Stimulated Interaction between
and
Subunits of Tryptophan
Synthase from Hyperthermophile Enhances Its Thermal Stability*
Kyoko
Ogasahara
,
Masami
Ishida§, and
Katsuhide
Yutani¶
From the
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 |
Tryptophan synthase from hyperthermophile,
Pyrococcus furiosus, was found to be a tetrameric form
(
2
2) composed of
and
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
and
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
subunit from the
hyperthermophile was enhanced by 17 °C due to the formation of the
2
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 |
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
2
2 is a multifunctional and allosteric
enzyme. This
2
2 complex has an



arrangement (23) and can be isolated as the
monomer and
2. The
and
2 subunits catalyze
inherent reactions (for reviews, see Refs. 24-28). When the
and
2 subunits associate to form the
2
2 complex, the enzymatic activity of
each subunit is enhanced by 1 to 2 orders of magnitude (for reviews,
see Refs. 24-28). The
/
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
2
2, and the enzymatic activities of the
and
2 subunits separated in their active forms were
stimulated by the formation of the
2
2
complexes as well as the reported mesophilic prokaryotic bacterial
tryptophan synthases (for reviews, see Refs. 24-28). The thermal
stability of the
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
and
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 |
Expression and Purification of
,
2, and
2
2 from P. furiosus--
The
subunit
(Pf
) from P. furiosus was expressed in the
E. coli strain JM109/p
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-
-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 Pf
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 Pf
2
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
Pf
2 and Pf
2
2.
The
subunit from E. coli was purified as already
described (31). The
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
were 6.92 for Pf
, 10.18 for
Pf
2 subunit, and 9.94 for
Pf
2
2. These values were
determined based on protein assay by the Lowry method using bovine
serum albumin as the standard protein. The concentrations of
Ec
, Ec
2, and
Ec
2
2 were determined using OD
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 Pf
, 0.743 for Pf
2, and
0.747 for Pf
2
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
and
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
2 subunit at a high concentration was
injected into the 1.3155-ml sample cell containing the
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 |
Confirmation of Association States of Recombinant
2
and
2
2 from P. furiosus--
Pf
, 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
chain is comprised of 388 residues and the calculated molecular weight is 42,500 (30). The
Mr app of the recombinant
was
84,000-88,000 in the pH region above 4.7, indicating that the
chain exists in a dimeric form (Pf
2). The
Mr app of the recombinant complex of
with
subunits was almost nearly equal to 2-fold (140,000) the calculated
value for 
around pH 7. These results show that tryptophan
synthase from P. furiosus forms a complex of
2
2
(Pf
2
2) as observed for
prokaryotic tryptophan synthases from mesophiles (24-28) and from the
hyperthermophile (37, 38). The Mr app of the
Pf
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
Pf
2
2 decreased with decreasing
pH between pH 5 and 4, although that of Pf
2
did not change.

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Fig. 1.
pH dependence of apparent molecular weight of
Pf 2 and
Pf 2 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
Pf 2 2 below pH 5 were estimated
by fixing the molecular weight (27,500) of Pf using the
software analyzing association system (Beckman). Closed
circles and open triangles are the values of
Pf 2 2 and
Pf 2, respectively.
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|
Binding Titration of
with
2 Subunits by
Isothermal Titration Calorimetry--
To examine the inherent feature
of the interaction between Pf
and
Pf
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 Pf
with Pf
2 were estimated.
The titration in this study was performed by injecting the
2 subunit into the
subunit, in the calorimetry cell,
at various temperatures and pH 7.0, because the solubility of
Pf
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
subunit with the
2 subunit at 40 °C. The binding of Pf
with Pf
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
subunit. The ITC titration curves for both PfTSase and
EcTSase fitted well to a model of one set site (
+

) (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,

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Fig. 2.
Typical isothermal titration calorimetry for
the association of an subunit with a
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 2 subunit into the sample cell containing the subunit. 1, Pf 2 (0.160 mM as a monomer) was injected into Pf
(0.015 mM); 2, Ec 2
(0.174 mM as a monomer) was injected into
Ec (0.0189 mM). B, integration of
the thermogram yielded a binding isotherm that fits a model of one set
site ( +  ) 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) ( / ), and
enthalpy change ( H) upon formation of the
2 2 complex obtained were 1.6 × 108 M 1, 1.1, and 26.0 kJ/mol of
subunit, respectively, for PfTSase and 3.6 × 106 M 1, 1.4, and 129.0 kJ/mol
of subunit, respectively, for EcTSase.
|
|
|
(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 subunit with the
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
subunit.
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|
The stoichiometry (molar ratio of
/
) of association between
Pf
and Pf
was similar to unity and did not
depend on temperature. The stoichiometry for EcTSase was
1.5. In a previous study in which Ec
is injected into the
Ec
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
subunit with PLP, because both Ec
and
Ec
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
H for the
interaction between Pf
and Pf
were smaller
that those in EcTSase (Table I and Fig. 3B). In
both cases of PfTSase and EcTSase, the
H values linearly correlated with temperature (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 mole of
subunit for PfTSase and
EcTSase, respectively. Fig. 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 using 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.

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Fig. 3.
Temperature dependences of the association
constant and enthalpy change upon association of the
subunit with the subunit
at pH 7.0. A and B display the temperature
dependence of K and 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
H and temperature. Cp values obtained from
the slope of a line were 1.96 and 5.56 kJ/K mol of the 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
with subunits at pH
7.0. A and B display the temperature
dependence for PfTSase and EcTSase, respectively.
Open triangles, H; open circles,
T S; closed circles,
G.
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|
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
Pf
, Pf
2, and
Pf
2
2 at pH 9.3-9.4. The
Pf
exhibited a DSC curve with a single peak at 87.2 °C
(curve a in Fig. 5A). For
Pf
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-Pf
2 and
apo-Pf
2 removing cofactor PLP, respectively.
In the case of Pf
2
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 Pf
2, because the peak
temperature (112.5 °C) was quite similar to that of
Pf
2 alone (112.2 °C). Therefore, the peak
temperature at the lower temperature could be considered to arise from
Pf
. 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 Pf
alone was lower by
25 °C than that of Pf
2. However, the
Td value of Pf
was enhanced by
17.4 °C due to the complex formation. The Td
value of Pf
2 did not change due to the
complex formation. On the other hand, the Td value of Ec
(53.0 °C) slightly increased by 1.7 °C
due to the
2
2 complex formation at pH 8.4 (curves a and c in Fig. 5B and Table
II). The Td value of
Ec
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 Pf
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
St
2
2 at pH 8.0 showed two
separate peaks at the denaturation temperatures of isolated
St
and St
2. Each of the
Td values of Ec
and
Ec
2 was similar to those of the reported
St
and St
2. Td values of Pf
and
Pf
2 were drastically higher by 34.2 and 31.9 °C than those of Ec
and
Ec
2, respectively.

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Fig. 5.
DSC curves of the
, 2, and
2 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, Pf (0.824 mg/ml) at pH
9.37; b, Pf 2 (0.688 mg/ml) at pH
9.30; c, Pf 2 2
(0.613 mg/ml) at pH 9.30. In panel (B), a,
Ec (1.679 mg/ml) at pH 8.40; b,
Ec 2 (1.207 mg/ml) at pH 8.21; c,
Ec 2 2 (0.999 mg/ml) at pH 8.40. In panels (A) and (B), Cp values for
2 and 2 2 were normalized
by the molar concentration of the monomer and  dimer,
respectively. In panel (C), a, complex of
Pf with Ec 2 at pH 9.50;
b, complex of Pf 2 with
Ec 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 and subunits from PfTSase and
EcTSase.
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Table II
Denaturation temperature (Td) of and 2 subunits
in the isolated forms and in the 2 2 complexes for
both P. furiosus and E. coli proteins
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ITC and DSC Measurements of Hybrid Complex between Pf Subunits and
Ec Subunits--
To explore which of the
and (or)
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
Pf
2-Ec
and Ec
2-Pf
associations,
respectively, compared with that for the Pf
-Pf
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
Pf
2Ec
2 and
Ec
2Pf
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
Pf
2
2 (Fig. 5A and Table II).
 |
DISCUSSION |
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
and
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
Cp of the subunit association for
PfTSase (
1.96 kJ/K mol of
) was less than half of that
for EcTSase (
5.56 kJ/K mol of
) (Table I and Fig.
3B). In the cases of TSases from mesophiles, Hiraga and
Yutani (39) 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
/
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
/
subunit
association in EcTSase and StTSase (39),
according to the method of Spolar and Record (48).
Anp and
Ap in the
/
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
). 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 pre-existing complementary surface (48). 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 (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
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
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 °C were drastically decreased relative to those for
PfTSase and EcTSase, resulting in the
decreases in the negative
G (Table I). The negative
H for the hetero subunit associations also decreased.
These results indicate that the
and
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
and
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 St
2
2 complex
form (23, 50-52) and an isolated Pf
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
Pf
and Ts
. The crystal structure of
Pf
alone (29) is the same topological pattern to that of
St
in the St
2
2
complex form (23). Pf
, St
, and
Ec
consist of 248, 268, and 268 residues, respectively.
The sequence identities between Pf
and St
and between Ec
and St
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 St
, which play an important role in
the catalysis and allosteric communication between the active sites of
the
and
subunits, contact with St
(23, 50-52). The B-factor averaged for the main-chain atoms of the loop 2 in Pf
is considerably lower than that in St
(29), indicating that the loop 2 is less mobile in Pf
than in St
. The loop 6 of St
is highly
mobile and 12 residues in the loop 6 have not been determined due to a
weak electron density (23). In Pf
, only three residues
are not determined, although Pf
does not form a complex
with the
subunit, indicating that the number of mobile residues in
Pf
is drastically reduced (29). The amino acid residues
of the loop 6 in Pf
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
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
and
subunits in PfTSase except
for the Asn
104-Gly
292 pair in
StTSase. The remarkable deviations in the root mean square deviations of the C
atoms between Pf
and
St
are found in the loop 2 and in the residues of
Val119, Phe120, and His121 in
Pf
(29), which are the residues in St
forming hydrogen bonds with the residues in St
. From
these observations, it seems that the conformations of the subunit
interface in Pf
substantially differ from those in
St
, and the rigidly ordered conformations of the loops 2 and 6 in Pf
might contribute to creating the key part of
the interface responsible for the strong subunit association in
PfTSase.

View larger version (12K):
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[in a new window]
|
Fig. 6.
The amino acid sequences for
Pf , Ec
and St in the interfaces of
the and subunits. Bolds show residues that differed from
St . Residues numbers of Pf and
St 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,
|
(Eq. 2)
|
where To and T are the melting
temperatures in the absence and presence of a ligand, respectively,
L is the ligand concentration,
H° 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 with the enthalpy change of the transition (57). Because the
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 Pf
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
and
subunits,
respectively. Table III shows the values of
T and
n calculated for
PfTSase from Equation 2 using the K values
obtained at various temperatures. The
n values were estimated to be 0.9-1.2 for PfTSase. The
n
values obtained are rational as a number of
subunits bound to
subunits in the PfTSase. This indicates that Equation 2 is
applicable for the calculation of
T in
PfTSase. The
T values for Pf
calculated from Equation 2, assuming a
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,
T was only 4.9 °C. This agrees with the fact that the
enhancement in Td of Pf
is due to
the
2
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 Pf
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.
View this table:
[in this window]
[in a new window]
|
Table III
The estimations of increment in denaturation temperature ( T) of
Pf due to Pf 2 2 complex formation using the
association constant (K) at different temperatures
n is the molar ratio of the association (Pf
per Pf ). T and n were
evaluated according to Equation 2. In Equation 2, T0
and T are the denaturation temperatures of Pf
alone (360.35 K) and of Pf (377.75 K) in
Pf 2 2 complex form, respectively (Fig.
5). L is the concentration (8.72 × 10 6
M) of monomer Pf under DSC measurements.
|
|
The Td values of the Pf
monomer in
the acidic region remarkably decreased with lower pH
levels,2 suggesting the
importance of the
-
subunit interaction for the higher stability
of Pf
2 compared with Pf
. The
Td (80.3 °C) value of
Ec
2 at pH 8.4 was higher than that of
Ec
and comparable to that of Pf
at pH 9.4 (Fig. 5, A and B). The interaction between the
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) Pf
and
Pf
2 tightly bind by the K value of
the order of 108 M
1; 2) The
negative values of
Cp and
H on the subunit
association were low, indicating that the conformational change coupled
to the subunit association is low; 3) The Td of
Pf
was drastically enhanced by 17 °C in the
2
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;
Pf
, Pf
2, and
Pf
2
2,
,
2
subunits and
2
2 complex of
PfTSase, respectively;
Ec
, Ec
2 and
Ec
2
2,
,
2
subunits and
2
2 complex of
EcTSase, respectively;
St
, St
2 and
St
2
2,
,
2
subunits and
2
2 complex of
StTSase, respectively;
ITC, isothermal titration
calorimetry;
DSC, differential scanning calorimetry;
PLP, pyridoxal
5'-phosphate;
DTT, dithiothreitol.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.