Investigation of the Structural Basis for Thermostability of
DNA-binding Protein HU from Bacillus
stearothermophilus*
Shunsuke
Kawamura
,
Yoshito
Abe§,
Tadashi
Ueda§,
Kiyonari
Masumoto§,
Taiji
Imoto§,
Nobuyuki
Yamasaki
, and
Makoto
Kimura
¶
From the
Laboratory of Biochemistry, Faculty of
Agriculture, Kyushu University, Fukuoka 812-81 and
§ Graduate School of Pharmaceutical Sciences, Kyushu
University 62, Fukuoka 812-81, Japan
 |
ABSTRACT |
Site-directed mutagenesis was used to identify
amino acid residues essential for the thermostability of the
DNA-binding protein HU from the thermophile Bacillus
stearothermophilus (BstHU). Two mutants,
BstHU-A27S and BstHU-V42I, in which
Ala27 and Val42 in BstHU were
replaced by the corresponding amino acids Ser27 and
Ile42, respectively, in the homologue from a mesophile
B. subtilis (BsuHU), were less stable than the
wild-type BstHU (63.9 °C), showing Tm values of
58.4 °C and 60.1 °C, respectively, as estimated by circular
dichroism (CD) analysis at pH 7.0. The denaturation of two mutants was
further characterized using differential scanning calorimetry; the
Tm values obtained by calorimetric analysis were in good
agreement with those estimated by CD analysis. The results suggest that
Ala27 and Val42 are partly responsible for
enhancing the thermostability of BstHU. When considered
together with previous results, it is revealed that Gly15,
Ala27, Glu34, Lys38, and
Val42 are essential for the thermostability of thermophilic
protein BstHU. Moreover, five thermostabilizing mutations
were simultaneously introduced into BsuHU, which resulted
in a quintuple mutant with a Tm value of 71.3 °C, which is
higher than that of BstHU, and also resulted in
insusceptibility to proteinase digestion.
 |
INTRODUCTION |
A thorough understanding of molecular rules governing
thermostability of proteins should provide invaluable insight into
basic principles for a design of novel proteins by protein engineering. Both theoretical and experimental approaches have been undertaken to
examine the thermostability of proteins and many different structural
principles have been postulated for increased thermostability (for
reviews, see Refs. 1-3). One convincing approach is a comparative study on proteins that are available from organisms living under different temperature conditions. Recently, Vogt et al.
statistically compared the amino acid sequences and tertiary structures
of mesophilic and thermophilic organisms and suggested that the
increased hydrogen bonds and ion pairs may provide the most general
explanation for thermostability in proteins (4). We have pursued the
investigation of the structural basis of thermostability of proteins
using a histone-like bacterial DNA-binding protein HU
(HU)1 as a model protein.
The HU is a small basic polypeptide chain composed of 90-92 amino
acids and occurs as a homotypic dimer in solution (5). The HU binds to
DNA in a sequence-independent manner and has been thought to play an
important role in the structure of the bacterial nucleoid, being
involved in replication (6), inversion (7), transposition (8), and
repair (9) as a DNA chaperon (10). Bacillus
stearothermophilus HU (BstHU) is composed of 90 amino acids, and its tertiary structure has been extensively studied by both
x-ray crystallographic (11, 12) and NMR spectroscopic (13, 14) methods.
The BstHU has two distinct halves; the N-terminal half
consists of two
-helices (
1 and
2), which are connected by a
broad turn to create a V-shaped supersecondary structure (HTH motif),
while the C-terminal half consists mainly of a three-stranded antiparallel
-pleated sheet. In a dimeric form, a pair of HTH motifs
are entangled with each other to form a tightly packed hydrophobic core
domain. In addition to BstHU, we have isolated three
homologous HUs from mesophilic (Bacillus globigii and
Bacillus subtilis) and thermophilic (Bacillus
caldolyticus) bacilli, and their amino acids have been sequenced
(15, 16). On the basis of sequence comparison of the four HUs and the
known three-dimensional structure of BstHU, the relative
thermostability with respect to amino acid differences between the four
proteins was discussed (16). This study revealed 11 amino acid
substitutions between the thermophilic and mesophilic proteins, which
are almost all restricted to the molecular surface not implicated in
the mode of DNA binding. Thus, it could be expected that these amino
acid substitutions might give rise to additional hydrogen bonds and/or salt bridges that would contribute to the thermostability of the thermophilic HU. To assess the contribution of the individual amino
acids to the thermostability of BstHU, we constructed
BstHU mutants, in which the amino acids were individually
replaced with the corresponding amino acids in B. subtilis
HU (BsuHU) and evaluated their thermostability. Previously,
it has been shown that Gly15 in the bend between two
-helices (
1 and
2) and Glu34 and Lys38
occurred at the molecular surface, significantly contributing to the
thermostability of BstHU (17, 18).
To this end, we extend this comparative study to the remaining six
amino acid replacements: BstHU Ala27 to
BsuHU Ser27, Ser31 to
Thr31, Val42 to Ile42,
Ala56 to Ser56, Met69 to
Ile69, and Lys90 to
Ala90-Gly91-Lys92. In the crystal
structure of BstHU (Fig. 1),
Ala27 resides on the outside of the second
-helix (
2)
and is exposed to solvent. Since the Ala residue is a strong
helix-forming amino acid, it could be suggested that Ala27
in BstHU would contribute to thermostability by stabilizing
the second
-helix (
2). Ser31 in BstHU is
located on the solvent-facing surface of the second
-helix, and its
side chain forms a hydrogen bond to the main chain carbonyl of
Ala27. Since an equivalent hydrogen bond is probably formed
through the side chain of the Thr27 residue in the
mesophile BsuHU, the replacement from Ser27 to
Thr was thought to be neutral. The Val42 in
BstHU is in the
1-strand and is buried in the interior of the protein. It was therefore suggested that the larger side chain Ile
in BsuHU would produce structural strain in the tightly
packed core, resulting in destabilization of the mesophilic protein. Ala56 and Met69 in BstHU are
substituted by Ser and Ile, respectively, in BsuHU. The
former residue is on the outgoing
-ribbon closed to the disordered arms of the BstHU, and the latter is in the returning part
of the disordered arm in BstHU. These residues are expected
to be accessible to solvent and, therefore, were thought to have little effect on the thermostability of the protein. The C-terminal
Lys90 in BstHU is replaced by the tripeptide
Ala90-Gly91-Lys92 in
BsuHU. It is shown that the C terminus is disposed on the molecular surface, and thus the significance of this replacement was
unclear.

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Fig. 1.
Tertiary structure of the monomer of
BstHU. The protein model is from Tanaka et
al. (11). Side chains are shown only for residues where the
contribution to the thermostability of BstHU was examined in
this study. It should be noted that the residues in the distal region
of the arm between strands 2 and 3 (59-70) were tentatively included
in this figure.
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In the present study, we reveal that Ala27 and
Val42 are essential for the thermostability of the
BstHU. Furthermore, on the basis of the present and previous
results, we construct a quintuple mutant HU using the BsuHU
gene as a prototype and discuss its hyperthermostability and resistance
to proteolytic degradations.
 |
EXPERIMENTAL PROCEDURES |
Genetics--
We previously described the cloning, sequencing,
and expression of the genes encoding BstHU and
BsuHU (19). All genetic procedures, including site-directed
mutagenesis were performed as described earlier (17, 18). The
oligonucleotide primers used in this study are as follows: for
BstHU-A27S, 5'-GCCGTTGATTCGGTGTTTGATTCG-3'; for
BstHU-S31T, 5'-GTGTTTGATACGATTACAGAAGCGC-3'; for
BstHU-V42I, 5'-GCGAAAAGGCGATAAAATCCAATTAATCGG-3'; for
BstHU-A56S, 5'-CGCGAGCGCTCCGCCCGGAAAGGACG-3'; for
BstHU-M69I, 5'-GGCGAAGAAATTGAAATTCCGGCAAGC-3'; for
BstHU-K90AGK, 5'GCATTGAAAGATGCCGTCGCCGGAAAGTAAAAGCTTGG-3';
for BsuHU-S27A, 5'-CGTATCAAAACAGCGTCAACTGC-3'; and for
BsuHU-I42V, 5'-CCGATCAGTTGGACTTTATCACCG-3'.
Purification and Characterization of Proteins--
All
procedures used for production, purification, and SDS-PAGE analysis of
the recombinant proteins BstHU and BsuHU and the mutants thereof have been described previously (17, 18).
Thermostability of the protein was determined by monitoring the change
in circular dichroism (CD) at 222 nm as a function of temperature, and
thermodynamic parameters were obtained as described in Refs. 17 and 18. Since it was reported that the protein BsuHU showed the
strong dependence of the CD spectral properties and stability under a variety of conditions (20, 21), we measured all proteins using the same
procedure and the same type of spectrophotometer, as described in
previous papers (17, 18).
Differential Scanning Calorimetry (DSC)--
Calorimetric
measurements were carried out with a VP-DSC (MicroCal Inc.,
Northampton, MA) microcalorimeter with an personal computer. The scan
rate was 1.0 K/min. Sample solutions for DSC measurements were prepared
by dialyzing of HU proteins dissolved in water against 0.05 M phosphate buffer at pH 7.0 exhaustively. The protein
concentrations were 22-33 µM. The concentrations of the
protein solutions were determined using amino acid analysis of proteins
after acid hydrolysis. Data analysis was done using the Origin software
(MicroCal).
NMR Spectra--
1H NMR spectra were recorded at 600 MHz with a Varian Unity Plus spectrometer. All NMR measurements were
carried out at pD 7 and 25 °C. Dioxane was employed as the internal
standard (3.743 ppm). The pD values were the pH meter readings without
adjustment for isotope effects.
Proteolysis--
Tryptic and chymotryptic digestions of the
proteins (1 mg/ml) were carried out at 37 °C in 0.1 M
Tris-HCl, pH 8.0, with an enzyme:substrate ratio of 1:1000 and 1:5000
(w/w), respectively. Proteolytic digestion was assayed by measuring the
change in the amount of the uncleaved protein, which was separated by
reverse-phase HPLC on a YMC-gel C4 column (4.6 × 250 mm)
equilibrated with 0.1% trifluoroacetic acid. The protein was eluted
with a linear gradient of 0-56% acetonitrile in 0.1% trifluoroacetic
acid for 30 min. The effluents were monitored by absorption at 220 nm.
 |
RESULTS AND DISCUSSION |
Overproduction and Characterization of Mutant Proteins--
Six
BstHU mutant proteins, designated as BstHU-A27S,
BstHU-S31T, BstHU-V42I, BstHU-A56S,
BstHU-M69I, and BstHU-K90AGK, were engineered, in
which Ala27, Ser31, Val42,
Ala56, Met69, and Lys90 were
changed to the corresponding amino acid residues in BsuHU. Expression of the mutated cDNAs was performed in Escherichia
coli BL21 (DE3) cells, using the T7 system as described previously (17, 18). All mutant proteins were purified from the soluble fractions
of cells so as to give a single band on SDS-PAGE. Their behaviors
during the purification steps were almost identical with that of the
wild type protein. The yields of protein from a 1-liter culture were
15-20 mg. The integrity of the mutant protein was confirmed by
measurements of far-ultraviolet CD as described previously. The CD
spectrum of each mutant was almost indistinguishable from that of the
wild type, indicating that none of these mutations affected the
backbone conformation (data not shown).
Thermostabilities of Mutant Proteins--
To define the amino acid
replacements responsible for the thermostability of BstHU,
the thermostabilities of the mutant proteins were analyzed by
monitoring the change in the CD value at 220 nm as a function of
temperature. Fig. 2A shows the
thermal denaturation curves of the wild type and the mutant proteins.
In all cases, the unfolding transitions appeared to be monophasic,
suggesting the absence of a folding intermediate in thermal denaturing
process of global structure. Thus, on the basis of the assumption that the wild type BstHU and its mutants are denatured with a
two-state model, thermodynamic parameters were calculated from the
thermal denaturation curves, as summarized in Table
I. Of five mutant proteins, the mutants
BstHU-A27S and BstHU-V42I were less stable by
5.5 °C (
G =
4.97 kJ/mol) and
3.8 °C
(
G =
3.48 kJ/mol), respectively, in Tm
than the wild type BstHU. This result suggests that
Ala27 and Val42 are the key residues to provide
the extra thermostability to BstHU.

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Fig. 2.
Thermal unfolding curves for
BstHU, BsuHU, and their mutant proteins.
A, temperature dependences of [ ]222 nm
values of BstHU ( ) and its mutants, BstHU-A27S
( ), BstHUS31T ( ), BstHU-V42I (+),
BstHU-A56S (×), BstHU-M69I ( ), and
BstHU-K90AGK ( ). B, temperature dependences of
[ ]222 nm values of BsuHU ( ) and its
mutants, BsuHU-S27A ( ) and BsuHU-I42V
( ).
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Table I
Parameters characterizing the thermal denaturations of BstHU,
BsuHU, and their mutants
Thermodynamic parameters were calculated from the thermal denaturation
curves described in Fig. 2. The table summarizes the results of four
independent experiments.
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The contributions of Ala27 and Val42 to the
thermal stabilization of BstHU were corroborated by
constructing the mesophilic mutants BsuHU-S27A and
BsuHU-I42V, in which Ser27 and Ile42
in BsuHU were conversely replaced by Ala and Val,
respectively, and the resulting mutants were characterized in terms of
their thermostabilities. As shown in Fig. 2B and Table I,
the Tm values of BsuHU-S27A and
BsuHU-I42V were increased by 5.6 °C
(
G = 3.07 kJ/mol) and 4.0 °C
(
G = 2.19 kJ/mol), respectively, compared with
that of the wild-type BsuHU, demonstrating that these two amino acids, Ala27 and Val42, are both
responsible for enhancing the thermostability of the BstHU.
In contrast, the stabilities of BstHU-A56S and
BstHU-M69I were almost identical to that of the wild type,
and the mutants BstHU-S31T and BstHU-K90AGK were
slightly more stable than the wild type. Two residues Ala56
and Met69 are located in the solvent-exposed arm region. In
the crystal structure of BstHU, the top part of the arm is
not visible because of its flexible nature. It was therefore concluded
that these substitutions in a mobile, solvent-exposed environment do
not affect the conformational stability. The Tm values of the
mutants BstHU-S31T and BstHU-K90AGK were higher
by 1.9 °C and 1.8 °C than that of the wild type BstHU.
The crystal structure of the BstHU shows that
Ser31 resides on the solvent-facing surface, clustering
together with Thr13 and Ala27, and forms a
hydrogen bond to the main chain carbonyl of Ala27.
Therefore, the increased stability of BstHU-S31T might be
due to the rearrangement of a hydrogen bond formed between the side chain oxygen of the substituted Thr31 and the main chain
carbonyl of Ala27. Another plausible interpretation is that
the introduction of the larger side chain might give some preferential
van der Waals contacts with Thr13 and/or Ala27.
The mutant BstHU-K90AGK was also stable as compared with the wild type. At present, no explanation for thermostable property of this
mutant has been obtained.
In order to understand the molecular basis for destabilization of the
mutant proteins, BstHU-A27S and BstHU-V42I, their
structural features were examined by 1H NMR spectroscopic
analysis. As a result, the chemical shift change of some resonances
were observed around aromatic regions at 6.2, 6.26, and 7.0 ppm in
BstHU-A27S and 6.2, 6.28, and 6.49, and 7.0 ppm in
BstHU-V42I, as compared with that of the wild-type HU (data
not shown). Although the assignment of these resonances in the
BstHU has still not been reported, Kakuta classified these to be derived from Phe residues (Phe29, Phe47,
Phe50, and Phe79) in BstHU by
DQF-COSY.2 It is therefore
likely that the replacement of Ala27 or Val42
by Ser or Ile, respectively, may cause rearrangement of some Phe
residues occupying the interior of the molecule.
DSC Measurements of BstHU and Its Mutant Proteins--
The present
study, together with previous studies (17, 18), suggests that five
amino acid residues, Gly15, Ala27,
Glu34, Lys38, and Val42, are
essential for thermostability of BstHU. To corroborate their involvement in thermostability, the thermal denaturations of five mutants (BstHU-G15E, BstHU-A27S,
BstHU-E34D, BstHU-K38N, and
BstHU-V42I) as well as the wild type were further
characterized by DSC measurements. A typical excess heat capacity curve
of the wild type BstHU is shown in Fig.
3. The examined proteins, as is the case
for the BstHU, gave single peaks in calorimetric
measurements; the Tm values of the wild type and its five
mutants could be calculated from these curves, as given in Table
II. This measurement clearly showed the
thermal destabilization of five mutant proteins as compared with the
wild type and demonstrated the involvement of five amino acid residues
in the thermostability of BstHU. Interestingly, the
Tm values obtained by DSC measurement for the wild type and its
mutant proteins were well coincident with those estimated by CD
analysis. This observation, together with a single peak in calorimetric
measurements, strongly suggests that BstHU as well as its
mutants are denatured with a two-state model involving the native dimer
and unfolded monomers.

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Fig. 3.
Typical excess heat capacity curve of the
wild type BstHU. The increments of excess heat
capacity were 5 kJ/mol·K.
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Table II
The Tm values of the wild type BstHU and its mutants obtained
by DSC measurement and CD analysis
DSC measurements of proteins were carried out as described under
"Experimental Procedures" and the same values estimated by CD
analysis were reported in Refs. 16 and 17.
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Contribution of Thermostabilizing Mutations--
In the foregoing
work, it was shown that the combination of the three thermostabilizing
substitutions (Glu15 in BsuHU to Gly,
Asp34 to Glu, and Asn38 to Lys) significantly
increased the thermostability of the mesophilic protein
BsuHU by 15.9 °C in Tm (18). Taking the present result, the five thermostabilizing mutations (Glu15 to Gly,
Ser27 to Ala, Asp34 to Glu, Asn38
to Lys, and Ile42 to Val) were simultaneously introduced
into the mesophilic protein BsuHU, and the thermostability
of the resultant mutant (quintuple mutant) was examined, exactly in the
same manner as described above. The thermal denaturation curves of the
wild type BsuHU and the quintuple mutant as well as those of
the five single mutant proteins with the constituent amino acid
substitutions are shown in Fig. 4. The
parameters characterizing the thermal denaturation are summarized in
Table III. The simultaneous introduction
of the five mutations greatly increased the thermostability of the
protein with the Tm value of 71.3 °C compared with
48.6 °C for the wild-type BsuHU. The stabilization energy
arising from the quintuple mutations seems to be somewhat greater than
the sum of the constituent single substitutions but only by about 8.9% of the calculated value. This result indicated that the effects of the
mutations on the thermostability are roughly independent of each other
and nearly additive. This result suggests that the individual local
reinforcements derived from the amino acid residues that reside far
from each other contribute to the greater thermostability of the
thermophilic protein BstHU. Interestingly, the Tm value of the quintuple mutant of BsuHU was higher than that
of BstHU. As shown in Table I, the mutation of amino acids
in BstHU to those of BsuHU such as
BstHU-S31T was not always less stable, indicating that the
amino acid sequence of BstHU was not optimized for thermal
stability. Considering this idea, the higher stability of the quintuple
mutant of BsuHU than BstHU may be reasonable.

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Fig. 4.
Thermal unfolding curves for
BsuHU and its mutant proteins. Temperature dependences
of [ ]222 nm values of BsuHU (X) and its
mutants, BsuHU-E15G ( ), BsuHU-S27A ( ),
BsuHU-D34E/N38K ( ), BsuHU-I42V (+), and the
quintuple mutant ( ).
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Table III
Stability of the mesophilic protein BsuHU and its five mutants
Thernodynamic parameters were calculated from the thermal denaturation
curves described in Fig. 2. The table summarizes the results of four
independent experiments.
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Susceptibility to Proteolytic Degradation--
As reported on the
tryptophan synthase
-subunit (22), kanamycin nucleotidyltransferase
(23), and RNase HI (24), the correlation was observed between the
thermostability and the resistance to proteolytic degradation. To
examine whether or not this rule is applicable to the protein HU, we
analyzed the susceptibility of the quintuple mutant and wild-type
BsuHU to chymotrypsin and trypsin, as described under
"Experimental Procedures." As shown in Fig.
5, the rates of chymotryptic and tryptic
degradations of the quintuple mutant were 200 and 11 times slower than
that of the wild type BsuHU, respectively. Taking into
account the substrate specificity of chymotrypsin and trypsin, the
resistance of the quintuple mutant to proteolytic degradation seems to
be independent of the substrate specificity of the protease. Imoto et al. (25) reported that digestion by proteases proceeds
mainly via the unfolded state of proteins. The stabilization of the
quintuple mutant of BsuHU may depend on the stabilization of
the folded state rather than the destabilization of the unfolded state,
because mutations were involved in the formation of salt bridge or the introduction of favorable intermolecular interaction. Therefore, it is
likely that the thermostabilizing mutations contribute to the decrease
in the rate constant of unfolding.

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Fig. 5.
Susceptibility of the quintuple mutant and
the wild type to proteolysis by chymotrypsin and trypsin. The
fraction of uncleaved protein is shown as a function of incubation
time. , BsuHU; , quintuple. The amount of uncleaved
protein was determined by subjecting the digests to reverse phase HPLC,
by which the uncleaved protein could be separated from the chymotryptic
and tryptic peptides. Rate constants were determined from the slope of
semilog plots of the fraction uncleaved versus the
incubation time, and then relative k values (0.005 and
0.091) were calculated for chymotrypsin and trypsin, respectively, by
dividing the k values of the quintuple mutant by that of the
wild type BsuHU.
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Conclusion--
This series of studies on the thermostability of
BstHU revealed that its extra thermostability relative to
the mesophilic protein BsuHU seems to be achieved mainly by
stabilization of two
-helices (
1 and
2) with
Gly15, Ala27, and Glu34 and by
improvement of the intermolecularly close packing in the hydrophobic
core with Ala27 and Val42 (Table
IV). Moreover, the additional salt bridge
between Glu34 and Lys38 on the hydrophobic
surface is found to be also responsible for enhancing the
thermostability of BstHU (Table IV). The amino acid residues
found to be responsible for thermostability of BstHU are
mapped in the crystal structure of BstHU (Fig.
6).

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Fig. 6.
Tertiary structure of the dimer of
BstHU. The protein model is from Tanaka et
al. (11), and side chains are shown only for residues that are
found to be essential for the thermostability of
BstHU.
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The Gly15 residue locates on the bend between
1- and
2-helices and is found to stabilize the conformation of the HTH
motif. The replacement of Gly15 by the corresponding
residue Glu in BsuHU caused a remarkable decrease in the
thermostability of the BstHU-G15E:
Tm =
9.9 °C by CD analysis (17) and
Tm =
10.1 °C by DSC
measurement (Table II). Further, Ala27 and
Glu34 locate at the second
-helix (
2) and may be
involved in stabilization of its conformation because of their enhanced
-helix structural propensity. Two
-helices (
1 and
2) in
BstHU adopt the HTH motif, which is structurally similar to
that found in the operator/repressor family of DNA-binding proteins,
such as CAP and
-cro, where the motif is directly involved in DNA
binding. Thus, the study on the thermostability of BstHU
indicates the unique role of the HTH motif in the BstHU. The
topology found in BstHU, where the helical core domain
consists of a tightly entangled pair of the HTH motif from two
protomers, is unique, and no similar structure has been found. However,
the recent tertiary structural study of the B. stearothermophilus ribosomal protein S7 (a primary 16 S
rRNA-binding protein) revealed a similar motif in its N-terminal half
region (26). Although a function of the HTH motif found in S7 is still
not known, it is likely that the HTH motif may have been selected for
stabilization of some nucleic acid-binding proteins as a scaffold.
Glu34, in addition to involvement in the stabilization of
the second
-helix, is also found to be responsible for enhancing the
thermostability of BstHU by forming an extra salt bridge
with Lys38. The replacement of Glu34 by the
corresponding residue Asp34 in BsuHU caused a
decrease in the thermostability of the BstHU-E34D:
Tm =
2.3 °C by both CD analysis (18) and DSC
measurement. The contribution of a salt bridge to the thermostability
of proteins is still controversial. It has been reported that the
engineered electrostatic interaction between pairs of mobile,
solvent-exposed charged residues on the molecular surfaces of proteins
contributes little to protein stability (27-30). In contrast, Vogt
et al. (4) reported that the salt bridge, together with the
hydrogen bond, is the main explanation for the thermostability of
proteins. In the crystal structure of the BstHU (Fig. 1),
the side chains of these two residues are also exposed to the solvent
and are mobile, the average B values for atoms within the side chains
being about 100 A2 (11, 12). However, as described above,
Glu34 may stabilize the second
-helix by its intrinsic
-helix forming property. Since Glu34 is the salt bridge
partner of the Lys38 residue, the salt bridge formed
between these two residues may somewhat stabilize the conformation of
the second
-helix (
2), thereby contributing to the
thermostability of the BstHU.
This series of studies has shown that some replacements of the amino
acid residues in the BstHU by the corresponding amino acids
in the BsuHU, such as Thr13 to Ala,
Ser31 to Ala, and Thr33 to Leu, resulted in
thermostabilized proteins; the resulting mutants were more stable than
the thermophilic parent protein BstHU (17). In this regard,
it is suggested that the thermophilic protein BstHU has not
evolved to optimize the protein structure in terms of thermostability.
As a result, the quintuple mesophilic mutant containing five
thermostable mutations has a Tm that is 7.4 °C higher than
the thermophilic protein BstHU. It is generally known that
many proteins have been selected in evolution to be marginally stable.
It is thus assumed that B. stearothermophilus, as is the
case for other organisms, has selected the present form of the
BstHU to coordinate the normal living temperature. This finding strongly supports an idea that a novel protein with
hyperthermostability can be constructed by protein engineering using
existing proteins in nature as prototypes.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. Isao Tanaka and Dr.
Yoshimitsu Kakuta of Hokkaido University for a helpful discussion
during the course of this study.
 |
FOOTNOTES |
*
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. Tel./Fax:
092-642-2854; E-mail: mkimura{at}agr.kyushu-u.ac.jp.
The abbreviations used are:
HU, DNA-binding
protein HU; BstHU, DNA-binding protein HU from B. stearothermophilusBsuHU, DNA-binding protein HU from
B. subtilisDSC, differential scanning calorimetryHTH, helix-turn-helixHPLC, high performance liquid chromatographyPAGE, polyacrylamide gel electrophoresis.
2
Y. Kakuta, unpublished results.
 |
REFERENCES |
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Vieille, C.,
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