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INTRODUCTION |
Prokaryotic tryptophan synthase, which catalyzes the last
processes in the biosynthesis of tryptophan, is a multienzyme
2
2 complex composed of nonidentical
-
and
-subunits. The separate
- and
2-subunits
catalyze inherent reactions termed
and
reactions, respectively.
When the
- and
2-subunits combine to form the
2
2 complex, the enzymatic activity of
each subunit is stimulated by 1 to 2 orders of magnitude (1). The
2
2 complex has been studied as an
excellent model system for seeking answers to important questions in
protein-protein interaction, especially in multifunctional enzymes. In
1988 (2) the three-dimensional structure of the tryptophan synthase
2
2 complex from Salmonella typhimurium was determined by x-ray analysis. However, the
structure of the
-or
2-subunit alone has not yet been
determined. To elucidate the molecular basis of the mutual activation
of the subunit interaction due to the formation of the
2
2 complex, we need to know the structures of the
- or
2-subunits alone as well as
that of the complex. Although the crystallization of each subunit from
S. typhimurium and Escherichia coli has been
tried for many years (3), the report of the x-ray structure has not yet
appeared. Recently, the structures of a number of proteins from
hyperthermophiles have been successfully determined by x-ray analysis.
This seems due to the facts that proteins from hyperthermophiles are
unusually stable and more easily form better crystals. Therefore, the
trpA and trpB genes, coding for tryptophan
synthase, were cloned from Pyrococcus furiosus and their
products were overexpressed in E. coli. The purified
-subunit from P. furiosus easily grew a suitable crystal
for x-ray analysis, and the crystal structure could be determined at a
resolution of 2.0 Å as described in this paper.
On the other hand, a fundamental understanding of the conformational
stability of proteins still remains elusive. Proteins from
hyperthermophiles should retain their native structures under extreme
conditions, whereas the homologous proteins from mesophiles completely denature. Therefore, comparative studies of extremely thermostable proteins with their homologs might help us understand the
stabilization mechanism of a general globular protein. Recently, structures and physicochemical properties of proteins from
hyperthermophiles have been extensively studied. In several
hyperthermophile proteins, an increased number of ion pairs and ion
pair networks have been observed (4-13), which have been explained as
the intrinsic changes for protein stability in hyperthermophiles. In
the case of the tryptophan synthase
-subunit from P. furiosus, many ion pairs were also found. However, the effect of a
surface salt bridge on the stability remains controversial even today;
some reports have shown little contribution of a surface salt bridge to
stability (14-19), whereas others have shown a favorable contribution
(20-24). There are also reports on some hyperthermophile proteins
without additional ion pairs (25-28). The conformation of general
globular proteins is marginally maintained by the combination of many
positive (such as hydrophobic interaction and hydrogen bond) and
negative (such as entropic effect and steric hindrance) factors for
stabilization (29). The enhanced stability of hyperthermophile proteins
might originate from several attractive forces (30). However, it still remains unclear what causes the dramatic stabilization of proteins from
hyperthermophiles. Understanding the molecular origin for stabilization
of hyperthermophile proteins provides valuable insights into the
problems of protein stability, protein folding, and protein engineering.
In this paper, to elucidate the stabilization mechanism of a protein
from a hyperthermophile, the stability of the
-subunit from P. furiosus
(Pf-
-subunit)1
was examined by differential scanning calorimetry (DSC) and its structure by x-ray crystallography. The DSC data indicated that the
higher stability of this protein was not caused by an enthalpic factor
as compared with homologous mesophile proteins. The stabilization mechanism of the Pf-
-subunit is discussed on
thermodynamic grounds based on structural information from x-ray analysis.
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EXPERIMENTAL PROCEDURES |
Purification of the
-Subunit from P. furiosus--
The
-subunit of tryptophan synthase from P. furiosus
(Pf-
-subunit) was overexpressed in E. coli
strain JM109/p
1974 containing only the trpA gene from
P. furiosus.2 The
E. coli strain was routinely grown in 15 liters of LB medium supplemented with ampicillin of 100 mg/liter of culture medium. The
production of the Pf-
-subunit was induced by addition of isopropyl-
-D(
)-thiogalactopyranoside at a
concentration of 1 mM to the culture medium after 1-h
incubation at 37 °C with shaking. Then the culture was continued for
about 20 h at 37 °C with shaking.
Pelleted cells (wet weight of about 25 g) collected by
centrifugation (5000 rpm for 10 min) were suspended in 100 ml of 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 5 mM DTT. The cells were disrupted
by sonication on ice for 5 min with cooling intervals for 10 min and
repeating three times. The homogenized solution was heated in a water
bath for 10 min at 75 °C (the temperature of the solution), under
which the majority of the E. coli proteins precipitated,
leaving the Pf-
-subunit in the soluble fraction. The
resultant suspended solution was centrifuged at 15,000 rpm for 30 min
at 4 °C to remove cell debris and denatured E. coli
proteins. The supernatant solution was treated with ammonium sulfate of
40% saturation and centrifuged, and the precipitate was discarded. The
Pf-
-subunit was precipitated by addition of ammonium
sulfate at 90% saturation. The precipitate was dissolved in 50 ml of
25 mM potassium phosphate buffer (pH 7.0) with 1 mM EDTA and 5 mM DTT and dialyzed against the
same buffer overnight at 4 °C.
The dialyzed sample was applied on a column (2.5 × 20 cm) of
DEAE-Sephacel (Amersham Pharmacia Biotech): the enzyme was eluted with
a linear gradient (25-200 mM) of potassium phosphate
buffer (pH 7.0) containing 1 mM EDTA and 5 mM
DTT. Active fractions of the eluted solutions were concentrated using
an Amicon PM10 membrane filter. Next, the protein was separated by gel
filtration (Superdex TM200 26/60, Amersham Pharmacia Biotech). The
active fractions collected were finally purified by ion exchange
chromatography (SP-Sepharose 26/16, Amersham Pharmacia Biotech) with a
linear gradient of 25-100 mM phosphate buffer (pH 7.0)
containing 1 mM EDTA and 5 mM DTT. The purified
protein showed a single band on SDS-polyacrylamide gel electrophoresis.
Protein Concentration--
The protein concentration was
estimated from absorbance at 278.5 nm at pH 7.0, OD1% = 6.92, using a cell with a light path length of 1 cm. The value was
determined based on a protein assay by the Lowry method using bovine
serum albumin as a standard protein.
Differential Scanning Calorimetry--
DSC was carried out with
a differential scanning calorimeter, MicroCal VP-DSC (Northampton) at a
scan rate of 1 K/min. Prior to the measurements, the protein solution
was dialyzed against the buffer used. The dialyzed sample was filtered
through a 0.22-µm pore size membrane and then degassed in vacuum. The
buffers used were 20 mM glycine-HCl in the acidic region
and 20 mM glycine-KOH in the alkaline region. The protein
concentrations under measurement were 0.4-1.4 mg/ml. The DSC curves
were analyzed by the Origin software from MicroCal (Northampton).
Crystallization and Data Collection--
Crystals suitable for
data collection were grown by the hanging drop vapor diffusion method
at 10 °C from a reservoir solution containing 0.1 M
MES-NaOH, pH 6.5, and 12% polyethylene glycol 20000. X-ray data
from the native and mercury derivative crystals were collected at the
beam line 18B of the Photon Factory, Tsukuba using a Weissenberg camera
(32) at 293 K. The data set of the platinum derivative was collected on
a Rigaku R-axis IV imaging plate using nickel-filtered double-mirror
focused Cu-Ka radiation from a Rigaku RU-200. The
native and derivative data were processed and integrated by DENZO and
scaled by SCALEPACK (33). The space group is
C2221, and the unit cell dimensions are
a = 73.013, b = 78.997, and
c = 170.964 Å (see Table II below). There were two
molecules in the asymmetric unit with a volume per unit of molecular
weight of the protein of 2.30 Å3/Da and a
calculated solvent content of 53.4% (34).
Structure Determination and Refinement--
Native, mercury, and
platinum data sets were used for phase calculation by MIRAS (see Table
II). The mercury position was identified in the difference Patterson
map. Heavy atom sites in the platinum derivative were found in the
difference Fourier maps calculated by using the phases from the first
derivative with the program MLPHARE (35). The heavy atom parameters
were refined, and the phases were calculated to 2.2 Å with the program
SHARP (36). The initial MIR phases were improved by solvent flattening and noncrystallographic 2-fold symmetry averaging at the same resolution with the program SOLOMON (37). An initial model was built
using the program O (38). Several cycles of rigid-body refinement,
positional refinement, and simulated annealing were performed at 2.2-Å
resolution with X-PLOR (39). The refinements were continued at 2.0 Å using CNS (40). Successive refinement with temperature factors and
addition of solvents resulted in an R-value of 19.8% and an
Rfree of 24.9% for reflections in the resolution range 40-2.0 Å. Rfree was
calculated with 5% of the reflections. During refinements NCS
restraints were enforced. The current model includes residues 1-166,
174-248 for molecule a and 1-169, 173-248 for molecule b, and 421 water molecules. Model geometry was analyzed with PROCHECK (41), and
96.4% of the nonglycine residues were in the most favorable region of
the Ramachandran plot and 3.3% in the additionally allowed region. The
final coordinates have been deposited in the Protein Data Bank
(accession number 1GEQ).
Calculation of Electrostatic Interaction--
The electrostatic
free energy Gel of the protein-solvent system is
generally described by a sum of the direct Coulombic energy and the
dielectric shielding from the solvent due to the reaction field
(42),
|
(Eq. 1)
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The potential energy
react(ri)
due to the dielectric shielding at the ith charge position
ri, can be given as the difference between the
electrostatic potentials
sol(ri) and
vac(ri), both of which were
calculated by numerically solving the Poisson equations,
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(Eq. 2)
|
assuming
p = 10 and
p=
s = 80, respectively. Here, qi is the
ith charge and
p and
s are the
dielectric constants
(r) in the protein region and in the
solvent region, respectively.
s in the solvent region
was always assumed to be 80. The divergence of the electrostatic
potential due to the self-Coulombic energy can be completely avoided,
because the delta function in the right-hand side in eq. 2 is replaced
by the smooth charge density in the actual numerical calculations
(42).
In the continuum model for the native state, only the ionic charges
were assumed as qi to be located on both carboxyl oxygen atoms of the side chains of Asp and Glu, on the N
atoms of
Lys, on the C
atoms of Arg, and on an N atom of the N terminus, respectively. When a strong ion pair is formed within 3 Å between the
two heavy atoms,
e charge was assumed on one of the two carboxyl oxygen atoms of Asp or Glu, which is the closest to the positively charged residue, and +e charge was on N
1, N
2, or N
, which is the closest to the negatively charged residue, in the case of Arg.
Then, the above Poisson equations were numerically solved twice for the
different
p values.
For the denatured state, the Coulombic term was calculated by using the
root mean square distance
r
1/2 between
the ith and jth charges in the different residues
as the distance between those charges. We evaluated the mean square distance between the corresponding C
atoms for a simplified protein chain model, which was approximated by a Gaussian chain composed of
only the connected C
atoms, with the mean inter-C
bond length 3.8 Å and the angle between the successive three C
atoms 106.3° (43).
Adding the squared distances between the C
atom and the charge
position in each extended side chain for the ith and
jth charges, respectively,
r
in the denatured state was
estimated. As the value of
(
sol(ri)
vac(ri)) in the denatured state, the
Poisson equations were solved for the models of the extended amino acid
trimers, Ala-Xaa-Ala, where Xaa is Asp, Glu, Lys, or Arg. No charges
were assumed on the N or C termini in the trimer models. The
interaction term between the ith and jth charges
in the reaction field calculation was neglected in the denatured
states. A similar calculation was also performed for the N terminus
using the extended Met-Ala model, assuming +e charge on the N atom of Met.
 |
RESULTS |
Thermal Stability of the
-Subunit of Tryptophan Synthase from P. furiosus--
To examine the thermal stability of the
Pf-
-subunit, DSC measurements were carried out at various
pH values. Fig. 1 shows excess heat
capacity curves of both the Pf-
-subunit and
-subunits from E. coli (Ec-
-subunit) near pH 9.5. The
denaturation temperature of the Pf-
-subunit was about
33 °C higher than that of the Ec-
-subunit at the same
conditions, indicating that the protein from a hyperthermophile has
extremely high thermostability. In the acidic region from pH 3 to 4, the heat denaturation of the Pf-
-subunit was almost completely reversible, but the Ec-
-subunit was
acid-denatured at room temperature. In Fig.
2 the specific enthalpy changes upon denaturation (
h, the enthalpy value per a gram of
proteins) obtained from DSC curves of the Pf-
-subunit at
various pH values are plotted against the denaturation temperature at
each pH. Line (a) represents a least-square fit to the
experimental points of the Pf-
-subunit. The slope
corresponds to the specific heat capacity change of denaturation
(
cp). The
cp
value (0.52 J K
1 g
1) of the
Pf-
-subunit was slightly smaller than those of the
Ec-
-subunit and the
-subunit from S. typhimurium (St-
-subunit), which are similar to each
other (0.64 J K
1 g
1 from the slope of
line 2 in Sugisaki et al. (44)). The figure indicates that
h values of the Pf-
-subunit
were considerably lower than those of mesophile proteins at each
temperature shown in the figure: for example,
h was 10.3 J/g and 21.4 J/g for those from the Pf-
-subunit and
mesophile proteins, respectively, at 60 °C. The thermodynamic
parameters for heat denaturation as a function of temperature can be
calculated using the following equations,
|
(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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assuming that
cp does not depend on
temperature (45). The temperature functions of
g for the
Pf-
-subunit and the Ec-
-subunits at pH 9.5 obtained from eq. 5 are shown in Fig. 3,
indicating that the Pf-
-subunit is more stable than the
Ec-
-subunit under all the temperatures shown. The
difference in
g was remarkable at higher temperatures.
Because the denaturation enthalpies of the Pf-
-subunit
under all the temperatures as shown in Fig. 2 are lower than those of
the Ec-
-subunit, the increase in
g of Pf-
-subunit comes from the decrease in
s as shown in
Fig. 3.

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Fig. 1.
Typical excess heat capacity curves of
tryptophan synthase -subunits from P. furiosus and E. coli near pH 9.5 in 20 mM Gly-KOH buffer. (a),
Pf- -subunit, at pH 9.60 and protein concentration of 0.40 mg/ml; (b), Ec- -subunit, at pH 9.54, and
protein concentration of 0.93 mg/ml.
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Fig. 2.
Specific enthalpy change upon denaturation
for tryptophan synthase -subunit from the
P. furiosus as a function of denaturation
temperature. Filled circles represent each experimental
point of Pf- -subunit obtained at various pHs. Line
(a) was given by a least-square fit to experimental points.
Line (b) is a reported one for the Ec- -subunit
and the St- -subunit (44).
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Fig. 3.
Specific Gibbs energy change
( G) and specific entropy change
(T S) upon denaturation of tryptophan
synthase -subunits from P. furiosus
and E. coli as a function of temperature at pH
9.5. (a) and (a') represent
G and T S of
Pf- -subunit, respectively. (b) and
(b') represent G and
T S of Ec- -subunit,
respectively.
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Amino Acid Compositions and Overall Structure of the
-Subunit
from P. furiosus--
The Pf-
-subunit consists of 248 residues,2 but the St-
-subunit has 268 (2).
Table I shows the amino acid compositions of both
-subunits. Hydrophobic residues of the
Pf-
-subunit were reduced from 157 (58.6%) to 133 (53.6%) as compared with those of the St-
-subunit:
especially, Ala was 40 (14.9%) to 22 (8.9%). On the other hand, the
hydrophilic residues were increased from 64 (23.9%) to 80 (32.3%):
especially, Lys was 8 (3.0%) to 20 (8.1%). Jaenicke et al.
(46) have reported that the ranking of the five most frequent amino
acid exchanges from mesophiles to hyperthermophiles is seen to be Lys
Arg, Ser
Ala, Gly
Ala, Ser
Thr, and Ile
Val. In the
case of the Pf-
-subunit, however, Lys was remarkably increased and Ala decreased.
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Table I
Comparison of amino acid compositions of tryptophan synthase
-subunits from P. furiosus and S. typhimurium
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For the structure determination of the Pf-
-subunit, the
molecular replacement method using the structure of the
-subunit in
the
2
2 complex of tryptophan synthase
from S. typhimurium (2) as a search model, was tried using
AMoRe (47) or X-PLOR (39), because the sequence identity of both
proteins was 31.5%. But all attempts to refine the solution failed.
Therefore, the structure of the Pf-
-subunit was solved by
the method of multiple isomorphous replacement using two derivatives
with K2PtCl4 and CH3HgCl (Table
II). It was found that the
Pf-
-subunit adopts an 8-fold
/
barrel fold, first
observed in a triose-phosphate isomerase barrel (48), similar to that
of the St-
-subunit (2), as shown in Figs.
4 and 6. The helices and strands
corresponding to canonical
/
barrel elements have been numbered
consecutively 1 to 8. In the structure of the
St-
-subunit, an extra helical segment, designated helix
0, precedes the first strand and acts as a cap in the bottom of the
barrel (2). However, in the Pf-
-subunit the helix 0 was
not observed because of the missing 12 residues in the N-terminal
region as compared with the St-
-subunit. Six residues in
the C-terminal helix 8 were also deleted in the
Pf-
-subunit. Fig. 5 shows
the secondary structure-based sequence alignment using the secondary
structure elements assigned using DSSP (49). This alignment also
suggests that another two residues corresponding to positions 41 and 95 in the St-
-subunit were deleted in the Pf-
-subunit. These deletions in the
Pf-
-subunit might cause the decrease in entropy in the
denatured state as compared with those from the mesophiles, resulting
in stabilization of a protein conformation, as described under
"Discussion."

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Fig. 4.
Schematic stereo view of the
-subunit of tryptophan synthase from P. furiosus. Protein figures were prepared using the programs
of MOLSCRIPT (76) and Raster3D (31).
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Fig. 5.
Sequence alignments with secondary structures
of -subunits of tryptophan synthase from
P. furiosus and S. typhimurium. The
first line represents the alias of secondary segments named
by Hyde et al. (2). The second and seventh
lines represent the residual numbers of the
St- -subunit and the Pf- -subunit,
respectively. The third and sixth lines represent
amino acid sequences of St- -subunit and
Pf- -subunit, respectively. The fourth and
fifth lines represent secondary structural elements of
St- -subunit (1BKS) and Pf- -subunit,
respectively, judged from the secondary structure definition as
established by DSSP (29). H, E, B,
G, T, and S in the secondary structure
elements represent -helix, -strand, -bridge, 3-helix, turn,
and bend, respectively. The residual names with underlines
in the third line represent hydrogen-bonding residues in the
St- -subunit with the -subunit. The residual names with
underlines in sixth line represent residues in
the Pf- -subunit whose C atoms are deviated more than
2.5 Å as compared with those in the St- -subunit (see
Fig. 7).
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Structural Comparison of Pf-
-subunit and
St-
-subunit--
The structures of the Pf-
-subunit
and St-
-subunit (1BKS) could be superimposed with the
root mean square deviation of 2.82 Å between 234 equivalent C
atoms
in both subunits (Fig. 6a). The seven remarkable deviations are found in Fig.
7. The three big deviations are indicated
by peaks I, VI, and VII in
Fig. 7. The deviation of peak I is caused by 12 missing residues in the N-terminal (the deletion of helix 0), where the
bottom of the barrel is capped by a short loop in the place of helix 0 in St-
-subunit as shown in Fig. 6. Peaks VI
and VII correspond to helices 6 and 8, respectively. The
deviation at helix 8 (peak VII) is caused by missing six
residues in the C terminus, resulting in a shorter helix with 11 residues from a long helix (18 residues) of the St-
-subunit. Residues 178-189 in a loop between strand 6 and helix 6 of the St-
-subunit are highly mobile, thus
the positions are not determined due to weak electron density. This
loop plays an important role in the intersubunit communication for
enzymatic functions (50). In the case of the Pf-
-subunit,
only three residues (170) could not be determined in contrast to
12 corresponding residues (164) of the St-
-subunit,
indicating that the number of the mobile residues in the
Pf-
-subunit was remarkably reduced. When the
St-
-subunit in the
2
2 form
binds with the substrate inhibitors of the
-subunit,
indole-3-propanol phosphate (50, 51) and 5-fluoroindole propanol
phosphate (52), the disordered residues in their complexes have been
reported to be reduced by two (190) and six (188) from 12 residues (178), respectively. These results indicate that the
ligand binding induces conformational changes to less mobile forms. On
the other hand the structure of the corresponding loop region of the
Pf-
-subunit was remarkably different from that of the
St-
-subunit with inhibitors (Fig. 6). This suggests that
the loop region of the Pf-
-subunit might drastically
change the conformation when the substrate (or inhibitor) binds to the
protein.

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Fig. 6.
Schematic stereo view of the
-subunits of tryptophan synthase from P. furiosus and from S. typhimurium
superimposed. Red and blue represent
the Pf- -subunit and the St- -subunit (1BKS),
respectively. H0 through H8 represents helices 0 through 8 in the St- -subunit, respectively.
(a) represents the whole structures of both -subunits.
(b) and (c) represent stereo drawings of the main
chains in the N- and C-terminal regions of tryptophan synthase
-subunits superimposed, respectively.
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Fig. 7.
Root mean square deviations (Å) of
C atoms between the
Pf- -subunit and the
St- -subunit after a least
squares fit of corresponding C atoms.
Peaks I through VII represent only a
discrimination peak of large differences. Residual numbers are for the
Pf- -subunit.
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Peaks II and IV in Fig. 7 are close to each
residue corresponding to positions 41 and 95, respectively, in the
St-
-subunit, which were deleted in the
Pf-
-subunit. Peak III corresponds to helix 2'.
In the case of the St-
-subunit a prominent deviation from
the canonical triose-phosphate isomerase barrel is an insertion of 26 residues (positions 53-78) between strand 2 and helix 2. These
residues, including helix 2', are highly conserved in
-subunits among many species. Two catalytic residues (Glu49 and
Asp60) in the St-
-subunit are involved
in or near the insertion region. The corresponding residues,
Glu36 and Asp47, in the
Pf-
-subunit were also found at coordinates similar to those from the mesophile protein. Helix 2' might be important in
connection with enzyme function, although catalytic residues are not
involved. In the region of peak V in the
St-
-subunit, there are hydrogen-bonding residues
(Val133, Glu134, and Glu135)
interacting with the
-subunit (Fig. 5), which are important residues
in the complex formation (53). The deviations of the peaks
I, II, IV, and VII were caused by
the deletion of residues, but other peaks of deviations
(III, V, and VI) were related to enzymatic function, suggesting that the conformation of these portions
might be affected when the
-subunit forms the complex with the
2-subunit.
High resolution structures of proteins from hyperthermophiles have
shown that the number of ion pairs in most of the hyperthermophile proteins is higher than in mesophile counterparts (4-13), although additional ion pairs were not observed in some hyperthermophile proteins (25-28). Table III lists the
ion pairs formed within 4 Å for the Pf-
-subunit and
St-
-subunit. The number (0.05) of ion pairs per a residue
for the Pf-
-subunit is relatively higher than that (0.02)
for the St-
-subunit but is comparable to that (0.04) of
the average number of mesophile proteins (54). In the
Pf-
-subunit, 24 residues are involved in the formation of 13 ion pairs: five ion pairs for nine residues in the
St-
-subunit. Only one ion pair,
Arg57-Asp15, in the Pf-
-subunit
is conserved in the St-
-subunit
(Arg70-Asp27). Ion pairs in the
Pf-
-subunit form between the following structural segments: five intra-helical, three inter-helical, two between helix
and loop, one between helix and
-strand, one between
-strands, and one between
-strand and loop (Table III).
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Table III
Ion pairs (salt bridges) in tryptophan synthase -subunits from P. furiosus and S. typhimurium within 4 Å
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The volume of the cavities in the subunits of glutamate dehydrogenase
from three different species decreases significantly with increasing
thermal stability of the enzymes (9). For two
-subunits of
tryptophan synthase, the cavity volume was determined by attempting to
insert a probe sphere of 1.4-Å radius (assuming a water molecule)
(55). In the case of the Pf-
-subunit, seven cavities were
found and the total volume was 226.8 Å3; in the
St-
-subunit eight cavities were found with 318.1 Å3 volume (Table IV). The
energy term of
G due to changes in the cavity
size can be expressed in terms of the cavity volume (100-140 J
mol
1 Å
3) (56). Using this parameter (120 J
mol
1 Å
3) the increment in stabilization of
the Pf-
-subunit due to the decrease in cavity volume
could be calculated to be 11.0 kJ/mol (Table IV), as compared with that
of the St-
-subunit.
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Table IV
Estimate of the difference in stability between -subunits from P. furiosus and S. typhimurium on the basis of structural information
GHP, GCAV,
GENT (= T S), and
GE1 represent G values due to
hydrophobic interaction, cavity volume, entropic effect, and
electrostatic interaction, respectively.
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Because the B-factor in x-ray analysis can correlate with
the flexibility of atoms, B-factors averaged for the
main-chain atoms of the Pf-
-subunit were
examined along with those of the St-
-subunit. Both data
were collected at room temperatures (293-300 K). The difference in
B-factors between both proteins (Fig.
8) shows that two parts of the
Pf-
-subunit, corresponding to peaks I and
II in Fig. 8, are more mobile than those of the
St-
-subunit. The residues of peaks I and
II are Tyr93 and Phe120,
respectively. The corresponding residues in the
St-
-subunit are Phe107 and
Glu134, which are the residues (or neighbor residues)
hydrogen bonding with the
2-subunit in the
2
2 complex. This suggests that these residues (Tyr93 and Phe120) in the
Pf-
-subunit might become less flexible when the
Pf-
-subunit associates with the
2-subunit.
The B-factor values in other parts of the Pf-
-subunit
were relatively lower than those of the St-
-subunit. This
may be due to enhanced thermal stability of proteins from hyperthermophiles in the standard state at 25 °C, caused by enhanced conformational rigidity in their folded native state (30).

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|
Fig. 8.
Differences in B-factors
(Å2) averaged for the main-chain atoms versus
residue number between -subunits of
tryptophan synthases from P. furiosus and S. typhimurium. B-factor subtracts
the values of the St- -subunit from those of the
Pf- -subunit. Peaks I and II
correspond to Tyr93 and Phe120 of the
Pf- -subunit, respectively.
|
|
 |
DISCUSSION |
The
-subunit of tryptophan synthase from the hyperthermophile,
P. furiosus, was remarkably stable as compared with those from mesophiles, E. coli and S. typhimurium.
Calorimetric results indicated that the higher stability is caused by
an entropic effect. This stabilization mechanism was investigated on
the basis of structural differences between
-subunits of P. furiosus and S. typhimurium.
Contribution of the Hydrophobic Interaction--
The hydrophobic
interaction is one of the important stabilizing forces of folded
protein structures (57). The hydrophobic effects have been extensively
analyzed by studies with site-directed mutagenesis, and it has been
revealed that hydrophobic residues in the interior of a protein
contribute to the conformational stability (58-63). Takano et
al. (64) have found a general rule for the relationship between
hydrophobic effect and conformational stability of a protein, using a
series of hydrophobic mutants of human lysozyme. Furthermore, the
change in unfolding Gibbs energy (
G) due to
hydrophobic effect between wild-type and mutant proteins
(
GHP) could be expressed using
changes in accessible surface area (ASA) of nonpolar and polar atoms
due to denaturation (65),
|
(Eq. 6)
|
where 
ASAnonpolar and 
ASApolar
represent the differences in
ASA of nonpolar and polar atoms of all
residues in a protein, respectively, upon denaturation between
wild-type and mutant proteins. Using the stability/structure data base
for a series of mutant human lysozymes, the parameters,
and
,
have been determined to be 0.178 and
0.013 kJ mol
1
Å
2, respectively (65). ASA values of proteins in the
native state can be calculated using x-ray crystal structures, and
those in the denatured state using their extended structures. For the
calculation of ASA, C/S atoms in residues were assigned to
ASAnonpolar and N/O to ASApolar.
We tried to estimate the difference in contribution of the hydrophobic
interaction between Pf- and St-
-subunits
(
GHP). ASA values using x-ray
crystal structures of the Pf- and St-
-subunits in the native state were calculated using the procedure of Connolly (55). The ASA values in the denatured states were calculated from their
extended structures of both proteins, which were generated from the
native structures using Insight II. As shown in Table IV, the
G values due to hydrophobic interaction
(
GHP) of the Pf-
-subunit was lower than that of
St-
-subunit: the

GHP value was
79.8 kJ/mol.
This means that the higher stability of the Pf-
-subunit
is not caused by the hydrophobic interaction. This fact could be
speculated from the comparison of the contents of hydrophobic residues
(Table I): the percent content of hydrophobic residues for the
Pf-
-subunit (53.6%) was lower than that for the
St-
-subunit (58.65%). Makhatadze and Privalov (66) have reported that protein folding is an enthalpically driven process caused
by van der Waals interactions between nonpolar groups in the interior
of a protein. In the case of the Pf-
-subunit, the higher
stability was not caused by enthalpic effects, suggesting that the
hydrophobic interactions due to nonpolar groups in the interior of the
protein do not dominate as compared with those of the
St-
-subunit. This is also confirmed by the result that the
Cp value of the
Pf-
-subunit (0.52 J K
1 g
1)
was lower than that for the St-
-subunit (0.64 J
K
1 g
1).
The ratio of hydrophobic residues (Val, Leu, and Ile) is generally
higher in hyperthermophile proteins than in mesophile ones (67). But it
is not certain that hydrophobic interactions are strengthened at higher
temperatures. Thermodynamic analysis of hydrophobic interactions (66)
indicates that at high temperature the entropy of hydration of a
nonpolar group decreases to zero and the Gibbs energy of the
hydrophobic interaction becomes completely enthalpic. This results in
the Gibbs energy of the hydrophobic interactions reaching its maximum
strength around 75 °C, which is lower than the optimum growth
temperatures of hyperthermophiles. This suggests that the hydrophobic
interaction does not always act efficiently for the stabilization of a
protein at extremely high temperatures over 100 °C.
Contribution of Ion Pairs (Salt Bridge)--
It seems that ion
pairs (salt bridges) have important roles in protein stability, because
they occur frequently in hyperthermophile proteins. However, Xiao and
Honig (23) have reported that the roles of individual ion pairs and ion
pair networks are variable, and among them some enhance protein
stability, whereas others reduce it. The contribution depends strongly
on the detailed environment of a particular network. Recently, using
computation of ionic interaction energies by solving the Poisson
equation in a continuum solvent medium, the ion pairs of glutamate
dehydrogenase from P. furiosus have been reported to be
highly important whereas those in the mesophilic homologs are only
marginally stabilizing (13). These reports suggest the necessity to
evaluate the strength of each ion pair in a protein. Therefore, we
calculated electrostatic free energies in the Pf-
-subunit
and the St-
-subunit solving the Poisson equations
numerically for their continuum.
As shown in Table IV, the electrostatic free energy in the native state
contributed by the direct interactions among ionic charges was much
less in the Pf-
-subunit than that in the
St-
-subunit. Because the Pf-
-subunit has
more ionizable residues than the St-
-subunit, the
reaction field energy in the native state of the
Pf-
-subunit was also much less than that of the
St-
-subunit. Thus, the ion pairs of the
Pf-
-subunit greatly contribute to the conformational
stability in the native state, as compared with those of the
St-
-subunit.
However, the stability of the protein should be accounted for by the
difference between the native state and the denatured state. Suppose
that almost all the protein atoms in the denatured states are well
exposed to the solvent molecules with the extended peptide chain
conformations. The reaction field energy should become a very large
negative value as the number of the ionizable residues increases.
Namely, the Pf-
-subunit in the denatured state was also
more stable than the St-
-subunit in the denatured state
due to the reaction field energy. Consequently, as shown in the
right-hand column of Table IV, 
E
between
E in the Pf-
-subunit and
that in the St-
-subunit is only 2 kJ/mol, which may not
explain all of the causes for the difference in the conformational
stabilities of the two proteins.
In this continuum model, both absolute free energy values in the native
states and in the denatured states were so large that even a slight
deviation in the parameters associated with the model would give a
large error without revising the parameter values. In the current
computation, it is not the comparison between the wild-type protein and
the point mutants as once calculated by Kumar et al. (13)
who neglected the electrostatic free energies in the denatured states.
But it is the comparison between proteins from different species with
very many substitutions of amino acid residues, and so the
electrostatics in the denatured state cannot be neglected.
Recently, Takano et al. (24) have estimated the strength of
an ionic interaction using a series of ion pair mutants of human lysozyme. Each contribution is not equivalent, but they have found that
the contribution correlates linearly with the solvent-inaccessibility of the ion pairs; the contribution of ion pairs is small when 100%
accessible, whereas it is about 9 kJ/mol if 100% inaccessible. Then,
using accessibility of ion pair atoms in Table III, the contribution due to electrostatic interactions of the Pf-
-subunit and
the St-
-subunit was calculated to be 80.4 and 37.7 kJ
mol
1, respectively
(
GEL in Table IV). This suggests
that ion pairs in the Pf-
-subunit play an important role
in conformational stability.
Contribution of the Entropic Effect--
One way of increasing the
stability of a protein (
G =
H
T
S) is to
lower its denaturation entropy (
S). The
substitutions of other residues by less flexible residues, Pro or Gly
(68-70), and the introduction of a disulfide bond (71) are confirmed to increase the stability of proteins. The conformational entropy of a
protein in the denatured state can be also lowered by shortening the
polypeptide chain. For thermophile proteins, a shortening of the N and
C termini and the reduction of loop sizes have been observed
(10, 28, 72, 73).
In the case of the Pf-
-subunit, 12 residues in the
N-terminal, six in the C-terminal, and one residue each in two loops
are deleted compared with the sequence of the St-
-subunit
(Fig. 5). Oobatake and Ooi (74) have estimated the difference in
thermodynamic parameters of each amino acid residue upon denaturation.
Using these parameters the difference in contribution due to
conformational entropy upon denaturation
(
T
S) between both proteins was calculated to
be 51.2 kJ mol
1 and 57.2 kJ mol
1, at 25 and
60 °C, respectively (
GENT in
Table IV). A great negative value of difference in enthalpy between
both proteins (
H =
330.2 kJ
mol
1 at 60 °C from Fig. 2) could be partly compensated
for by the difference in entropic effect (57.2 kJ mol
1).
This indicates that the entropic effect due to these deletions plays an
important role in the stabilization of the Pf-
-subunit. Recently, by analyzing many homologous relationships among many translated open reading frames of DNA from the 20 proteomes,
Thompson and Eisenberg (75) have reported that there is a general trend in nature for thermophile sequences to be shorter than their mesophile homologs. This finding has not yet received as much attention as have
ion pair networks (75). Present experimental results strongly support
this transproteomic evidence.
The N-terminal segments of most prokaryotic tryptophan synthase
-subunits are approximately the same length and contain a helix 0. However,
-subunits from thermophiles, Bacillus
stearothermophilus and Thermotoga maritime, appear to
lack eight and 16 residues, respectively, at their N termini, similar
to deletion of 12 residues in the Pf-
-subunit. These
deletions might be related to the higher stability due to entropic effect.
Conclusion--
The Pf-
-subunit from
hyperthermophile, P. furiosus, was especially stable in the
high temperature region as compared with homologous counterparts from
mesophiles. DSC experiments indicated that its higher stability was
caused by entropic factors. The tertiary structure of the
Pf-
-subunit could be analyzed to interpret these
thermodynamic characteristics showing thermostabilization of the
hyperthermophile protein. On the basis of structural information of the
Pf-
-subunit and the St-
-subunit, it could
be concluded that 1) the contribution of hydrophobic interaction of
Pf-
-subunit to stability was considerably lower than that
of the St-
-subunit, and 2) the higher stability of the
Pf-
-subunit was caused by a decrease in cavity volume in
the interior of the molecule, increase in ionic interaction (salt
bridge), and entropic effects due to shortening of the polypeptide chain.