A differential scanning calorimetry study of tetracycline repressor
Sylwia Ke
˛
dracka-Krok and Zygmunt Wasylewski
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland
Tetracycline repressor (TetR), which constitutes the most
common mechanism of bacterial resistance to an antibiotic,
is a homodimeric protein composed of two identical sub-
units, each of which contains a domain possessing a helix–
turn–helix motif and a domain responsible for binding tetra-
cycline. Binding of tetracycline in the protein pocket is
accompanied by conformational changes in TetR, which
abolish the specific interaction between the protein and
DNA. Differential scanning calorimetry (DSC) and CD
measurements, performed at pH 8.0, were used to observe
the thermal denaturation of TetR in the absence and pres-
ence of tetracycline. The DSC results show that, in the
absence of tetracycline, the thermally induced transitions of
TetR can be described as an irreversible process, strongly
dependent on scan rate and indicating that the protein
denaturation is under kinetic control described by the simple
kinetic scheme: N
2
À!
k
D
2
,wherek is a first-order kinetic
constant, N is the native state, and D is the denatured state.
On the other hand, analysis of the scan rate effect on the
transitions of TetR in the presence of tetracycline shows that

resistance protein TetA. If Tc enters a resistant bacterial cell,
it binds with high affinity to TetR [1]. This binding is
accompanied by conformational changes in TetR, which
abolish the specific interaction with DNA, reduces the
binding affinity for operator DNA by 6–8 orders of
magnitude [2] and finally induces the release of the TetR–
[Mg–Tc]
+
ternary complex, thereby initiating expression of
TetA [3,4]. Regulation of TetR by binding of [Mg–Tc]
+
takes place in the core of the repressor, which is formed by
helices a5toa10 of both subunits. Study of the crystal
structure of the TetR homodimer in complex with its
palindromic DNA operator shows that after [Mg–Tc]
+
insertion into the binding tunnel in the repressor core, Tc is
anchored by hydrogen bonds between its functional groups
and the C-terminal side chains of helix a4, and helices a5
and a6. This initiates conformational changes starting with
C-terminal unwinding and shifting of the short helix a6in
each monomer. Subsequently, it forces a pendulum-like
motion of helix a4, which increases the separation of the
attached DNA-binding domains by 3 A
˚
[5].
As TetR–tetO is the most efficient inducible system of
transcriptional regulation known so far, it is often used as a
tool for selective target gene regulation in eukaryotes [6,7].
From the studies of Backes et al. [8], it is known that

protein.
(Received 26 June 2003, revised 15 September 2003,
accepted 29 September 2003)
Eur. J. Biochem. 270, 4564–4573 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03856.x
SO
À
3
650 (M) was from Merck, and Q Sepharose Fast Flow
and Sephacryl S-200 HR were from Amsterdam Pharmacia
Biotech. The nutrients for bacterial growth were from Life
Technologies. All other chemicals were products of analyt-
ical grade from Polish Chemical Reagents (Gliwice,
Poland). Buffers in water purified by the Millipore system
were used throughout this work.
Protein purification
The wild-type Tet repressor was overproduced in Escheri-
chia coli strain RB 791 (a gift from W. Hillen, Universitat
Erlangen-Nurnberg, Germany).
Protein purification in general followed the scheme
described by Ettner et al. [13] with a few modifications
[14]. After the purification procedure, the protein was highly
pure (> 97%) as judged by SDS/PAGE and Coomassie
Brilliant Blue staining. The dimer repressor concentration
was determined spectrophotometrically using an excitation
coefficient e
280nm
¼ 30 · 10
3
M
)1

the protein in the absence of Tc, in buffer with and without
Mg, does not show any differences in these values.
Differential scanning calorimetry
DSC experiments were performed on a Calorimetry Sciences
Corporation (CSC) 6100 Nano II differential scanning
calorimeter with a cell volume of 0.3228 mL, interfaced with
a personal computer (IBM-compatible). Different concen-
trations of the protein samples within the 0.4–4.0 mgÆmL
)1
range and different scan rates of 0.1–2.0 KÆmin
)1
were used.
Before the measurements, the protein samples were exhaust-
ively dialyzed against buffer A, and the samples with Tc
against buffer B. The samples and reference solutions were
degassed for at least 5 min at room temperature and
carefully loaded into the cells to avoid bubble formation.
Cells were carefully cleaned before each experiment. A
constant pressure of 304 kPa was maintained to prevent
possible degassing of the samples on heating. A background
scan recorded with the buffer in both cells was subtracted
from each test scan. The reversibility of thermal transitions
was checked by examining the reproducibility of the
calorimetric trace in the second heating of the sample
immediately after fast cooling from the first scan.
The excess molar heat capacity was calculated using the
molecular mass of the Tet repressor of 46 708 Da and the
partial specific volume of the protein equal to 0.73 mLÆg
)1
,

2
max
C
exc;max
p
DH
cal
ð1Þ
where C
exc,max
p
is the excess of molar specific heat capacity
over the baseline value at maximum transition, T
max
is the
denaturation temperature in Kelvin, DH
cal
is the total molar
enthalpy change during the denaturation process, R is the
gas constant, and A is equal to 4.0 for monomer or for
nondissociated dimer [19].
Circular dichroism
CD measurements were performed on a Jasco-710 spectro-
polarimeter equipped with water-jacketed cell holder and a
Julabo F25 circulator bath with programmable temperature
controller. The actual temperature inside the quartz cell
(with path length of 1 mm) was measured with Digi-sense
thermocouple thermometer. Protein thermal denaturation
was monitored by following the changes in ellipticity at
222nm with a scan rate of 1KÆmin

B
T=6pgD
T
ð2Þ
where k
B
is the Boltzman constant, T is temperature in
Kelvin, and g is the solvent viscosity. The theoretical
hydrodynamic radius (R
theo
H
) can be obtained from the
following formula:
Ó FEBS 2003 DSC study of tetracycline repressor (Eur. J. Biochem. 270) 4565
R
theo
H
¼½ð3Mðm þ hÞÞ=ð4pN
A
Þ
1=3
where N
A
is the Avogadro constant, m is the partial
specific volume, h is the hydration, and M is the molar
mass of the protein. The ratio R
H
=R
theo
H

concentration range and the same scan rate as for samples
without the ligand. The typical denaturation curves for
TetR and its complex with Tc are presented in Fig. 1. Fig. 2
shows the dependence of T
max
on the concentration of the
protein. In the case of TetR, the T
max
obtained decreases
with increasing protein concentration, which indicates that
TetR unfolds without dissociation. Indeed, if multimeric
proteins undergo unfolding with simultaneous dissociation
into monomers, T
max
should increase with the total protein
concentration [20,21]. The DSC profiles obtained for TetR
alone are highly asymmetrical, and the ratio DH
cal
/DH
vH
is much below unity, i.e. between 0.55 and 0.76
(Table 1), which indicates some oligomerization, which is
independent of protein concentration over the range used in
these studies. The calorimetric enthalpy, DH
cal
, although
determined with some inaccuracy because of the existence of
an exothermic peak, increases a little with a rise in TetR
concentration (Table 1).
The enthalpic effect that accompanies the aggregation

HCl buffer, pH 8.0, containing 150 mM NaCl and 2 m
M
dithiothrei-
tol) at a scan rate of 1 KÆmin
)1
and buffer B (10 m
M
Tris/HCl buffer,
pH 8.0, containing 150 m
M
NaCl, 2 m
M
dithiothreitol and 10 m
M
MgCl
2
)atascanrateof1KÆmin
)1
, for TetR alone and for the com-
plex of TetR with Tc, respectively. Protein concentration in both cases
was 0.4 mgÆmL
)1
. The ratio of concentrations was 5 mol Tc/mol TetR
dimer.
Fig. 2. Effect of concentration on transition temperature (T
max
). The
circles correspond to T
max
for TetR obtained from DSC measurements

% 10.0 °C. The ligand binding leads to a doubling of the
denaturation enthalpy value (Table 1).
Effect of the scan rate
Thermal denaturation of TetR was carried out at a protein
concentration of 0.6 mgÆmL
)1
andscanrate(v)of
0.1–2.0 KÆmin
)1
. Measurements of liganded protein were
performed in buffer B, at a protein concentration of
0.4 mgÆmL
)1
and at fivefold molar excess of Tc per mol of
the dimer. The denaturation enthalpy values of the proteins
(in the absence and presence of ligand) as a function of scan
rate are shown in Table 2. A small decrease in denaturation
enthalpy was observed on a rise in scan rate for the repressor
in the absence of Tc, whereas for the ligated protein, a
slight increase was noted.
The T
max
is the increasing linear function of the scan rate
of TetR (Fig. 3). These results indicate that denaturation of
TetR protein occurs as a kinetically controlled process,
which cannot be described by equilibrium thermodynamics
[21–23]. This kind of denaturation process is assumed to be
a first-order reaction with a rate constant, k, that changes
with temperature, according to the Arrhenius equation:
k ¼ Aexp À

exc
p
¼
1
m
DHexp
E
a
R
1
T
Ã
À
1
T

ÂÀ
1
m
Z
T
T
0
exp
E
a
R
1
T
Ã

c
(mgÆmL
)1
)
DH
cal
(kJÆmol
)1
) DH
cal
/DH
vH
0.40 397.84 0.59 0.30 954.40 0.89
0.60 411.54 0.55 0.40 1058.14 0.92
0.90 502.76 0.68 0.50 1230.56 1.08
1.50 517.59 0.71 1.00 1031.91 0.88
2.00 520.23 0.76 2.00 1134.11 1.03
3.00 515.79 0.57 2.80 1025.80 0.91
4.00 527.98 0.58 3.40 1080.94 0.89
– – 4.00 976.98 1.03 –
– – 4.00 988.80 1.13 –
Mean (± SD) – 484.82 (55.39) 0.64 (0.08) – 1053.53 (86.40) 0.97 (0.10)
Table 2. Apparent thermodynamic transition parameters of TetR and complex of TetR with Tc at various heating rates. The buffer for TetR was
10 mM Tris/HCl, 150 mM NaCl, 2 mM dithiothreitol, pH 8.0. The buffer for TetR + Tc was 10 mM Tris/HCl, 150 mM NaCl, 2 mM
dithiothreitol, 10 mM MgCl
2
,pH8.0.
TetR TetR + Tc
m
(KÆmin

Ó FEBS 2003 DSC study of tetracycline repressor (Eur. J. Biochem. 270) 4567
where DH is the enthalpy difference between the denatured
and native state, m ¼ dT/dt is the scan rate, and E
a
is the
activation energy.
The thermal dependence of heat capacity (C
exc
p
)forTetR
was fitted to the experimental curves. The results are
presented in Fig. 4. The mean value for the activation
energy, E
a
, was calculated as 409.14 ± 30.5 kJÆmol
)1
,and
the mean value of temperature T*wasdeterminedas
61.53 ± 0.9 °C. The results are presented in Table 3.
To check further the validity of the two-state kinetic
model, proposed for denaturation of TetR alone, the
following equations proposed by Kurganov et al. [24] were
used:
d ln C
exc
p

d1=T
¼
1

p
Q
t
À Q

E
a
R
ð5Þ
The estimated Arrhenius equation parameters obtained
from Eqns (3), (4), and (5) are in good agreement with
each other and clearly support the proposed model of
TetR denaturation in the absence of Tc.
The temperature dependence of excess molar heat
capacity of TetR in the presence of Tc, at various scan
rates is presented in Fig. 5, and the dependence of the
transition temperature, T
max
,onscanrateforTetR–
[Mg–Tc]
+
complexes is shown in Fig. 3. The T
max
rapidly
increases in the range of low scan rates, but for higher scan
rates, it reaches a noticeable plateau. Such a relationship
between T
max
and scan rate indicates that this type of
equilibrium thermodynamic analysis can be employed

)1
in buffer A.
Solid lines are the best fit to each curve according to Eqn (3). Protein
concentration was always 0.6 mgÆmL
)1
.
Fig. 3. Effect of scan rate on transition temperature. Data obtained
from DSC experiments. (d) T
max
for TetR; (m) T
max
for the complex
ofTetrepressorwithTc(5molexcessofTcover1molofthedimer
was applied). The continuous lines have no theoretical meaning and
are shown to guide the eye.
Table 3. Arrhenius equation parameters estimated from the two-state irreversible model of thermal denaturation of TetR according to Eqns (3), (4) and
(5). The buffer was 10 mM Tris/HCl, 150 mM NaCl, 2 mM dithiothreitol, pH 8.0.
TetR
m
(KÆmin
)1
)
DH
(kJÆmol
)1
)
E
a
(kJÆmol
)1

insufficient signal-to-noise ratio of the experimental data.
The thermodynamic parameters obtained are summarized
in Table 4.
Thermal transition of the TetR–[Mg–Tc]
+
complex was
analyzed according to a two-state model. The best fit for
scan rate curves above 1.0 KÆmin
)1
is shown in Fig. 5.
This model assumes that the total excess capacity is
the sum of n independent thermal transitions. The heat
capacity associated with thermal transition is deter-
mined by a temperature derivative of enthalpy changes,
as given by:
C
p
ðTÞ¼
dH ðTÞ
dT
ð6Þ
The enthalpy change is determined by the total enthalpy
of the transition, which is assumed to be a constant
multiplied by the fraction of the molecules that are
unfolded: H(T) ¼ f
u
(T)DH. The fraction of unfolded
molecules is determined by the equation:
f
u

was calculated from the spectral parameter used to follow
denaturation (y) before the minimization procedure accord-
ing to the relation:
F
U
¼ðy À y
N
Þ=ðy
U
À y
N
Þ
y
N
¼ a
1
+a
2
T and y
U
¼ b
1
+b
2
T are the means of y,
characteristic of the native and denatured conformation,
respectively. They were obtained by linear regression of the
pre-transition and post-transition baselines. The parameter
used to follow denaturation, y, can be expressed as a
function of the kinetic parameters according to the follow-

(kJÆmol
)1
)
TetR Scan rate 418.40 ± 15.74 61.41 ± 0.56 – –
TetR + Tc Concentration – – 3.06 ± 0.25 1077.2 ± 86.2
Scan rate – – 3.10 ± 0.08 1067.1 ± 36.3
a
Values from fitting one-transition two-state model, according to Eqn (6).
Ó FEBS 2003 DSC study of tetracycline repressor (Eur. J. Biochem. 270) 4569
y¼y
U
À½y
U
Ày
N
exp À
1
m
Z
T
T
0
exp
E
a
R
1
T
Ã
À

; a ¼
K
1 þ K
) K ¼
f
u
1 À f
u
where, f
u
is the degree of advancement of the denaturation
process which refers to the unfolded fraction of a protein,
resulting from normalization of thermal CD profiles. The
denaturation CD profiles for the complex of TetR with Tc
were analyzed according to the following equation:
fðTÞ¼
½f
n
þm
n
Tþ f
u
þm
u
ðTÞ exp
DH
mH
R
1
T

vH
and T
m
values obtained are
917.78 ± 5.99 kJÆmol
)1
and 69.97 ± 0.01 °C, respectively.
DLS measurements
The buffer conditions in the DLS experiments were the
same as in the DSC measurements. The curves showing the
estimated hydrodynamic radius of wild-type TetR and its
complex with Tc as a function of protein concentration are
presented in Fig. 8. For evaluation of R
theo
H
,hydrationwas
estimated to be 0.2 g H
2
O per g protein and the partial
specificvolumetobe0.73cm
3
per g protein [18,27]. From
the comparison of R
H
values obtained from linear extra-
polation to zero concentration for wild-type TetR (2.98 ±
0.01 nm) and its complexes with Tc (3.04 ± 0.06 nm), it
appears that there are no pronounced differences in
hydrodynamic radii. The R
H

+
inducer to TetR can influence the
gross structure of the protein and the repressor stability in
solution. Here we studied the TetR
B
homodimer variant,
which is believed to have a similar structure to the TetR
D
variant [3]. Crystallographic studies of TetR
D
have shown
that binding of [Mg–Tc]
+
is accompanied by conforma-
tional changes in TetR, which in turn can abolish the specific
interaction of the protein with the DNA operator
sequences [28]. [Mg–Tc]
+
binds to the two tunnel-like
cavities, which, in the absence of the inducer, are filled with
disordered water molecules, and interact by both hydrogen
bonding and hydrophobic interactions with the protein
moiety [29]. Our previous CD studies in solution showed
that, in the case of TetR
B
,[Mg–Tc]
+
binding does not lead
to dramatic changes in the secondary structure of the
protein [30]. However, it has been suggested that a small

slightly to 2.98 nm on binding of [Tc–Mg]
+
in the protein
pocket. It should be pointed out that binding of the inducer
to TetR does not lead to any changes in the global structure
of the protein. However, a much stronger tendency to
protein aggregation has been observed in the case of TetR
B
alone than for the complex of the protein with [Tc–Mg]
+
inducer. The DLS measurements indicate that at 20 °C
TetR alone, as well as in the presence of [Mg–Tc]
+
,was
dimeric. As the binding of two molecules of [Mg–Tc]
+
to
TetR leads to changes in the tertiary structure of the protein,
one can expect that these changes may lead to a decrease in
the tendency of the protein to aggregate.
The DSC thermograms of TetR alone, presented in
Fig. 1, show irreversible thermal unfolding of the protein,
assuming an asymmetrical shape. The observed decrease in
T
max
on increasing TetR concentration (Fig. 2) indicates
that the dimeric protein aggregated at higher TetR concen-
tration. The DH
cal
/DH

to fit of the experimental data. The values of the average
energy of activation, E
a
, presented in Table 3, together with
T*(temperatureatrateconstant,k, equal to 1 min
)1
)arein
good agreement and further support the idea that the two-
state irreversible model offers a good explanation of the
TetR denaturation process. The model is also supported by
the observation that the T
max
of dimeric TetR does not
change significantly when the protein concentration is
increased. Such behavior would be expected from a
multimeric protein if its dissociation into monomers does
not take place before the rate-determining step and the
irreversible process shows first-order kinetics [21]. The
average activation energy, estimated to be 414 ± 15 kJÆ
mol
)1
for TetR, is equal to (8.9 ± 0.3) · 10
)3
kJÆmol
)1
after re-counting per gram of protein. This can be compared
with the value of (7.1 ± 5.8) · 10
)3
kJÆmol
)1

mean temperature of TetR denaturation, T
max
,determined
for the protein concentration range 0.4–4.0 mgÆmL
)1
is
60 °C. This value and that for denaturation enthalpy
changes are very close to those determined for the structur-
ally similar protein, cAMP receptor protein (CRP) (61 °C
and 503 kJÆmol
)1
, respectively) [42]. CRP, which has a very
similar molecular mass to TetR, is a homodimeric molecule
with a larger domain responsible for the cAMP binding and
a smaller domain, which possesses HTH structure, respon-
sible for the interactions with DNA sequences [32]. TetR
Ó FEBS 2003 DSC study of tetracycline repressor (Eur. J. Biochem. 270) 4571
undergoes reversible chemically induced denaturation by
urea, with simultaneous dissociation to monomers, charac-
terized by a Gibbs free energy change DG (H
2
O, 25 °C) of
75 kJÆmol
)1
[8]. It has been shown that CRP, which is
structurally similar to TetR, undergoes reversible denatur-
ation by guanidine hydrochloride, characterized by more
rapid dissociation into monomers followed by co-operative
unfolding of CRP monomers. The overall process of CRP
unfolding is characterized at 20 °CbyaDG (H

equal to 1 (Table 2).
In similar cases, where the T
max
was independent of scan
rate in the high range of the heating ratio, the irreversible
denaturation of annexin V E17G [25] and human phenyl-
alanine hydrolase and human phenylalanine hydrolase with
L
-Phe [26] was described by application of the equilibrium
thermodynamic analysis.
A two-state reversible model was used to describe the
thermal transition of the complex of wild-type TetR with Tc
(at high scan rate). This model is based on the general
Lumry–Erying model [21], simplified by excluding the
kinetic irreversible step, which is negligible at a scan rate
over 1 KÆmin
)1
:
N
2
Tc
2
!
K
2Tc þ U
2
This two-state model assumes that the total excess heat
capacity is a sum of n independent two-state thermal
transitions. As can be seen in Fig. 5, fitting of one-
transition two-state model seems to be satisfied (Table 4).

N
2
Tc
2
!
K
2Tc þ U
2
À!
k
D
According to this model, the dimeric native TetR in the
presence of Tc undergoes two-state reversible unfolding
with simultaneous dissociation into monomers U and
ligand loss. The unfolded species thus obtained, U
2
,
undergoes an irreversible alteration to yield a final,
denaturated state D. It is assumed that chemical equilib-
rium between species N
2
Tc
2
and U
2
is always established in
such a way that the differences between the heat capacity
of the unfolded and native state (DC
p
) is negligible, and

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