The crystal structure of the tryptophan synthase b
2
subunit
from the hyperthermophile
Pyrococcus furiosus
Investigation of stabilization factors
Yusaku Hioki
1,2
, Kyoko Ogasahara
1
, Soo Jae Lee
1
, Jichun Ma
1
, Masami Ishida
3
, Yuriko Yamagata
4
,
Yoshiki Matsuura
1
, Motonori Ota
5
, Mitsunori Ikeguchi
6
, Seiki Kuramitsu
2
and Katsuhide Yutani
7,8
1
Institute for Protein Research, Osaka University, Japan;
2
Department of Biology, Graduate School of Science, Osaka University,
Japan;
3
Tokyo University Marine Science and Technology, Japan;
4
Graduate School of Pharmaceutical Sciences, Kumamoto
University, Japan;
5
Global Scientific Information and Computing Center, Tokyo Institute of Technology, Japan;
6
Graduate School of
Integrated Science, Yokohama City University, Japan;
7
Kwansei Gakuin University, Graduate School of Sciences, Hyogo, Japan;
8
RIKEN Harima Institute, HTPF, Hyogo, Japan
The structure of the tryptophan synthase b
2
subunit (Pfb
2
)
from the hyperthermophile, Pyrococcus furiosus,wasdeter-
mined by X-ray crystallographic analysis at 2.2 A
˚resolu-
tion, and its stability was examined by DSC. This is the first
report of the X-ray structure of the tryptophan synthase b
2
subunit alone, although the structure of the tryptophan
synthase a
2
b
2
complex from Salmonella typhimurium has
already been reported. The structure of Pfb
2
was essentially
similartothatoftheb
2
subunit (Stb
2
)inthea
2
b
2
complex
from S. typhimurium. The sequence alignment with secon-
dary structures of Pfband Stbin monomeric form showed
that six residues in the N-terminal region and three residues
in the C-terminal region were deleted in Pfb, and one residue
at Pro366 of Stband at Ile63 of Pfbwas inserted. The
denaturation temperature of Pfb
2
was higher by 35 Cthan
the reported values from mesophiles at pH 8. On the basis
of structural information on both proteins, the analyses of
the contributions of each stabilization factor indicate that:
(a) the higher stability of Pfb
2
is not caused by either a
hydrophobic interaction or an increase in ion pairs; (b) the
number of hydrogen bonds involved in the main chains of
Pfbis greater by about 10% than that of Stb, indicating that
the secondary structures of Pfbaremorestabilizedthan
those of Stband (c) the sequence of Pfbseems to be better
fitted to an ideally stable structure than that of Stb,as
assessed from X-ray structure data.
Keywords: calorimetry; crystal structure; hyperthermophile;
tryptophan synthase b
2
subunit; stability.
Prokaryotic tryptophan synthase (EC 4.2.1.20) is an a
2
b
2
complex composed of nonidentical aand bsubunits [1,2].
The a
2
b
2
complex with an abba arrangement [3] can be
isolated as the amonomer and b
2
subunits. The aand b
2
subunits catalyse inherent reactions, termed the aand b
reactions (Eqns 1 and 2), respectively. The physiologically
important reaction catalysed by the a
2
b
2
complex, termed
the ab reaction (Eqn 3), is the sum of the aand breactions:
areaction
indole-3-glycerol phosphate $indole
þd-glyceraldehyde 3-phosphate ð1Þ
breaction
l-serine þindole !l-tryptophan þH2Oð2Þ
ab reaction
l-serine þindole 3-glycerol phosphate !
l-tryptophan þd-glyceraldehyde 3-phosphate þH2O
ð3Þ
When the aand b
2
subunits associate to form the a
2
b
2
complex, the enzymatic activity of each subunit is syn-
chronically enhanced by one to two orders of magnitude [2].
The tryptophan synthase is a typical allosteric enzyme
whose activity is affected by the ligands [3–6]. Prokaryotic
tryptophan synthase has been studied extensively as an
excellent model system for investigating protein–protein
interaction mechanisms [2,7–10].
In order to elucidate the structural basis of the subunit
communication and mutual activation of the functions of
each subunit resulting from the formation of the a
2
b
2
complex, it is necessary to determine the three-dimensional
Correspondence to K. Yutani, RIKEN Harima Institute, HTPF,
Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
Fax: +81 791 58 2917, Tel.: +81 791 58 2937,
E-mail: yutani@spring8.or.jp
Abbreviations: ASA, accessible surface area; Eca, tryptophan synthase
asubunit from Escherichia coli;Ecb
2
, tryptophan synthase b
2
subunit
from E. coli;Pfa, tryptophan synthase asubunit from Pyrococcus
furiosus;Pfb
2
,tryptophansynthaseb
2
subunit from P. furiosus;Pfb,
monomer of tryptophan synthase b
2
subunit from P. furiosus; PLP,
pyridoxal 5¢-phosphate; Sta, tryptophan synthase asubunit from
Salmonella typhimurium;Stb
2
, tryptophan synthase b
2
subunit from
S. typhimurium;Stb, monomer of tryptophan synthase b
2
subunit
from S. typhimurium; RMSD, root mean square deviation.
Enzymes: prokaryotic tryptophan synthase (EC 4.2.1.20).
(Received 21 January 2004, revised 25 March 2004,
accepted 28 April 2004)
Eur. J. Biochem. 271, 2624–2635 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04191.x
structures of the aor b
2
subunits alone as well as that of the
complex. The three-dimensional structure of the tryptophan
synthase a
2
b
2
complex from Salmonella typhimurium was
determined by X-ray analysis in 1988 [3]. However, the
determination of the structure of the aor b
2
subunit alone
has not yet succeeded, although much effort has expended
on obtaining good quality crystals of the subunits from
mesophiles. Recently, the structure of the asubunit alone of
tryptophan synthase from a hyperthermophile, Pyrococcus
furiosus, was determined by X-ray analysis [11]. In this
report we describe the crystal structure of the b
2
subunit
alone of tryptophan synthase from P. furiosus.
Proteins from hyperthermophiles are remarkably stable
compared with homologous proteins from mesophiles
[12,13]. Three-dimensional structures of many proteins
from hyperthermophiles have been analysed to determine
the structural bases of unusually high stability [14–17].
Structural features of hyperthermophile proteins compared
with their mesophilic homologues vary depending on the
individual proteins. Hydrophilic factors such as ion pairs
and hydrogen bonds are superior in some proteins
[11,14,18–23], and hydrophobic interaction is favoured in
others [24,25]. The internal cavity decreases in hyperthermo-
phile proteins [25]. An entropic effect has been reported to
be important for enhanced stability [11].
However, the cause of the extremely high stabilization of
proteins from hyperthermophiles still remains unclear.
Elucidating the structural basis of the ultra-thermostability
of proteins is an important for understanding protein
folding problems, aspects of biotechnological applications,
and progress in structural genomics. Using mutant human
lysozymes Funahashi et al. [26,27] have proposed the
parameters of various stabilization factors estimated by
a unique equation, considering the relationship between
stability and conformational changes due to the mutations.
Using these parameters, the stabilization mechanism of
pyrrolidone carboxyl peptidase from P. furiosus has been
elucidated on the basis of its X-ray structure [17]. In this
report, the stabilization mechanism of the hyperthermophilc
b
2
subunit will be discussed on the basis of the crystal
structures, compared with the structural features of the
hyperthermophile and mesophile proteins.
Materials and methods
Purification of proteins
The b
2
subunit of tryptophan synthase from P. furiosus
(Pfb
2
) was overproduced in Escherichia coli strain JM109
(pb1837) [28]. Pfb
2
and the a-subunit of tryptophan
synthase from P. furiosus (Pfa) were purified as described
[29,11]. The equivalent subunits from E. coli (Eca,Ecb
2
)
were purified also [10,30,31]. All of the purified proteins
showedasinglebandonSDS/PAGE.
The protein concentrations were determined from the
absorbance at 278.5 nm using A
1%1cm
¼6.92 for Pfaand
10.18 for Pfb
2
[29], 4.4 for Eca[32] and 6.5 for Ecb
2
[33].
Enzymatic activity assay
The bactivity was measured by the disappearance of indole
using a phenol reagent [1] instead of the direct spectropho-
tometric assay ordinarily used [33], because temperature
control of the spectrophotometer was difficult above 80 C.
The assay was carried out in the presence of a 3 : 1 molar
excess of the asubunit over the bsubunit monomer. One
unit of activity is defined by the formation of 0.1 lmol of
product in 20 min at the indicated temperature [33].
DSC
DSC was carried out using an adiabatic differential
microcalorimeter, VP-DSC (Microcal) at a scan rate of
1CÆmin
)1
. Before making measurements, the protein
solution was dialysed against buffer with the composition
10 m
M
Gly/KOH, 1 m
M
EDTA, 0.02 m
M
pyridoxal
5¢-phosphate (PLP) (as described in Fig. 1). The dialysed
sample was filtered through a 0.22-lm pore size membrane
and then degassed in a vacuum. Protein concentrations
during the measurements were 0.5–1.5 mgÆmL
)1
.
Protein crystallization and data collection
The crystals were grown by a hanging drop vapour diffusion
at 10 C, by mixing 2 lL of the protein solution with 2 lL
of a reservoir solution containing 12% (w/v) PEG 20 000
and 100 m
M
Mes, pH 6.5. The concentration of Pfb
2
was
10–12 mgÆmL
)1
in 20 m
M
Tris/HCl pH 8.5 containing
100 l
M
dithioerythritol and 20 l
M
PLP.
Diffraction experiments with the Pfb
2
crystal were
performed at the beam line, BL44XU and BL411XU at
SPring8. The crystal belonged to the orthorhombic space
group of P2
1
2
1
2
1
with unit cell dimensions of a ¼84.8, b¼
110.5, c¼160.0 A
˚. The value of the Matthews coefficient is
2.2 A
˚
3
ÆDa
)1
for two Pfb
2
per asymmetric unit, correspond-
ing to a solvent content of 44.0%. The crystals were flash-
cooled in a cold nitrogen gas stream immediately after
cryoprotection by addition of the reservoir solution con-
taining 25% (w/v) glycerol to the crystallization buffer at
Fig. 1. pH dependence of the denaturation temperature of Pf b
2
.The
denaturation temperature, T
d
, represents the peak temperatures of
DSC curves observed at a scan rate of 1 CÆmin
)1
.d,sand m
represent Pfb
2
,Ecb
2
,andStb
2
, respectively. The buffer conditions were
10 m
M
Gly-KOH with 1 m
M
EDTA and 0.02 m
M
PLP. The pH
indicates the values after DSC measurements. The data for Ecb
2
and
Stb
2
are those reported in [29] and [35] respectively.
FEBS 2004 Crystal structure of tryptophan synthase b
2
subunit alone (Eur. J. Biochem. 271) 2625
100 K. This crystal diffracted to a maximum of 2.2 A
˚and
was suitable for structure determination.
The data collected were processed and integrated by
DENZO
andscaledby
SCALEPACK
[34]. Data collection
statistics are summarized in Table 1.
Structure determination and refinement
The dimeric structure (Stb
2
)ofthebsubunit in the
tryptophan synthase a
2
b
2
complex (Sta
2
b
2
)from
S. typhimurium (1BKS) [3] provided the initial model for
molecular replacement solutions using
AMORE
. The cross-
rotation function showed two peaks for the two-dimer
molecules. The model was subjected to cycles of rigid body
refinement using noncrystallographic symmetry (NCS): the
four bsubunit molecules in the asymmetric unit were refined
using NCS restraints. The experimental map at 2.2 A
˚was of
high quality and allowed unambiguous modelling of all
residues 1–388. The model was built using O and refined by
energy minimization, simulated annealing and restrained
B-factor refinement procedures with NCS. Successive
refinement with temperature factors and addition of
solvents resulted in an R-value of 22.0% and an R
free
of
26.4% for all reflections in the resolution range 100–2.2 A
˚.
R
free
was calculated with 10% of the reflections. The current
model consists of four chains of residues 1–388 of Pfband
193 water molecules per asymmetric unit. All residues are
within the most favoured (89.7%) and additional allowed
regions (10.3%) of the Ramachandran plot. Refinement
statistics are summarized in Table 1. The final coordinates
have been deposited in the Protein Data Bank (PDB
accession no. 1V8Z).
Results
Thermal stability and enzymatic activity of
Pf
b
2
Figure 1 shows the pH dependence of the denaturation
temperatures of Pfb
2
measured by DSC. The heat denatur-
ation of Pfb
2
was not reversible. The peak temperatures of
the DSC curves above pH 6.5 were around 115 C
independent of pH, which were higher by about 35 Cthan
those reported for mesophilic proteins [29,35]. DSC meas-
urements could not be carried out between pH 6 and 4,
because the protein became turbid on heating. Below pH 4,
the denaturation temperatures decreased markedly. Ultra-
centrifugation analysis of Pfb
2
indicates that the apparent
molecular weight of the protein, which exists in a dimeric
form in solution around pH 7, decreases with decreasing
pH below 4.0, resulting in dissociation to a monomer at
pH 3.0 [29]. This suggests that the decreased denaturation
temperature below pH 4.0 is correlated with the dissociation
from a dimer to a monomer. The mesophilic protein of
E. coli (Ecb
2
)wasdenaturedintheacidicregion.
The enzymatic activities of Pfb
2
and Ecb
2
were measured
at various temperatures in the presence of excess asubunit
from P. furiosus or E. coli (Fig. 2B). The activity for Ecb
2
rapidly decreased at temperatures above 55 C. This
decrease might be due to thermal denaturation of Ecain
a
2
b
2
complex, because the denaturation temperature of Eca
is around 55 C, although Ecb
2
denatures at 80 C [29]. It
has also been reported that Stain the complex is inactivated
by 50% at 55 C, whereas 50% inactivation of Stb
2
occurs
at 80 C [4]. The activity of Pfb
2
at the physiological
temperature of mesophiles was negligible, although the
specific activity for Pfb
2
around 90 C was comparable with
that of Ecb
2
around 50 C. This was in marked contrast to
the result with a hyperthermophilic pyrrolidone carboxyl
peptidase from P. furiosus, which exhibits higher specific
activity over a broad range of temperature than the
corresponding mesophilic protein [13].
The Arrhenius plots of the activity for Pfb
2
were clearly
divided into two lines at a boundary around 45 C
(Fig. 2A). The low-temperature portion showed a much
higher slope than the high-temperature portion. The
Arrhenius activation energies (Ea) of Pfb
2
calculated from
the slopes were 215.4 and 54.6 kJÆmol
)1
for the low- and
high-temperature portions, respectively. The Ea values of
Pfb
2
, especially in the low-temperature portion, were higher
than that for Ecb
2
(135.7 and 43.0 kJÆmol
)1
, respectively)
which also showed biphasic Arrhenius plots (Fig. 2A). The
Ea values for Ecb
2
were similar to those of bactivity of
tryptophan synthase (Sta
2
b
2
)fromS. typhimurium reported
[5,36]. Based on the effect of temperature on the catalytic
properties for Sta
2
b
2
and Stb
2
in the presence of monova-
lent cations and an allosteric ligand, Fan et al. have shown
that biphasic Arrhenius plots are caused by a temperature-
dependent conformational change from a low-activity
openconformation to a high-activity closedconformation
[36].ItseemsthatthePfb
2
is also converted from a low
activity conformation to a highly active one by increasing
temperature.
Table 1. Data collection and refinement statistics of the tryptophan
synthase bsubunit from P. furiosus.
Characteristics of the crystals
Space group P2
1
2
1
2
1
Cell parameters
a(A
˚) 84.8
b(A
˚) 110.5
c(A
˚) 160.0
Z16
V
m
(A
˚
3
ÆD
a
)1
) 2.2
Solvent content (%) 44
Data collection
Resolution (A
˚) 2.2
No. of unique reflections 75 098
Average redundancy 9.2
R
merge
(%)
a,b
5.3 (27.6)
Completeness (%)
a
98.7 (97.0)
Refinement statics
Resolution (A
˚) 67–2.2
No. of reflections 75 065
R
factor
(%)
c
20.8
R
free
(%)
d
26.3
RMSDs
RMSD lengths (A
˚) 0.006
RMSD angles () 1.3
a
Values within parentheses are for the last shell of data.
b
R
merge
¼
S
h
S
i
|(I
h
–I
hi
)|/S
h
S
i
I
hi
* 100.
c
R
factor
¼S||F
obs
|–|F
calc
||/S|F
obs
|*
100.
d
R
free
¼S||F
obs
|–|F
calc
||/S|F
obs
| * 100 where |F
obs
| are test set
amplitudes (10%) not used in refinement.
2626 Y. Hioki et al. (Eur. J. Biochem. 271)FEBS 2004
Amino acid composition of the bsubunit from
P. furiosus
Table 2 shows the amino acid compositions of both b
monomers of Pfb
2
and Stb
2
(Pfband Stb, respectively). Pfb
consists of 388 residues, but Stbhas 397. The content (%) of
hydophobic residues for Pfbwas similar to that for Stb,
although the number of hydrophobic amino acid residues of
Pfbwas slightly lowered. The number of hydrophilic
residues increased from 110 (27.71%) to 121 (31.19%) in
Pfb, compared with that of Stb. The number of neutral
residues of Pfbwas largely reduced from 73 (18.39%) to
57 (14.69%). In the case of the asubunit of tryptophan
synthase from P. furiosus, hydrophobic residues were
remarkably reduced from 58.58% to 53.93%, compared
with those from S. typhimurium [11].
Overall structure of the bsubunit from
P. furiosus
The structure of the bsubunit from P. furiosus was observed
as a dimeric form in which the two bsubunits are tightly
associated over a broad surface. The buried surface at the
interface between the two subunits was estimated to be
3945 A
˚
2
(Table 3). The dimer structure is depicted by the
ribbon drawing in Fig. 3A. The subunit structure consists
of two domains, N (residues 1–46, 81–200) and C (residues
47–80, 201–388) domains of almost equal size. The
N-terminal (1–200), and the C-terminal (201–388) residues
are coloured red and blue, respectively. The core of the N
domain is formed from four strands which are surrounded
by seven helices. The core of the C domain constitutes six
strands with five parallel strands and one antiparallel strand.
A short piece (residues 47–80) of the N-terminal residues
intrudes into the C domain, forming the first two strands of
ab-sheet at the centre of the C domain. A helical structure
(residues 58–64) between the first two strands is clearly
observed in Pfbalthough it is not reported in Stb. Arrows in
Fig. 3A point to the first two strands and one helical
structure (residue 58–64) that intrude into the C domain.
The coenzyme PLP is located in the deep cleft between the
two domains. PLP forms a Shiff base with the e-amino
group of Lys82 in Pfb, corresponding to Lys87 in an active
site of Stb. The overall topology of Pfbwas equivalent to
the bsubunit monomer in the Sta
2
b
2
complex reported by
Hyde et al.[3].
Structural comparison of
Pf
band
St
b
Fig. 4 shows the secondary structure-based sequence align-
ment using the secondary structure elements assigned by
Fig. 2. Temperature dependence of the specific enzymatic activities of
the breaction for the Pfb
2
subunit (d) and the Ecb
2
subunit (s) at
pH 7.0. Activities for Pfb
2
and Ecb
2
were measured in the presence of
an excess of the asubunit from P. furiosus and E. coli, respectively.
(A) Arrhenius plots of the data from (B). (B) Comparison of the
activities of Pfb
2
and Ecb
2
.
Table 2. Comparison of the amino acid compositions of the tryptophan
synthase bsubunit monomers from P. furiosus and S. typhimurium.
Values within parentheses are for the percentage of residue per total
number of residues.
Residue
Pfb
a
Stb
b
D(Pfb–Stb)
Residue
number
Residue
number
Differences in
residue number
Hydrophobic 210 (54.12) 214 (53.90) )4()0.22)
Gly 42 (10.82) 43 (10.83) 1 ()0.01)
Ala 38 (9.79) 43 (10.83) )5()1.04)
Val 30 (7.73) 19 (4.79) 11 (2.94)
Leu 35 (9.02) 38 (9.57) )3()0.55)
Ile 23 (5.93) 24 (6.05) )1()0.12)
Met 11 (2.84) 15 (3.78) )4()0.94)
Phe 13 (3.35) 13 (3.27) 0 (0.08)
Trp 3 (0.77) 1 (0.25) 2 (0.52)
Pro 15 (3.87) 18 (4.53) )3()0.66)
Neutral 57 (14.69) 73 (18.39) )16 ()3.70)
Ser 15 (3.87) 19 (4.79) )4()0.92)
Thr 16 (4.12) 21 (5.29) )5()1.17)
Asn 13 (3.35) 11 (2.77) 2 (0.58)
Gln 12 (3.09) 17 (4.28) )5()1.19)
Cys 1 (0.26) 5 (1.26) )4()1.00)
Hydrophilic 121 (31.19) 110 (27.71) 11 (3.48)
Asp 17 (4.38) 18 (4.53) )1()0.15)
Glu 30 (7.73) 28 (7.05) 2 (0.68)
Lys 30 (7.73) 19 (4.79) 11 (2.94)
His 11 (2.84) 14 (3.53) )3()0.69)
Arg 16 (4.12) 19 (4.79) )3()0.67)
Tyr 17 (4.38) 12 (3.02) 5 (1.36)
Total number
of residues
388 397 )9
Amino acid compositions were taken from:
a
Ishida et al. [28] and
b
Hyde et al. [3].
FEBS 2004 Crystal structure of tryptophan synthase b
2
subunit alone (Eur. J. Biochem. 271) 2627
DSSP [37]. The sequence homology between Pf band Stb
is 58.5%. The alignment indicates that six residues in the
N-terminal domain and three residues in the C-terminal
domain were deleted in Pfb. Pro366 of Stband Ile63 of Pfb
were inserted in each protein. Fig. 5 shows a schematic
stereo view of superimposed bmonomer structures of the
tryptophan synthase b
2
from P. furiosus and S. typhimu-
rium. The most different part is an a-helical structure
around position 60 of Pfbin place of a turn structure in Stb
(anarrowinFig.5).
Structures of N and C domains. The structures of Pfb
and Stb(1BKS) could be superimposed with a root
mean square deviation (RMSD) of 1.181 A
˚between 385
equivalent Caatoms in both monomers (Fig. 6). The
RMSD values of only the N domain (168 residues) and
C domain (185 residues) were 0.596 and 1.003 A
˚,
respectively. These results indicate that the structures of
both bmonomers show a smaller deviation compared
with that of the Pfasubunit (RMSD ¼2.82 A
˚)[11],
especially for the N domain of the bsubunits, because of
higher sequence identity. The sequence identities between
Pfband Stbin the N and C domains are 64.5 and 54.1,
respectively, while that of Pfaand Stais 31.5%. As
shown in Fig. 6, two large deviations are found in peaks
II and IV. In the case of peak II, Ile63 is inserted in Pfb
and the region from Lys57 to Ile63 of Pfbclearly forms
the a-helix, although the corresponding region of Stbis
judged to be in a turn. There is no sequence identity
except for one residue (Thr) in this region (Fig. 4). At
peak IV, one residue of Pfbat Pro366 of Stbis deleted
in a turn region, and there is also no sequence identity
between residues 360 and 367 of Pfb(Fig. 4). The
deviations of the other two peaks I and III are not great,
less than about 3 A
˚. These regions are slightly decreased
in sequence identity compared with the others.
The core region of the N domains of Stbhas been
reported to have a conformation similar to that of the C
domain [3]. To estimate the structural similarity between
the N and C domains in Pfb, the RMSD values of the
structurally homologous region of the two domains were
calculated using 73 Capairs corresponding to the residues
of Stb, which are reported to deviate by less than 4.0 A
˚
between both domains. The values were 2.7 and 2.4 A
˚for
Pfband Stb, respectively. That for Stbwas quite similar to
that reported (2.2 A
˚)[3].AsshowninFig.3B,theoverall
topology of the N and C domains in Pfbis similar, and
especially, a four-stranded b-sheet structure is well super-
imposed. In order to superimpose the C domain on the N
domain, the C domain had to be rotated 165.2about an
axis and moved by 26.0 A
˚between the centroids of the two
domains for Pfband 160.5and 26.6 A
˚for Stb, respectively.
This slight difference might be due to the differences in the
structures of the b
2
subunit alone and the a
2
b
2
complex,
although the complex structure from P. furiosus has not yet
been solved.
Active site. The X-ray crystal structure of the Sta
2
b
2
complex indicates the presence of a 25-A
˚long hydrophobic
tunnel connecting aand bactive sites through which the
metabolic intermediate of the areaction, indole, would be
transferred from the asubunit to the bsubunit. The residues
Table 3. Estimate of the difference in stability between tryptophan synthase bsubunits from P. furiosus and S. typhimurium on the basis of structural
information. ASA values were calculated for Pfband Stbwithout PLP. DDG
HP,
DDG
HB,
DDG
CAV,
and DDG
ENT
represent the difference of DG
values between Pfband Stb,due to hydrophobic interaction, hydrogen bond, cavity volume, and entropic effect, respectively. Monomer/dimer
represents the values calculated using monomer and dimer forms of bsubunit, respectively. The positive value of DGmeans that the protein from
P. furiosus is more stable than the other.
PfbStbD(Pfb–Stb)
Total number of residues 388 397 )9
ASA value (N-state)
C/S atoms (monomer/dimer) 8095/13 700 A
˚
2
7788/13 208 A
˚
2
307/419 A
˚
2
N/O atoms (monomer/dimer) 7404/13 353 A
˚
2
7259/13 266 A
˚
2
145/87 A
˚
2
ASA value (
D
-state)
C/S atoms (monomer) 33 838 A
˚
2
34 093 A
˚
2
)255 A
˚
2
N/O atoms (monomer) 19 935 A
˚
2
20 161 A
˚
2
)226 A
˚
2
DASA value (D–N)
C/S atoms (monomer/dimer) 25 743/53 976 A
˚
2
26 305/54 905 A
˚
2
)562/)929 A
˚
2
N/O atoms (monomer/dimer) 12 531/26 517 A
˚
2
12 902/27 056 A
˚
2
)371/)539 A
˚
2
Surface area buried at b/binterface
C/S atoms 2490 A
˚
2
2295 A
˚
2
195 A
˚
2
N/O atoms 1455 A
˚
2
1252 A
˚
2
203 A
˚
2
Cavity volume (monomer/dimer) 292/595 A
˚
3
343/734 A
˚
3
)51/)139 A
˚
3
Secondary structure content
(a-helix/b-sheet) 44.3/19.6% 40.3/19.4% 4.0/0.2%
Contribution of various factors to the stability
DG
HP
(monomer/dimer) )76.9/)129.1 kJ mol
)1
DDG
HP b/binterface
24.7 kJ mol
)1
DDG
HB
(monomer) 291.0 kJ mol
)1
DDG
CAV
(monomer/dimer) 2.7/7.2 kJ mol
)1
DDG
ENT
(–TDS) (25/100 C) 101.7/127.3 kJ mol
)1
2628 Y. Hioki et al. (Eur. J. Biochem. 271)FEBS 2004
