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INTRODUCTION |
Plants are exposed to various environmental stresses, such as low
or high temperatures, dehydration, high salt, infection, injuries, etc.
Many protective mechanisms have been evolved by plant cells to overcome
the stress. Two major pathways have been suggested in the dehydration
and low temperature-responsive processes (1). One is an abscisic acid
(ABA)1-dependent
pathway, which involves an ABA-responsive element (2-4) and an
ABA-responsive element-binding protein with a bZIP motif (5), and the
other is an ABA-independent pathway (6-8). The C-repeat/dehydration-responsive element (CRT/DRE) (9-11) and
CRT/DRE-binding factor 1 (CBF1) (12) are thought to be important
elements in the ABA-independent pathway. CRT has a 5-base pair core
sequence of CCGAC and is present in the promoters of several
cold-regulated plant genes, including COR15a (10) and
COR78/RD29A (9) from Arabidopsis and
BN115 from Brassica napas (11). DRE consists of
the sequence TACCGACCT in the RD29A promoter (9).
Yamaguchi-Shinozaki and Shinozaki (9) showed that CRT/DRE was not
responsive to the ABA level. Stockinger et al. (12) isolated
an Arabidopsis thaliana cDNA encoding CBF1 by
using the yeast one-hybrid method. Jaglo-Ottosen et al. (13)
reported that overexpression of CBF1 in
Arabidopsis induced cold-regulated (COR) gene
expression and increased the freezing tolerance of the
Arabidopsis plant. Recently, it has been reported that CBF1
belongs to a small family, which includes CBF2 and CBF3 (14, 15).
The CBF1 protein sequence deduced from the DNA sequence of
CBF1 is composed of 213 amino acids, and can be divided into
four regions (12), the N-terminal region (amino acid residues 1-32), the potential nuclear localization signal (33-44), the AP2 domain (48-106), and the acidic region (107-213), as shown in Fig.
1a. The AP2 domain, composed of about 60 amino acid
residues, is a DNA-binding motif that has been found only in plant
proteins thus far (16). Ohme-Takagi and Shinshi (17) reported that the
AP2 domain bound to the cis-acting ethylene-responsive element
designated the GCC-repeat. The AP2 domain is present in the APETALA2
(18), AINTEGUMENTA (19, 20), TINY (21), and AtEBP (22) proteins and in
the RAP2 family of proteins (23) of Arabidopsis,
ethylene-responsive element-binding proteins of tobacco (17), and the
Glossy15 product of maize (24). The highly conserved core
region capable of forming an amphipathic
-helix is present in the
latter half of the AP2 domain.
Stockinger et al. (12) concluded that CBF1 is a
transcriptional activator, based on yeast transformation experiments.
The acidic region of CBF1 is thought to play the leading role in
transcriptional activation. Acidic activation domains are present, for
example, in the yeast transcription factors GAL4 (25) and GCN4 (26), the herpes simplex virus-1 VP16 protein (27-29), the human p53 tumor
suppresser gene product (30), and the RelA(p65) subunit of the cellular
transcription factor NF-
B (31, 32). It has been reported that acid
activation domains interact with the general transcription factor TFIIB
(33, 34), TFIID (35), the TATA box-binding protein (30), and the
transcriptional adaptor ADA2 (36). These results led us to expect that
the acidic region of CBF1 might also directly or indirectly interact
with general transcription factor(s).
In this study, we report the characterization of the CBF1 protein
purified from Escherichia coli transformants. Our purpose was to examine whether various responses of the CBF1 protein occurred when the temperature was lowered from normal to low, nonfreezing temperatures. CD spectra of the intact and truncated CBF1 proteins measured at various temperatures suggested that cold denaturation (37,
38) locally occurred in the CBF1 protein in aqueous solution without
denaturant and that thermal denaturation occurred in the AP2 domain.
This local cold denaturation is thought to be a characteristic property
of the CBF1 protein. We discuss the possible role of this local cold
denaturation of CBF1 in the cold acclimation response.
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EXPERIMENTAL PROCEDURES |
Cells and Plasmids--
Plasmids pET28a and pET29b, and E. coli BL21(DE3) were from Novagen, Inc. Competent cells of E. coli JM109 (recA1, endA1, gyra96, thi-1, hsdR17, supE44,
relA1,
(lac-proAB)/F[traD36,
proAB+, lacIq,
lacZ
M15]) for plasmid construction were from Takara Shuzo Co., Ltd. Cells were grown in Luria-Bertani medium (39) containing 50 mg/liter kanamycin.
Materials--
Restriction and DNA-modifying enzymes were from
Takara Shuzo Co., Ltd. The DNA ligation kit (ligation high) and KOD DNA
polymerase for the polymerase chain reaction (PCR) were from Toyobo
Co., Ltd. Lysyl endopeptidase (LEP),
isopropyl-
-D-thio-galactopyranoside (IPTG), and
guanidine hydrochloride (GdnHCl) were from Wako Pure Chemical
Industries, Ltd. Thrombin was from Sigma. The proteins used as
standards for gel filtration were from Bio-Rad. DNA oligomers were
synthesized by Sawady Technology Co., Ltd. Other chemicals were of
reagent grade.
Construction of the Expression Vector--
The CBF1
gene was cloned from an A. thaliana cDNA library by PCR
using the synthetic DNA oligomers
5'-GTACTCTGACATATGAACTCATTTTCAGC-3', 5'-GAGGATCCAATATTAGTAACTCCAAAGCGACACG-3', and
5'-TCGGATCCTCGAGGTAACTCCAAAGCGACACG-3' as primers.
Recognition sites of restriction endonucleases NdeI, BamHI, and XhoI are shown by the three underlined
areas, respectively. The sequences downstream of the NdeI,
BamHI, and XhoI sites of the primers are
complementary to the 5'- and 3'-sequences of CBF1, respectively. Thirty cycles of PCR were performed using a
Trio-Thermoblock (Biometra) apparatus with KOD DNA polymerase using the
procedures recommended by the supplier. The DNA product was digested
with NdeI and BamHI or with NdeI and
XhoI and ligated to the large NdeI-BamHI fragment of plasmid pET28a or to the
large NdeI-XhoI fragment of pET29b to generate
plasmid pETCBF1(a or b), in which the CBF1 gene, encoding
the CBF1 protein, is under the control of bacteriophage T7
transcription and translation signals. The sequence of the
CBF1 gene in pETCBF1 was confirmed by cycle sequencing using
ABI PRISMTM dye terminator cycle sequencing ready reaction
kits and ABI PRISMTM 310 genetic analyzer from
Perkin-Elmer. Truncation of the CBF1 gene was performed via
PCR with the synthetic DNA oligomers as primers and using KOD DNA
polymerase, according the procedures recommended by the supplier. The
nucleotide sequences of the truncated CBF1 genes were
confirmed as described above.
Overproduction of the CBF1 Protein--
Expression of the CBF1
protein was induced in E. coli BL21 (DE3) cells harboring
plasmid pETCBF1 by the addition of IPTG. Cultivation of the E. coli transformants was carried out at 30 °C. When the
absorbance of the culture at 600 nm reached 0.8, 1 mM IPTG
was added to the culture medium on ice, and cultivation was then
continued for an additional 5 h at 25 °C. Cells were harvested
by centrifugation and subjected to the purification procedures
described below. The production of the CBF1 protein in cells was
examined by analyzing the whole-cell extract by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) (40). The solubility of CBF1 in cells was
examined as follows: cells were suspended in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA, sonicated for 2 min on
ice, and centrifuged at 15,000 rpm (27,000 × g) for 20 min. The resulting supernatants and pellets were analyzed by SDS-PAGE
.
Purification--
All purification procedures were carried out
at 4 °C. Cells from a 250-ml culture were suspended in 25 ml of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM
-mercaptoethanol, sonicated on ice for 2 min, and
centrifuged at 15,000 rpm (27,000 × g) for 30 min. EDTA (1 mM) was present in the suspension to prevent the
digestion of the CBF1 protein by proteases in E. coli. The
resulting supernatant was supplemented with 10 mM imidazole
and then applied to a HiTrap chelating column with Ni2+
ions from Amersham Pharmacia Biotech. After the column was washed, the
CBF1 protein was eluted with a solution containing 300 mM imidazole, 0.5 M NaCl, 20 mM phosphate buffer,
pH 7.4, and 5 mM
-mercaptoethanol. The eluant was
diluted 10-fold with a buffer containing 10 mM Tris-HCl, pH
8.0, 1 mM EDTA, and 5 mM
-mercaptoethanol and applied to Q-Sepharose FF from Amersham Pharmacia Biotech. The CBF1
protein was eluted by a gradient from 0 to 0.8 M NaCl in a
buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 5 mM
-mercaptoethanol, and 10% glycerol. The
purity of the protein was analyzed by SDS-PAGE. CBF1 with a histidine
tag at the N terminus was used for gel shift assay, footprinting, CD
and fluorescence measurements, and gel filtration analyses. CBF1 with a
histidine tag at the C terminus was used as the substrate for the
digestion with LEP to generate LEP peptides. The N-terminal sequence of CBF1 was confirmed by ProciseTM protein sequencing system
from Applied Biosystems.
Gel Shift Assays and Footprinting--
A 21-base pair duplex
that was chemically synthesized and contained the sequence of CRT/DRE
was mixed with the CBF1 protein in 25 µl of a binding buffer
containing 20 mM Hepes, pH 7.5, 30 mM NaCl, 1 mM dithiothreitol, 2 mM EDTA, and 10%
glycerol, and the mixture was incubated for 20 min at room temperature
and then electrophoresed on a nondenaturing 5% polyacrylamide gel at
100 V for 3 h. The bands containing DNA were visualized by
ethidium bromide staining and UV illumination. DNase I footprinting was performed using a 74-base pair synthetic double-stranded DNA fragment that contained CRT/DRE and that had been labeled with 32P
by end-filling with T7 sequenase from Amersham Pharmacia Biotech. The
binding reaction was performed as described above in the presence of 1 µg of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). DNase I
digestion and gel analysis were carried out as described (41). The G/A
sequencing ladder was generated by performing Maxam-Gilbert chemical
reaction (42) using the same 74-base pair DNA fragment.
Circular Dichroism--
CD spectra (200-300 nm) were measured
on a J-720 automatic spectropolarimeter (Japan Spectroscopic Co.,
Ltd.). Spectra were obtained using solutions containing the proteins or
the peptides at 0.3 mg/ml in 20 mM sodium phosphate buffer
at pH 7.9 containing 0.5 mM dithiothreitol and 10%
glycerol or 20 mM sodium acetate buffer at pH 5.5. The mean
residue ellipticity [
], expressed in units of
deg·cm2·dmol
1, was calculated by using an
average amino acid molecular weight of 110.
Digestion with Lysyl
Endopeptidase--
S-Carboxymethylation of CBF1 was carried
out as follows: iodoacetic acid (10 mg) was added to a solution (4 ml)
of CBF1 (1.3 mg) in 200 mM Tris-HCl, pH 8.5, saturated with
N2. The tube containing the reaction mixture was sealed and
shaken at room temperature for 1 h in the dark. The resultant
solution was dialyzed against a buffer containing 50 mM
Tris-HCl, pH 9.0, and 10 mM NaCl at 4 °C overnight.
Carboxymethylated CBF1 (1.3 mg) was digested with LEP (25 µg) at
37 °C for 30 min to generate five peptides, which corresponded to
the amino acid residues 1-32, 40-58, 59-68, 70-119, and 120-213,
shown in Fig. 1b. These peptides were separated by reversed-phase high performance liquid chromatography using a TSK gel
ODS-120T column, 4.6 × 250 mm (Tosho, Tokyo, Japan) with a 60-min
linear gradient from 5 to 75% acetonitrile, containing 0.05%
trifluoroacetic acid at the flow rate of 1 ml/min. Identification of
the peptide in each peak was performed by comparison between the
measured and theoretical mass values summarized in Table
I and by N-terminal amino acid sequencing
by stepwise Edman degradation. The CBF1 protein has five cysteine
residues. The measured mass values of 3585.62 for LEP1 and 5609.96 for
LEP4 (Table I) were larger than the theoretical mass values by 116.87 and 104.66, respectively, which corresponds to the mass value of two
carboxymethyl groups. These results are consistent with the fact that
four cysteine residues, at positions 23, 30, 100, and 117, were
carboxymethylated. The mass spectrum of LEP5 could not be determined.
However, a peak from a carboxymethylated cysteine residue was detected
at step 14 of the Edman degradation of LEP5, indicating that the cysteine residue at position 137 was also carboxymethylated. Based on
these results, we concluded that the five cysteine residues in CBF1
were in the reduced form and were carboxymethylated with iodoacetic
acid.
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Table I
Measured and theoretical mass values for the identification of the LEP
peptides
The LEP digestion of CBF1 was performed as described under
"Experimental Procedures." The resultant LEP peptides were
subjected to mass spectrometry using matrix-assisted laser desorption
ionization time-of-flight mass spectrometry from PerSeptive Biosystems.
Each peptide was identified by N-terminal amino acid sequencing.
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Gel Filtration Chromatography of CBF1--
The gel filtration of
the intact or truncated CBF1 protein was performed using Superdex 75 PC
3.2/30 in a SMART system from Amersham Pharmacia Biotech with a buffer
containing 20 mM sodium phosphate, pH 7.9, 0.5 mM dithiothreitol, and 100 mM NaCl as the eluent with a flow rate of 30 µl/min at 4 or 25 °C.
Protein and Peptide Concentrations--
The protein
concentration of CBF1 with a tag was determined from the UV absorption
at 280 nm. The A2800.1% value of 1.43 for
CBF1(1-213) with a molecular weight of 25,994 was calculated by using
1,576 M
1·cm
1 for tyrosine
(×7) and 5,225 M
1·cm
1 for
tryptophan (×5) at 280 nm (43). A2800.1%
values of 1.57, 1.42, and 1.44 for CBF1(41-213), CBF1(41-157), and
CBF1(41-146), respectively, were also calculated as described for
CBF1(1-213). The concentrations of peptides were determined using the
following formula: Concentration (µg/ml) = (A224
A233.3) × 210 (44).
Secondary Structure Prediction--
The secondary structure of
CBF1 was predicted using a neural network system that was offered as a
service on the World Wide Web, the Predict Protein Server
(http://www.emble-heidelberg.de/predictprotein/predictprotein.html). The program was provided by Rost and Sander (45).
Analytical Ultracentrifugation--
Analytical
ultracentrifugation experiments were carried out in an Optima XL-I
analytical ultracentrifuge (Beckman Instruments Inc.). In velocity
sedimentation experiments, CBF1 (0.12 mg/ml) in a buffer containing 100 mM NaCl, 20 mM phosphate buffer, pH 7.8, and 1 mM dithiothreitol was centrifuged at 50,000 rpm and 25 or
3 °C, and radial scans at 230 nm were taken at 5-min intervals for
4 h. The sedimentation coefficient,
sT,b, and the diffusion coefficient,
DT,b, were calculated by the second moment
method with the Optima XL-A data analysis software supplied by Beckman.
The values from the data at 25 and 3 °C were corrected to the
standard conditions (water, 20 °C) to obtain the
s20,w values (46, 47). They are designated
as s25,b
20,w and
s3,b
20,w, respectively. The value
for the diffusion coefficient was corrected to standard conditions
(water, 20 °C) to obtain the D20,w
values. The frictional coefficient, f, of CBF1 was obtained
by the Svedberg equation (47). The value of the partial specific
volume,
, of CBF1 was estimated from its amino acid composition
(47) as 0.718 ml g
1. The degree of hydration,
, was
estimated using the method of Kuntz based on the amino acid composition
(47). The frictional coefficient of the hydrated sphere,
fsphere, was calculated by the equation from the
Stokes-Einstein relationship (47). The hydrodynamic parameters were
calculated according to the flow diagram by Waxman et al.
(48).
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RESULTS |
Overproduction and Purification--
The expression of the
CBF1 gene in the plasmid pETCBF1 was induced in E. coli by the addition of IPTG. The cultivation of the transformants
was performed at 25 or 37 °C to examine the dependence of the
solubility of CBF1 in cells on the temperature. The production level of
CBF1 was estimated to be ~4 mg/liter of culture at 25 °C and ~8
mg/liter of culture at 37 °C, from the intensities of Coomassie
Brilliant Blue-stained bands in SDS-polyacrylamide gels. The
corresponding bands in SDS-polyacrylamide gels of the supernatants and
pellets obtained after centrifugation of the suspension of sonicated
cells showed that about 80% of the protein was soluble when the
cultivation was carried out at 25 °C, whereas about 90% of the
protein was insoluble at 37 °C (data not shown). Based on these
results, the cultivation of E. coli transformants was
carried out at 25 °C after the addition of IPTG in order to obtain
the CBF1 protein in soluble form.
The CBF1 protein was efficiently and selectively trapped by the
chelating column when a histidine tag was attached to the N or C
terminus of CBF1. Elution with 300 mM imidazole solution yielded 3.3 mg of protein product containing CBF1 with 80% purity from
1 liter of culture. Subsequent ion exchange chromatography using
Q-Sepharose FF yielded homogeneous CBF1, which appeared as a single
band in SDS-PAGE analysis (data not shown). The final yield was 1.7 mg
of the pure CBF1 protein from 1 liter of culture.
Construction of Truncated Mutants--
The CBF1 protein is
composed of four regions, that is, the N-terminal region (1-32), the
potential nuclear localization signal (33-44), the AP2 domain
(48-106), and the acidic region (107-213) (12), as shown in Fig.
1a. In order to examine the
effect of each region on the DNA binding of CBF1 and the temperature
dependence of the structure of CBF1, three truncated CBF1 mutants were
constructed, as shown in Fig. 1a. Amino acid residues from
positions 1 to 40 in the N-terminal region were removed to construct
CBF1(41-213), and in addition, the residues from positions 158 to 213 and those from positions 147 to 213 in the acidic region were also
removed to construct CBF1(41-157) and CBF1(41-146), respectively
(Fig. 1a). The levels of expression of the truncated
proteins in E. coli were almost the same as that of the
intact protein. The purification of CBF1(41-213) and CBF1(41-157) was
performed by the procedure described above for CBF1(1-213). The
predicted isoelectric points for CBF1(1-213), CBF1(41-213),
CBF1(41-157), and CBF1(41-146) were 4.8, 4.6, 7.2, and 9.4, respectively. Because anion exchange chromatography was not appropriate
for the purification of CBF1(41-146), it was purified by chelating and
gel filtration column chromatography. The purified CBF1(41-213),
CBF1(41-157), and CBF1(41-146) proteins with a histidine tag at the N
terminus gave single bands in SDS-PAGE analysis (data not shown).

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Fig. 1.
Analysis of the CBF1 protein.
a, schematic drawing showing locations of the N-terminal
region, the potential nuclear localization signal (NLS), the
AP2 domain, and the acidic region of the intact and truncated CBF1
proteins CBF1(1-213), CBF1(41-213), CBF1(41-157), and CBF1(41-146).
Numbers indicate amino acid residues. b, schematic drawing
showing locations of LEP peptides. c, schematic drawing
showing locations of two -helices and six -sheets predicted in
the secondary structure. The solid and gray
regions correspond to the -helices and -sheets,
respectively. d, schematic drawing showing locations of the
regions estimated or suggested to exhibit cold denaturation and thermal
denaturation by CD spectra of the intact and truncated CBF1
proteins.
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DNA Binding of the Intact and Truncated CBF1 Proteins--
The
binding ability of the purified CBF1 protein to CRT/DRE was analyzed by
gel-shift assays, DNase I footprinting, and measurement of the
intrinsic fluorescence. Gel-shift experiments were carried out to show
that the CBF1 proteins bound to CRT/DRE (data not shown). No shift was
detected without CBF1. Mobility shifts resulting from complex formation
between CRT/DRE and the CBF1 proteins were observed in the assays. The
migration of the complex-containing band became faster with increasing
truncation of the CBF1 protein.
DNase I footprinting was performed to study whether the purified CBF1
protein was able to bind specifically to CRT/DRE. A 75-base pair
synthetic oligonucleotide probe containing CRT/DRE was digested with
DNase I in the presence or absence of CBF1(1-213) and analyzed on a
12% sequencing gel. As shown in Fig. 2,
regions on both strands surrounding CRT/DRE were protected by
CBF1(1-213) binding. On the bottom strand, 11 nucleotide residues
including GTCGGC, were protected, whereas on the top strand, 20 nucleotide residues, including GCCGAC, were protected.

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Fig. 2.
DNase I footprinting demonstrating the
specific binding of CBF1(1-213) to CRT/DRE. The CRT/DRE probes
were labeled on both strands (bottom and top) and
incubated with or without CBF1. The reactions were analyzed on a 12%
sequencing gel together with G/A sequencing ladders generated from the
same probes. and + refer to naked and complexed DNA fragments,
respectively, and G/A refers to the sequencing ladder. The
protected nucleotide residues are indicated by arrows.
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The intrinsic fluorescence spectra were measured to determine the
binding constants between the intact and truncated CBF1 proteins and
CRT/DRE. The intensity of the fluorescence peak at 338 nm was decreased
to about 60% of the maximum level by the addition of a double-stranded
synthetic DNA fragment containing CRT/DRE (data not shown). Titration
of CBF1 with a double-stranded CRT/DRE-containing oligomer caused
changes in the fluorescence anisotropy of CBF1 (Fig.
3). The binding curves (Fig. 3) showed a
stoichiometry of one protein bound per CRT/DRE either at 30 °C or at
1 °C. The apparent binding constant of CBF1(1-213) was (4.6 ± 0.9) × 107 M
1 at 1 °C,
(4.8 ± 0.9) × 107 M
1 at
10 °C, (6.2 ± 1.4) × 107
M
1 at 20 °C, and (6.4 ± 0.7) × 107 M
1 at 30 °C. The apparent
binding constants of the intact protein, CBF1(1-213) and the three
truncated proteins, CBF1(41-213), CBF1(41-157), and CBF1(41-146),
are summarized in Table II. The apparent
binding constant of CBF1(41-213), lacking 40 amino acid residues in
the N-terminal region, was (1.8 ± 0.4) × 108
M
1, suggesting that the N-terminal region is
not necessary for the binding of CBF1 to CRT/DRE. The possible
protein-protein interactions caused by the N-terminal region might
reduce the apparent binding constant of the intact CBF1 protein. Two
mutants, CBF1(41-157) and CBF1(41-146), lack 56 and 67 amino acid
residues, respectively, in the acidic region. CBF1(41-146) bound to
CRT/DRE less tightly than did CBF1(41-213), with a binding constant of
(4.0 ± 0.8) × 107 M
1
(Table II).

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Fig. 3.
Fluorescence quenching of the CBF1 protein by
the addition of CRT/DRE, showing a stoichiometry of 1 DNA fragment
bound per protein. Aliquots of a double-stranded DNA oligomer
containing CRT/DRE were added to a solution of CBF1 in binding buffer
at 1 °C ( ) or 30 °C ( ).
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Table II
Apparent binding constants of the intact and truncated CBF1 proteins at
20 °C
Aliquots of the double-stranded DNA oligomer containing CRT/DRE were
added to a solution of CBF1 in the binding buffer to determine the
binding constants at 20 °C by measuring the intrinsic fluorescence
of the protein on a Hitachi F-4500 fluorescence spectrophotometer. The
excitation wavelength was 279 nm, and the emission was monitored from
285 to 400 nm. The binding constant was calculated as described by
Kelly et al. (56). The values are the average of three
experiments with S.D.
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Secondary Structures of the LEP-Peptides--
Stockinger et
al. (12) reported that CBF1 had nine lysine residues at amino acid
positions 32, 33, 38, 39, 58, 68, 69, 119, and 123. In order to study
the structures of the peptides from CBF1, the CBF1 protein was digested
with LEP. Prior to the digestion with LEP, the cysteine residues in the
CBF1 protein were S-carboxymethylated, as described under
"Experimental Procedures," to avoid the formation of non-native
disulfide bonds during the protease digestion. Carboxymethyl-CBF1 was
then digested with LEP to generate five peptides, LEP1, LEP2, LEP3,
LEP4, and LEP5, shown in Fig. 1b. The measurement of the far-UV CD spectra of the peptides LEP1, LEP2, LEP3, LEP4, and LEP5 from
CBF1 in aqueous solution was carried out at intervals of 10 °C by
raising and lowering the temperature in the range between 2 and
60 °C. Fig. 4, a-e, shows
the far-UV CD spectra of the peptides at 60 and 2 °C and those in 3 M GdnHCl. Fig. 4f shows the temperature
dependence of the [
] value of each peptide at 220 nm. The CD
spectra of LEP4, which included the latter half of the AP2 domain, were
indicative of
-helicity (Fig. 4d). Its [
] value,
4300 at 220 nm at 60 °C, was the lowest among those of the five
peptides, and it was decreased to
6400 by lowering the temperature to
2 °C (Fig. 4, d and f). The helical content of
LEP4 was about 9% at 40-60 °C, and it was increased to about 17%
by lowering the temperature to 2 °C. The difference between the
[
] value at 220 nm in the spectrum of LEP4 at 60 °C and that in
3 M GdnHCl (Fig. 4d) suggested that LEP4 still
had a considerable amount of thermally stable secondary structure at
60 °C but was almost completely denatured in 3 M
GdnHCl.

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Fig. 4.
CD spectra and temperature dependence of the
LEP peptides. The far-UV CD spectra measured at 2 °C
(thick solid lines) and 60 °C (thin solid
lines) and in the presence of 3 M GdnHCl
(dot-dashed lines) of LEP1 (a), LEP2
(b), LEP3 (c), LEP4 (d), and LEP5
(e) and the temperature dependence (f) of LEP1
( ), LEP2 ( ), LEP3 ( ), LEP4 ( ), and LEP5 (×) are shown. The
reversibility was confirmed by lowering the temperature from 60 to
2 °C and then raising it from 2 to 60 °C.
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In contrast, the CD spectra of the other four peptides, LEP1, LEP2,
LEP3, and LEP5, were indicative of random coil, and the negative molar
ellipticity at 220 nm at 60 °C was lower than that at 2 °C (Fig.
4, a-e). The molar ellipticity for the four peptides at 220 nm increased as the temperature was decreased from 60 to 2 °C (Fig.
4f). The spectrum of LEP1 at 2 °C was very similar to
that of the same peptide denatured with 3 M GdnHCl (Fig.
4a). The similarity between the spectrum of the peptide at
2 °C and that of the peptide denatured in 3 M GdnHCl was
also observed for LEP2, LEP3, and LEP5 as shown in Fig. 4, b,
c, and e, respectively. These results suggest that
thermally stable secondary structure was present at 60 °C, decreased
with decreasing temperature, and was almost completely absent at
2 °C in the four peptides LEP1, LEP2, LEP3, and LEP5. These results
suggest that LEP1, LEP2, LEP3, and LEP5 exhibited cold denaturation; in
contrast, LEP4 exhibited cold stabilization, with an increase of
secondary structure, when the temperature decreased from 60 to 2 °C.
Secondary Structures of the Intact and Truncated CBF1
Proteins--
The far-UV CD spectra of the intact and truncated CBF1
proteins were measured at various temperatures in the range between
5
and 70 °C by lowering the temperature from 30 to
5 °C, raising it from
5 to 70 °C, and again lowering it from 70 to 2 °C, in steps of 10 °C (Fig. 5). The spectra
of the intact CBF1 protein, (1-213) and of the truncated proteins,
(41-213), (41-157), and (41-146), in the solution at pH 7.9 containing 10% glycerol at
5, 30, and 70 °C, and those in 3 M GdnHCl solution, are shown in Fig. 5, a-d,
respectively. The helical content of the protein, which was calculated
by the method of Wu et al. (49), increased with increasing
truncation of both the N-terminal and acidic regions in the following
order: CBF1(1-213) (13.8%) < CBF1(41-213) (15.5%) < CBF1(41-157)
(22.3%) < CBF1(41-146) (23.0%). The temperature dependence of the
[
] value at 220 nm for the proteins is shown in Fig. 5,
e-h. The negative molar ellipticity at 220 nm in the spectrum of CBF1(1-213) was
5090 at
5 °C,
5600 at 30 °C,
and
4300 at 70 °C (Fig. 5a). The ellipticity at 220 nm
was minimal (
5600) at 30 °C, increased by about 500 (to
5090) as
the temperature was decreased from 30 to
5 °C, and also increased
by about 1300 (to
4300) as the temperature was raised from 30 to
70 °C (Fig. 5e). Similar temperature dependence of the
ellipticity was observed in the spectra of CBF1(41-213) and
CBF1(41-157) (Fig. 5, b, c, f, and g). These
results strongly suggest that reversible cold denaturation of the
secondary structure occurred in the temperature range between 30 and
5 °C and that thermal denaturation also occurred in the range
between 40 and 60 °C in CBF1(1-213), CBF1(41-213), and
CBF1(41-157). The ratio of the change in the ellipticity at 220 nm
resulting from possible cold denaturation to that resulting from
thermal denaturation was 500/1300 for CBF1(1-213), 500/1600 for
CBF1(41-213), and 400/2700 for CBF1(41-157) (Fig. 5,
e-g). The ratio decreased with increasing truncation of the
protein in the following order: CBF1(1-213) > CBF1(41-213) > CBF1(41-157). An increase of the ellipticity as the temperature was
decreased from 30 to
5 °C was not observed in CBF1(41-146), as
shown in Fig. 5, d and h, suggesting that the
region exhibiting the cold denaturation was removed from the protein by
the truncation. The increase of the ellipticity between 40 and 60 °C
was still observed in the spectra of CBF1(41-146) (Fig. 5,
d and h). These results strongly suggest that the
region exhibiting the possible cold denaturation was diminished or
removed by the truncation of the protein, whereas the region exhibiting
the thermal denaturation remained despite the truncation. The change of
ellipticity was reversible in the temperature range between
5 and
30 °C in the spectra of all CBF1 proteins (Fig. 5, e-h).
These results suggest that the CBF1 protein has regions exhibiting cold
denaturation and thermal denaturation and that the regions exhibiting
cold denaturation are present in the N-terminal region and in the
acidic region, whereas the region exhibiting the thermal denaturation is present in the region that includes the AP2 domain. The data also
show that the secondary structure of CBF1 is most stable at around
30 °C.

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Fig. 5.
CD spectra and temperature dependence of the
intact and truncated CBF1 proteins. The far-UV CD spectra measured
at 5 °C (thick solid lines), 30 °C (thin solid
lines), and 70 °C (dashed lines) or in the presence
of 3 M GdnHCl (dot-dashed lines) of CBF1(1-213)
(a), CBF1(41-213) (b), CBF1(41-157)
(c), and CBF1(41-146) (d) are shown in the
top row. The plots showing the temperature dependence of
CBF1(1-213) (e), CBF1(41-213) (f),
CBF1(41-157) (g), and CBF1(41-146) (h) are
shown in the bottom row. The temperature was lowered from 30 to 5 °C ( ), raised from 5 to 70 °C ( ), and again
lowered from 70 to 2 °C (+) at intervals of 10 °C.
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The spectra of the CBF1 proteins in 3 M GdnHCl (Fig. 5,
a-d) exhibited slight negative ellipticity around 220 nm,
suggesting that the secondary structure of the protein was almost
completely denatured in 3 M GdnHCl. Comparison between the
spectra of the protein at 70 °C and those of the protein in 3 M GdnHCl suggests that the CBF1 proteins still had a
considerable amount of thermally stable secondary structure at
70 °C. The salt (100 mM NaCl) did not affect the CD
amplitude at around 220 nm or the temperature dependence of the molar
ellipticity at 220 nm (data not shown). The helical contents of CBF1
were calculated as 13.8% at 30 °C and 11.7% at 2 °C, by the
method of Wu et al. (49).
Secondary Structure Prediction of CBF1--
The predicted
secondary structure of CBF1 is summarized in Fig. 1c. The
analysis suggested that residues 4-10 and 79-94 had the propensity to
form
-helices, whereas residues 60-64, 70-75, 99-101, 136-140,
153-156, and 208-212 had the propensity to form
-sheet structures.
The prediction of the formation of the
-helix designated as helix 2 for residues 79-94 (Fig. 1c) agreed with the conclusions
about the secondary structure of CBF1 from the CD spectroscopy.
Gel Filtration of the Intact and Truncated CBF1 Proteins at 4 and
25 °C--
Gel filtration of CBF1(1-213), CBF1(41-213),
CBF1(41-157), and CBF1(41-146) was performed at 4 and 25 °C to
examine the change in the molecular shape and volume of the CBF1
protein caused by lowering the temperature. The values of the relative
retention volumes (Ve/Vo) for each protein at 4 and 25 °C are shown
in Fig. 6. The retention volume for each
protein was smaller at 4 °C than at 25 °C. The difference between
the retention volume for each protein at 4 °C and that at 25 °C
decreased with increasing truncation of the protein. The difference for
CBF1(41-146) was smallest and was almost the same as that for the
standard proteins. The apparent molecular weights for the proteins are
as follows: 38,900 at 4 °C and 33,100 at 25 °C for CBF1(1-213),
29,500 at 4 °C and 25,700 at 25 °C for CBF1(41-213), 15,800 at
4 °C and 15,500 at 25 °C for CBF1(41-157), and 9800 at 4 °C
and 9800 for 25 °C for CBF1(41-146). These results suggest that
extension of the CBF1 molecule was caused by cold denaturation. The
molecule was extended by 18, 15, 3, and 0% for CBF1(1-213),
CBF1(41-213), CBF1(41-157), and CBF1(41-146), respectively.

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Fig. 6.
Estimation of the apparent molecular weight
of the intact and truncated CBF1 proteins at 4 and 25 °C to examine
the change of the molecular shape caused by lowering the
temperature. A plot of log molecular weight versus
Ve/Vo for the protein standards is shown. The protein standards were
bovine gamma globulin (GG), ovalbumin (OA) from
chicken, myoglobin (MG) from horse, and vitamin B-12
(VB), with molecular weights of 158,000, 44,000, 17,000, and
1350, respectively. The retention volumes for CBF1(1-213) were
1.17 ± 0.00 ml at 4 °C and 1.20 ± 0.00 ml at 25 °C;
those for CBF1(41-213) were 1.22 ± 0.01 ml at 4 °C and
1.25 ± 0.00 ml at 25 °C; those for CBF1(41-157) were
1.35 ± 0.00 ml at 4 °C and 1.36 ± 0.00 ml at 25 °C;
and those for CBF1(41-146) were 1.44 ± 0.01 ml at 4 °C and
1.45 ± 0.01 ml at 25 °C. The values are the average of 3-6
experiments with S.D. Ve/Vo values obtained for the CBF1 proteins, 1.27 for CBF1(1-213) at 4 °C (arrow 1), 1.30 for CBF1(1-213)
at 25 °C (arrow 2), 1.33 for CBF1(41-213) at 4 °C
(arrow 3), 1.36 for CBF1(41-213) at 25 °C (arrow
4), 1.47 for CBF1(41-157) at 4 °C (arrow 5), 1.48 for CBF1(41-157) at 25 °C (arrow 6), 1.57 for
CBF1(41-146) at 4 °C (arrow 7), and 1.58 for
CBF1(41-146) at 25 °C (arrow 8), are indicated. Ve/Vo
values of the standard proteins and the CBF1 proteins obtained at
4 °C are indicated by open circles and open
arrows, respectively. Those obtained at 25 °C are indicated by
shaded circles and shaded arrows.
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Ultracentrifugation Studies--
Sedimentation velocity
experiments were carried out to determine the sedimentation and
diffusion coefficients, s20,w and
D20,w, respectively. The sedimentation
process of a 50,000 rpm centrifugation of CBF1 at 25 or 3 °C was
analyzed by following the movement of the boundary. Table
III summarizes the results from the
sedimentation velocity analysis. The corrected sedimentation
coefficient, s25,b
20,w, obtained
from the data at 25 °C was (1.71 ± 0.06) × 10
13
s, and that obtained from the data at 3 °C
(s3,b
20,w) was (1.42 ± 0.07) × 10
13 s (Table III). This sedimentation coefficient
change reflects the conformational transition of the CBF1 protein
caused by lowering the temperature from 25 to 3 °C. The increase of
the friction ratio f/fsphere from 1.52 ± 0.05 to 1.83 ± 0.09 caused by lowering the temperature from 25 to
3 °C suggests a change of the axial ratio of the molecule. The
decrease of the diffusion coefficient D20,w caused by lowering the temperature
(Table III) may be correlated with the conformational change of CBF1
caused by lowering the temperature.
 |
DISCUSSION |
Possible local cold denaturation suggested by the CD measurement
and gel filtration analyses is thought to be a characteristic property
of the CBF1 protein. The regions that caused cold denaturation suggested by the CD spectra of the intact and truncated CBF1 proteins were present in residues 1-40 and 147-213, as shown in Fig.
1d. The retention times of the intact and truncated CBF1
proteins in gel filtration (Fig. 6) and the results from the
sedimentation velocity analysis suggested the following: (i) the intact
and truncated CBF1 proteins were monomeric; (ii) the shape of
CBF1(1-213) was not spherical, but rather extended, whereas that of
CBF1(41-146) was likely to be spherical; (iii) the volume of a
molecule of CBF1(1-213) at 4 °C was larger than that at 25 °C;
and (iv) the difference between the molecular volume at 4 °C and
that at 25 °C decreased with increasing truncation of the protein,
suggesting that the local cold denaturation was accompanied by the
extension of the protein molecule.
CBF1 could bind to CRT/DRE both at normal and at near-zero temperatures
in vitro (Fig. 3). However, CBF1 in Arabidopsis
did not activate the transcription of the COR gene at normal
temperatures (12). It seems to be necessary for CBF1 bound to CRT/DRE
to be transformed from a repressor to an activator via the reduction of
temperature in Arabidopsis. Thus, we propose two possible
models to explain the transcriptional activation involving CBF1. The first model is that the local cold denaturation of CBF1 caused by the
low temperature initiates the transcriptional activation of the
COR gene, because extension of the molecule resulting from the cold denaturation facilitates interaction with the factor(s) that
activate or initiate transcription of the genes. The second model is
that other cofactor(s) associate with CBF1 for repression of the
transcription at normal temperatures, but these factors are released
from the locally denatured CBF1 protein, which results in the
transcriptional activation, at the low temperature. Jaglo-Ottosen et al. (13) reported that CBF1 overexpression induced
COR gene expression without a low temperature stimulus,
probably suggesting the shortage of some repressor to suppress the
transcription. This result seems to support the second model.
The content of secondary structure in the peptides LEP1, LEP2, LEP3,
and LEP5 was largest at 60 °C, in the temperature range between 2 and 60 °C, as shown in Fig. 4. The intact and truncated CBF1
proteins also still had considerable amounts of secondary structure at
70 °C, after thermal denaturation between 40 and 60 °C as shown
in Fig. 5, a-d. The residual structure was thermally stable
but was denatured in 3 M GdnHCl, suggesting that it was maintained by hydrophobic interactions (50).
Cold denaturation is thought to be caused by the interaction between
water and nonpolar groups exposed to the solvent (51). The nonpolar
groups are usually buried in a protein molecule by hydrophobic
interactions at normal temperatures. As the temperature is decreased,
the hydrophobic forces are reduced, and the resultant disruption of the
hydrophobic interactions results in the denaturation of the structure
at low temperatures. Cold denaturation has been observed in multimeric
enzymes, such as ATPases and fatty-acid synthetases, as well as in
proteins under destabilizing conditions, such as moderate denaturant
concentrations, low pH, or high pressure (51), and in proteins
destabilized by site-directed mutagenesis (52), as well as in the
artificially designed peptides (53). The possible cold denaturation of
CBF1 suggested by the CD spectra, gel filtration, and ultracentrifuge
analyses is thought to be a rare case, in which a monomeric protein
with a natural sequence possibly exhibits the cold denaturation under
physiological conditions. It seems likely that this cold denaturation
of CBF1 leads to initiation of the transcriptional activation, because
cold denaturation is thought to be a direct physical response of the
protein to the low temperature stimulus, and it seems reasonable that
the plant might utilize the physical properties of its constituents to
respond to this environmental change. Some physiological and
biochemical properties conferring tolerance to cold temperatures have
been reported, such as an increase of the degree of unsaturation of fatty acids in the membrane, and the accumulation of osmolytes. Murata
et al. (54) reported that the sensitivity of plants to cold
stimuli was correlated with the degree of unsaturation of fatty acids
of the membrane and that the tolerance was improved by introducing an
appropriate glycerol-3-phosphate acetyltransferase. Xin and Browse (55)
reported a mutant of Arabidopsis with constitutive freezing
tolerance conferred by the accumulation of proline.
The CD spectra of the intact CBF1 protein seemed to show that the
secondary structure content of CBF1 was relatively low (Fig. 5a). Secondary structure prediction suggested that there
were only two
-helices, one in the N-terminal region and one in the AP2 domain (Fig. 1c, helix1 and helix2). The
formation of helix2 in the AP2 domain was confirmed by the CD spectrum
of the peptide LEP4 (Fig. 4d). However, the CD spectra of
the LEP peptides other than LEP4 were mainly consistent with random
coil. In general, the structure of a peptide does not completely match
the structure of the corresponding region in the protein, because of
different environments or neighborhoods, but the structure of the
peptide does reflect a tendency to form a particular structure based on a particular amino acid sequence. The CD spectra of the LEP peptides (Fig. 4) suggest that the peptide sequences in both the N-terminal and
the acidic regions have a tendency to cause cold denaturation and that
the CBF1 protein has at least one
-helix in the AP2 domain, which
functions in DNA binding. The calculated helical content of the
protein, based on the CD spectra in Fig. 5, a-d, increased
with increasing truncation of both the N-terminal and acidic regions,
possibly suggesting that the
-helix could form in the AP2 domain
despite the truncation. It is very likely that the thermal denaturation
between 40 and 60 °C, shown in Fig. 5, e-h, is due to
the thermal destabilization of the AP2 domain. In addition, the spectra
shown in Fig. 5d and the thermal denaturation plot shown in
Fig. 5h suggest that the region around the AP2 domain does
not participate in the cold denaturation. The fact that the CBF1
protein could bind to CRT/DRE with same stoichiometry at 1 °C and at
30 °C in vitro (Fig. 3) is consistent with the fact that
the structure of the region around the AP2 domain was stable in the
temperature range between
5 and 30 °C, as shown in Fig. 5,
d and h. The amphipathic
-helix (Fig.
1c, helix2), which was previously predicted in
the AP2 domain (16), is thought to be a core structure of the CBF1 protein.
On the other hand, the formation of helix1 that was predicted as shown
in Fig. 1c was not clearly demonstrated. The plot of the
[
] values at 220 nm as a function of temperature showed that the
values for LEP1 were lower than those for LEP2, LEP3, and LEP5 by about
1000, and higher than those for LEP4 (Fig. 4f). The spectrum
of LEP1 at 60 °C (Fig. 4a) exhibited a slight hump around
220 nm. These results might suggest that LEP1, which corresponds to the
amino acid residues 1-32 in CBF1, included a small amount of helicity
(corresponding to helix1 in Fig. 1c). Limited proteolysis of
the CBF1·CRT/DRE complex with trypsin selectively released the
N-terminal fragment 1-41 (data not shown). The binding ability of CBF1
was improved by the removal of the region 1-40, as summarized in Table
II. These results suggest that the region from positions 1-40 is not
necessary for the binding of CBF1 to CRT/DRE. On the other hand, the
results of the present study and its location close to the nuclear
localization signal (Fig. 1a) might suggest some function
for the N-terminal region. Finally, the creation of transgenic
Arabidopsis plants that overexpress the intact or truncated
CBF1 will be helpful for determining the molecular components of the
cold stress-responsive pathway involving CBF1.