming from Pfβ2, because the peak temperature

(112.5 oC) was quite similar to that of Pfβ2 alone (112.2 oC). Therefore, the peak

temperature at the lower temperature could be considered to arise from Pfα. Table 2

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 Pfα alone was

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lower by 25 oC than that of Pfβ2. However, the Td value of Pfα was enhanced by 17.4

oC due to the complex formation. The Td value of Pfβ2 did not change due to the

complex formation. On the other hand, the Td value of Ecα (53.0 oC) slightly increased

by 1.7 oC due to the α2β2 complex formation at pH 8.4 (curves a and c in Fig. 5B, Table

2). The Td value of Ecβ2 (80.3 oC) at pH 8.2 did not change by complex formation

(curves b and c in Fig. 5B, Table 2). 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. Remeta et al. (40) have reported that the DSC curve of Stα2β2 at pH

8.0 showed two separate peaks at the denaturation temperatures of isolated Stα and Stβ2.

Each of the Td values of Ecα and Ecβ2 was similar to those of the reported Stα and

Stβ2.

Td values of Pfα and Pfβ2 were drastically higher by 32.5 and 31.9 oC than

those of Ecα and Ecβ2, respectively.

ITC and DSC Measurements of Hybrid Complex between Pf-Subunits and

Ec-Subunits -----To explore which of the α and (or) β 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 oC 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 Pfβ2-Ecα and Ecβ2-Pfα associations, respectively,

compared with that for the Pfα-Pfβ2 association. These results suggest that the

conformation of the subunit interface in PfTSase differs from that in EcTSase.

Figure 5C shows the DSC curves of the complexes with hetero subunits. The

peak positions for both the Pfα2Ecβ2 and Ecα2Pfβ2 appeared at temperatures

corresponding to each of the component subunits (Table 2), indicating that the

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interaction between the subunits in the hybrid complexes did not contribute to

enhancing the thermal stability of the subunits in contrast to the Pfα2β2 (Fig. 5A and

Table 2).

DISCUSSION

The Conformational Change upon the Subunit Association of PfTSase

-----The K values of PfTSase in the range from 40 to 60 oC were 8 orders and 2 orders

higher than those of EcTSase in the range from 20 to 40 oC (Table 1 and Fig. 3A). This

means that the interaction between the α and β 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 (41-44), barnase-barstar (45)

transthyretin-retinol binding protein (46) 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 ∆Cp of the subunit association for PfTSase (-1.96 kJ/K

mol of α) was less than half of that for EcTSase (-5.56 kJ/ K mol of α) (Table 1 and Fig.

3B). In the cases of TSases from mesophiles, Hiraga and Yutani (38) have reported that

a heat capacity change (∆Cpest) is estimated to be -1.05 kJ/K mol from the values of the

water accessible nonpolar (∆Anp) and polar (∆Ap) surface areas buried upon subunit

association in the α/β subunit interface in the crystal structure of the StTSase complex.

The negative values of ∆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 α/β subunit association in

EcTSase and StTSase (38), according to the method of Spolar and Record (47). ∆Anp

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and ∆Ap in the α/β 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 ∆Cp (-1.96 kJ/K mol of α). For a

rigid body association in which a specific site is recognized by a "lock and key"

interaction, an experimental ∆Cp value might be similar to the ∆Cpest predicted from

∆Anp and ∆Ap resulting from burial of the preexisting complementary surface (47).

Negative ∆Cp values of association corresponding to the concept of "induced fit" are

larger than the negative ∆Cpest predicted from ∆Anp and ∆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 (47). 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 ∆H of the subunit association for PfTSase relative to that

for EcTSase (Table 1 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 (48). In PfTSase,

the small values of negative ∆H and positive ∆S yield the negative value of ∆G for

driving the subunit association (Fig. 4A). The association constant between the hetero

subunits from PfTSase and EcTSase at 40 oC were drastically decreased relative to

those for PfTSase and EcTSase, resulting in the decreases in the negative ∆G (Table 1).

The negative ∆H for the hetero subunit associations also decreased. These results

indicate that the α and β subunits of PfTSase cannot strongly bind to each of subunits

from EcTSase.

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The thermodynamic parameters of the subunit associations revealed that the

binding between the α and β 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 Stα2β2 complex form (21, 49-51) and an isolated Pfα monomer (27) 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 Pfα and Tsα. The crystal structure of Pfα alone (27) is the same

topological pattern to that of Stα in the Stα2β2 complex form (21). Pfα, Stα and Ecα

consist of 248, 268 and 268 residues, respectively. The sequence identities between Pfα

and Stα and between Ecα and Stα 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 Stα, which play an important role in the catalysis and allosteric

communication between the active sites of the α and β subunits, contact with Stβ (21,

49-51). The B-factor averaged for the main-chain atoms of the loop 2 in Pfα is

considerably lower than that in Stα (27), indicating that the loop 2 is less mobile in Pfα

than in Stα. The loop 6 of Stα is highly mobile and 12 residues in the loop 6 have not

been determined due to a weak electron density (21). In Pfα, only 3 residues are not

determined, although Pfα does not form a complex with the β subunit, indicating that

the number of mobile residues in Pfα is drastically reduced (27). The amino acid

residues of the loop 6 in Pfα 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 α subunits (Fig. 6). In other regions of the subunit

interface in StTSase, six hydrogen bonds are formed (52). The corresponding residues

in PfTSase are presented in Fig. 6. The hydrogen bonding residues in StTSase are not

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conserved in both the α and β subunits in PfTSase except for the α104Asn-β292Gly

pair in StTSase. The remarkable deviations in the root mean square deviations (RMSD)

the of Cα atoms between Pfα and Stα are found in the loop 2 and in the residues of

119Val, 120Phe, and 121His in Pfα (27), which are the residues in Stα forming

hydrogen bonds with the residues in Stβ. From these observations, it seems that the

conformations of the subunit interface in Pfα substantially differ from those in Stα and

the rigidly ordered conformations of the loops 2 and 6 in Pfα might contribute to

creating the key part of the interface responsible for the strong subunit association in

PfTSase.

Correlation between Subunit Association Constant and Protein

Stability-----For dimeric phosphoribosylanthranilate isomerase (7) and tetrameric

pyrrolidone carboxyl peptidases (46) from hyperthermophiles, and also dimeric

3-isopropylmalate dehydrogenase from thermophiles (47), 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 has developed an equation for

the relationship between the binding constant of a ligand to a biopolymer and the

melting temperature of a biopolymer (56, 57) as follows.

∆T=T–To = [(TToR)/∆Ho] ln(1+KL)∆n (Eq. 2)

where To and T are the melting temperatures in the absence and presence of a ligand,

respectively, L is the ligand concentration, ∆Ho is the denaturation enthalpy in the

absence of a ligand, ∆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 to the enthalpy change of the transition (57). Since the ∆H values of the

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subunit association for PfTSase were small, although the values for EcTSase were large

(Table 1), we used this equation 2 to verify whether the enhancement in the Td value of

Pfα in the complex form (Fig. 5, Table 2) is due to the large K value of the subunit

association. In the present case, a biopolymer and a ligand are the α and β subunits,

respectively. Table 3 shows the values of ∆T and ∆n calculated for PfTSase from the

equation 2 using the K values obtained at various temperatures. The ∆n values were

estimated to be 0.9 to 1.2 for PfTSase. The ∆n values obtained are rational as a number

of β subunits bound to α subunits in the PfTSase. This indicates that equation 2 is

applicable for the calculation of ∆T in PfTSase. The ∆T values for Pfα calculated from

equation 2 assuming a ∆n of 1.0 were 15.6 to 19.5 oC. These values were near the

experimentally obtained ones (Tables 1 and 2, Fig. 5). If the K value is assumed to be

1.0x106 M-1, ∆T was only 4.9 oC. This agrees with the fact that the enhancement in Td

of Pfα is due to the α2β2 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 Pfα 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

towards 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.

The Td values of the Pfβ monomer in the acidic region remarkably decreased

with lower pHs (unpublished data), suggesting the importance of the β-β subunit

interaction for the higher stability of Pfβ2 compared to Pfα. The Td (80.3 oC) value of

Ecβ2 at pH 8.4 was higher than that of Ecα and comparable to that of Pfα at pH 9.4 (Fig.

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5A and B). The interaction between the β 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 to EcTSase. (1) Pfα and Pfβ2 tightly bind by the K value of the

order of 108 M-1. (2) The negative values of ∆Cp and ∆H upon the subunit association

were low, indicating that the conformational change coupled to the subunit association

is low. (3) The Td of Pfα was drastically enhanced by 17 oC in the α2β2 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.

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Table 1. Thermodynamic 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 7.0 Temperature (oC) n (β/α) Kasso (108 M-1) ∆H (kJ/mol) ∆G (kJ/mol) ∆S (J/K mol) ∆Cp (kJ/K mol) Pf αααα/ββββ association

35 1.2 0.7 -9.4 -46.3 119.6 1.96 40 1.1 1.6 -26.0 -49.2 074.

43.1 1.2 2.7 -27.2 -51.0 75.4 45 1.2 3.1 -33.2 -51.7 58.3 47 1.1 3.9 -38.4 -52.6 44.5 50 1.1 4.0 -43.9 -53.2 28.0 55 1.1 6.5 -53.4 -55.4 5.9 60 1.2 9.5 -58.7 -57.3 -4.3

Ec Ec Ec Ec αααα////ββββ associationassociationassociationassociation 21 1.5 0.0293 -15.4 -36.4 71.7 5.56 25 1.4 0.0286 -33.5 -36.9 11.4 30 1.5 0.0599 -56.1 -39.3 -55.5 35 1.5 0.0400 -87.4 -38.9 -157.1 37 1.5 0.0422 -101.4 -39.4 -200.0 37 1.4 0.034 -104.4 -38.8 -211.5 38 1.5 0.0322 -105.4 -38.8 -213.9

40.07 1.4 0.0356 -129.0 -39.3 -286.3 Hybrid αααα/ββββ association Pfα / Ecβ2 40 1.1 0.00470 -24.0 -34.0 32.0 Ecα / Pfβ2 40 0.7 0.000610 -2.9 -28.7 82.0 Parameters obtained by ITC are represented per molar concentration of α subunit.

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Table 2 Denaturation temperature (Td)of α and β2 subunits in the isolated forms and in the α2β2 complexes for both P. furiosus and E. coli proteins. ____________________________________________________________________________________________ Subunits Td (oC) ∆Td c) (oC) ______________________________________________________________________

Isolated form a) Homo complex form a) Hybrid complex form b) ____________________________________________________________________________________________ Pfα 87.2 (pH 9.37) 104.6 (pH 9.30) 86.4 (pH 9.50) 17.4 Pfβ2 112.2 (pH 9.30) 112.5 (pH 9.30) 111.6 (pH 9.49) 0.3 ____________________________________________________________________________________________ Ecα 53.0 (pH 8.40) 54.7 (pH 8.40) 53.5 (pH 9.49) 1.7 Ecβ2 80.3 (pH 8.21) 79.9 (pH 8.40) 71.5 (pH 9.50) -0.4 ____________________________________________________________________________________________ The Td values represent the peak temperature of the DSC profiles measured at scan rate of 1 K/min. a) From panels (A) and (B) in Fig. 5. b) From panel (C) in Fig. 5 for the hybrid complexes between the Pf-subunits and Ec-subunits. c) The differences in the Td values of individual subunits in the isolated and homo complex forms.

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Table 3 The estimations of increment in denaturation temperature (∆T) of Pfα due to Pfα2β2 complex formation using the association constant (K) at different temperatures. Temperature a) (oC)

40.0 43.1 45.0 47.0 50.0 55.0 60.0

∆T (oC) 15.6 16.8 17.1 17.6 17.6 18.7 19.5

∆n (β/α) 1.2 1.0 1.0 1.0 1.0 0.9 0.9

∆n is the molar ratio of the association (Pfβ per Pfα). ∆T and ∆n were evaluated according to equation (2). In the equation (2), T0 and T were the denaturation temperatures of Pfα alone (360.35 K) and of Pfα (377.75 K) in Pfα2β2 complex form, respectively (Fig. 5). L was the concentration (8.72x10-6 M) of monomer Pfβ under DSC measurements. a) Temperature under ITD measurements (Table 1). The K used in the equation (2) were obtained by ITC measurements at various temperatures in listed in Table 1. ∆T was calculated assuming ∆n of 1.0. ∆n was calculated using 17.4 oC as ∆T (=T-T0).

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FIGURE LEGENDS

Figure 1 pH dependence of apparent molecular weight of Pfβ2 and Pfα2β2.

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 (MWapp) of Pfα2β2 below pH 5 were estimated by fixing the molecular weight

(27.5 k) of Pfα using the software analyzing association system (Beckman). Closed

circles and open triangles are the values of Pfα2β2 and Pfβ2, respectively.

Figure 2 Typical isothermal titration calorimetry for the association of an α

subunit with a β2 subunit at 40.0 oC 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 β2 subunit into the sample cell containing

the α subunit. 1, Pfβ2 (0.160 mM as a β monomer) was injected into Pfα (0.015 mM). 2,

Ecβ2 (0.174 mM as a β monomer) was injected into Ecα (0.0189 mM). (B) Integration

of the thermogram yielded a binding isotherm that fits a model of one set site (α + β <->

αβ) by using variables K, n, and ∆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) (β/α) and enthalpy change (∆H) upon formation of the α2β2 complex obtained were

1.6x108 M-1, 1.1, and -26.0 kJ/mol of α subunit, respectively, for PfTSase and 3.6x106

M-1, 1.4, and -129.0 kJ/mol of α subunit, respectively, for EcTSase.

Figure 3 Temperature dependences of the association constant and enthalpy

change upon association of the α subunit with the β subunit at pH 7.0.

(A) and (B) display the temperature dependence of K and ∆H, respectively. Closed and

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open circles denote the values for PfTSase and EcTSase, respectively. In panel (B), the

linear lines show the linear correlation between ∆H and temperature. ∆Cp values

obtained from the slope of a line were -1.96 and -5.56 kJ/ K mol of the α subunit for

PfTSase and EcTSase, respectively.

Figure 4 Temperature dependencies of enthalpy change, entropy change and

Gibbs energy change upon association of the α with β subunits at pH 7.0.

Panels (A) and (B) display the temperature dependence for PfTSase and EcTSase,

respectively. Open triangles, ∆H, open circles; -T∆S; closed circles; ∆G.

Figure 5 DSC curves of the α, β2, and α2β2.

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, Pfα (0.824 mg/ml) at pH 9.37; b, Pfβ2 (0.688 mg/ml) at pH

9.30; c, Pfα2β2 (0.613 mg/ml) at pH 9.30. In panel B, a, Ecα (1.679 mg/ml) at pH 8.40;

b, Ecβ2 (1.207 mg/ml) at pH 8.21; c, Ecα2β2 (0.999 mg/ml) at pH 8.40. In panels (A)

and (B), Cp values for β2 and α2β2 were normalized by the molar concentration of the β

monomer and αβ dimer, respectively. In panel (C), a, complex of Pfα with Ecβ2 at pH

9.50; b, complex of Pfβ2 with Ecα at pH 9.49. Cp values for hybrid the 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 α and β subunits from

PfTSase and EcTSase.

Figure 6 The amino acid sequences for Pfα, Ecα and Stα in the interfaces of the α

and β subunits.

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Bolds show residues that differed from Stα. Residues numbers of Pfα and Stα are

shown.

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2 4 6 8 10

50

100

150

Fig. 1A

ppar

ent m

olec

ular

wei

ght (

kd)

pH

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0.0 0.5 1.0 1.5 2.0 2.5 3.0-150

-100

-50

0

2

1(B)

Time (min)

DH (

kJ/m

ol o

f asu

buni

t)

Molar ratio of b / a

0 20 40 60 80

Fig. 2

2

1

10 mc

al/s

ec (A)B

indi

ng h

eat

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20 30 40 50 60

-150

-100

-50

0(B)

DH (

kJ/m

ol)

Temperature (oC)

20 30 40 50 60

7

8

9 (A)

Fig. 3lo

g K

(M

-1)

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20 30 40 50 60-150

-100

-50

0

50

100(B)

(kJ/

mol

of a

sub

unit)

Temperature (oC)

10 20 30 40 50 60

-60

-40

-20

0 (A)

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40 60 80 100 120

(C)

a

b

Fig. 5C

p (I

ncre

men

t=50

kJ/

K m

ol)

Temperature (oC)

40 60 80 100 120

(B)

b

c

a

40 60 80 100 120

(A)

b

c

a

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Fig. 6

Loop2

Hydrogen bonds in St

Loop6

Resudue No. Pf Ec St Resudue No.

39 48 IPFSDPLADG IPFSDPLADG VPFSDPLADG

52 61

115 125 VDLPVFHAKEF ADVPVEESAPF ADVPVEESAPF

129 139

163 177 VSLYGTTGAREEIPK LSRAGVTGAENRAAL LSRSGVTGAENRGAL

177 191

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Additions and Corrections

Vol. 278 (2003) 11818–11827

The unfolded protein response is required for haploid tolerance in yeast.

Kyungho Lee, Lenore Neigeborn, and Randal J. Kaufman

Page 11818: The symbol for Dr. Neigeborn’s affiliation was incorrect. The corrected author list with affiliations is shown below:

Kyungho Lee‡§, Lenore Neigeborn¶, and Randal J. Kaufman‡�

From the ‡Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical Center, AnnArbor, Michigan 48109 and ¶Rutgers College, New Brunswick, New Jersey 08901

Vol. 278 (2003) 8922–8928

Stimulated interaction between � and � subunits of tryptophan synthase from hyperthermophile enhances itsthermal stability.

Kyoko Ogasahara, Masami Ishida, and Katsuhide Yutani

Page 8924, Fig. 2B: The line below the figure should read molar ratio of �/�. The denominator and the numerator weremistakenly reversed. The corrected figure is shown below.

FIG. 2B

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 22, Issue of May 30, p. 20444, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to becorrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract)services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

20444

Kyoko Ogasahara, Masami Ishida and Katsuhide Yutanifrom hyperthermophile enhances its thermal stability

Stimulated interaction between alpha and beta subunits of tryptophan synthase

published online January 6, 2003J. Biol. Chem. 

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