From the Programa de Biologia Estrutural, Departamento de
Bioquímica Médica-ICB, Centro Nacional de
Ressonância Magnética Nuclear de Macromoléculas,
Universidade Federal do Rio de Janeiro,
21941-590 Rio de Janeiro, Brazil
The pressure-induced dissociation of the dimeric
DNA binding domain of the E2 protein of human papillomavirus (E2-DBD)
is a reversible process with a Kd of 5.6 × 10
8 M at pH 5.5. The complete exposure of the
intersubunit tryptophans to water, together with the concentration
dependence of the pressure effect, is indicative of dissociation.
Dissociation is accompanied by a decrease in volume of 76 ml/mol, which
corresponds to an estimated increase in solvent-exposed area of 2775 Å2. There is a decrease in fluorescence polarization of
tryptophan overlapping the red shift of fluorescence emission,
supporting the idea that dissociation of E2-DBD occurs in parallel with
major changes in the tertiary structure. The dimer binds
bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding
by about 30%, in contrast with the almost complete loss of dye binding
in the urea-unfolded state. These results strongly suggest the
persistence of substantial residual structure in the high pressure
state. Further unfolding of the high pressure state was produced by low
concentrations of urea, as evidenced by the complete loss of
bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded
state is also apparent from the distribution of excited state lifetimes
of tryptophan. The combined data show that the tryptophans of the
protein in the pressure-dissociated state are exposed long enough to
undergo solvent relaxation, but the persistence of structure is evident
from the observed internal quenching, which is absent in the completely
unfolded state. The average rotational relaxation time (derived from
polarization and lifetime data) of the pressure-induced monomer is
shorter than the urea-denatured state, suggesting that the species
obtained under pressure are more compact than that unfolded by
urea.
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INTRODUCTION |
The interaction of proteins with DNA constitutes the basis for the
regulation of key biological functions such as gene expression, replication, and recombination. DNA-binding proteins recognize specific
stretches of DNA, and the molecular basis for the interactions is
currently the focus of intensive research (1, 2). The domains of
proteins that interact with DNA are highly variable in folding
topology, and thus they can accommodate a large number of functions for
the different complexes in both eukaryotic and prokaryotic systems (1,
3). One characteristic of many DNA-binding dimeric proteins is that the
structures of the monomers are often highly intertwined (4-8). Studies
on several complexes have revealed that both DNA and protein undergo
conformational changes upon interaction, especially at the interface,
and there is an important free energy coupling among folding,
dimerization, and DNA recognition (9-12). In cases where these
DNA-binding proteins have been studied, their unfolding by denaturing
agents occurs simultaneously with dissociation in a highly concerted
manner (13-17).
Human papillomavirus (HPV)1
infection of the anogenital tract is associated with several
premalignant and malignant lesions, especially dysplasia and carcinoma
of the uterine cervix (18). The E2 transcriptional transactivator
protein (E2-TA) participates in the regulation of the expression of
viral genes in papillomavirus (19, 20). The products of the E2 gene are
crucial to the life cycle of the virus because they regulate
transcription from all viral promoters, which makes E2-TA a potential
target for antiviral therapy. The E2 protein is comprised of an
N-terminal transactivation domain separated from the C-terminal DNA
binding and dimerization domain by a flexible region rich in proline
residues (7). The solution structure of the C-terminal DNA binding
domain (E2-DBD) from human papillomavirus strain-31 (HPV-31) was
recently determined by NMR spectroscopy (21). The urea-induced
denaturation of the recombinant E2-DBD from HPV-16 was shown to proceed
through a concerted two-state unfolding and dissociation process, with
no detectable intermediate species (17). However, investigation of its
kinetic folding pathway reveals the presence of a short lived monomeric
intermediate (22).
Noncovalent interactions can be reversibly perturbed using high
hydrostatic pressure, which allows a thermodynamic characterization of
protein folding, protein-protein, and protein-ligand interactions (23,
24). Hydrostatic pressure drives the structure of proteins to a
thermodynamic state of smaller volume (23, 25, 26). Protein folding and
protein-protein interactions are normally accompanied by an increase in
volume because of the combined effects of the formation of
solvent-excluding cavities and the release of bound solvent (24). Water
is released as nonpolar amino acid residues are buried, as well as when
salt linkages are formed. Arc repressor, a small DNA-binding dimer
protein from the bacteriophage P22, has been studied in detail by high
pressure, in an attempt to understand interrelationships among protein
folding, dimerization, and DNA recognition (9, 11, 15, 16).
In this paper, we study the reversible dissociation of HPV-16 E2-DBD
using high pressure in combination with fluorescence spectroscopic
techniques. We present evidence for a persistent residual structure in
the monomeric denatured state at high pressure and are able to
characterize it by Trp fluorescence spectra, polarization, lifetime
distribution, stability to urea unfolding, and binding of
bis(8-anilinonaphthalene-1-sulfonate) (bis-ANS). The existence of a
folded monomeric state of E2-DBD in the absence of denaturants may
represent an important target to drug development in addition to being
highly relevant to the understanding of the basic principles underlying
protein folding mechanisms.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All reagents were of analytical grade. bis-ANS
was purchased from Molecular Probes (Eugene, OR). Distilled water was
filtered and deionized through a Millipore water purification
system.
The C-terminal 80-amino acid (positions 286-365) DNA binding domain of
HPV-16 E2 protein was overexpressed in Escherichia coli and
purified as a soluble, folded dimeric protein (17). As shown
previously, the isolated C-domain still retains the ability to dimerize
and bind to the DNA (27). Protein concentration was determined using an
extinction coefficient of 41,900 M
1
cm
1 at 280 nm (28).
Spectroscopic Measurements under Pressure--
The high pressure
cell equipped with optical windows has been described (15) and was
purchased from ISS (Champaign, IL). Fluorescence spectra were recorded
on an ISSK2 spectrofluorometer (ISS Inc., Champaign, IL). Fluorescence
spectra at pressure p were quantified by the center of
spectral mass <
p>.
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(Eq. 1)
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where Fi stands for the fluorescence emitted
at wave number
i and the summation is carried out over the range of appreciable values of F.
The pressure was increased in steps of 200 bars. At each step, the
sample was allowed to equilibrate for 15 min prior to making measurements. There were no time-dependent changes in
fluorescence spectra between 10 and 30 min.
Unless otherwise stated, the experiments were performed at 22 °C in
the standard buffer: 50 mM bis-Tris-HCl containing 1 mM dithiothreitol and adjusted to the desired pH by the
addition of HCl.
Thermodynamic Parameters--
The degree of dissociation
(
p) is related to <
p> by the expression,
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(Eq. 2)
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where <
i> and <
f> are the initial and
final values of the center of spectral mass, respectively, while <
p> is the center of spectral mass at pressure
p.
The equilibrium constant, and therefore the Gibbs free energy for a
monomer-dimer association equilibrium, will depend on the standard
volume change (
V) of the reaction,
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(Eq. 3)
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(Eq. 4)
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where Kd(p) and
Kdo are the equilibrium constants for dissociation
at pressure p and at atmospheric pressure respectively;
V is the volume change,
p is the extent of
reaction at pressure p, and C is the protein
concentration as dimer. In a dissociation-association process, a change
in protein concentration from C1 to C2 results
in a parallel displacement
p of the plot of ln
Kd(p) versus p along the
pressure axis,
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(Eq. 5)
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where
Vc is the volume change of
association determined from changes in dissociating pressure with
concentration at a constant degree of dissociation, and n is
the number of subunits.
Lifetime Measurements--
Lifetime measurements were performed
in a multifrequency cross-correlation phase and modulation fluorometer
(ISS/K2), as described previously (29-31). Samples were excited at 295 nm with a 300-W xenon lamp, and emission was collected using 7-54 and
0-52 filters. For pressure experiments, light scattered from ficol
particles was used as reference (15). The choice of fitting with
Lorentzian components was based on
2 values and plots of
weighted residuals.
Fluorescence Anisotropy Measurements--
Values of fluorescence
anisotropy were measured according to the equation,
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(Eq. 6)
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where I
and I
are the
intensities of the emission when the polarizers are oriented parallel
or perpendicular to the direction of the polarizer of the excitation,
respectively. The errors for the polarization measurements were less
than ±0.005.
Average rotational relaxation times (<
>) were calculated from the
anisotropy values (A) and from the average lifetime
experimentally determined by using the Perrin equation,
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(Eq. 7)
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where Ao is the limiting anisotropy of the
fluorophore (0.240), and
is the fluorescence decay lifetime.
Urea Unfolding of the High Pressure State--
To a solution
containing the E2-DBD dimer at 1 µM, 50 mM
bis-Tris-HCl, 1 mM dithiothreitol, pH 5.5, and 5 µM bis-ANS at 22 °C, different concentrations of urea
were added. The mixture was introduced in the high pressure cell, and
after a 30-min period of incubation, fluorescence spectra of bis-ANS
were recorded. Next, the pressure was taken to 2860 bars, required for
attaining the maximum change in the center of spectral mass when
monitoring Trp fluorescence in pressure titrations in the absence of
bis-ANS. Since the decrease of the fluorescence of E2-bound bis-ANS at
high pressure is a slow reaction, we followed the changes in the total
fluorescence intensity with time until the completion of the
reaction.
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RESULTS |
Structural Considerations of the E2-DBD--
The crystal structure
of the E2-DBD from bovine papillomavirus strain-1 revealed a new class
of folding topology (7, 32), only shared by the EBNA1 DNA binding
domain from the Epstein-Barr virus with no amino acid sequence homology
(33). The fold consists of a dimeric eight-stranded
-barrel, with
each monomer contributing half of the barrel. Two helices/monomer
interact with the outside of the barrel, forming hydrophobic cores: a
major
-helix, which forms the DNA binding site, and a minor
-helix at the opposite side (Fig.
1A). The solution structure of
the E2-DBD from HPV-31 (80% homology with E2-DBD from HPV-16; Fig.
1B) has been solved recently by NMR methods and has
identical folding topology, although some significant differences
between solution and crystal structures were observed (21). The dimeric
interface is stabilized by intersubunit
-sheet hydrogen bonding and
by the packing of hydrophobic residues at the center of the barrel. The
formation of the dimer buries 2,567 Å2 of
solvent-accessible area in the bovine papillomavirus strain-1 domain,
which strongly suggests that any partly folded monomer would have to
undergo substantial accommodations in structure (7).

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Fig. 1.
A, ribbon representation of the solution
structure of E2-DBD of HPV-31. The monomeric subunits are colored
differently. The tryptophans (residues 37 and 39) and histidines
(residues 8, 38, 46) are shown in green and
yellow, respectively. The coordinates are from Liang
et al. (21). The schematic representation was produced by
using the program Rasmol. B, sequence alignment of the
E2-DBD proteins from human papillomaviruses 31 and 16 (Ref. 21).
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The HPV-16 E2-DBD has three tryptophan residues/monomer, two of which
face the central cavity of the barrel that forms the interface between
the two monomers in a stacked conformation as shown in the HPV-31
E2-DBD structure (Ref. 21; Fig. 1). The position of tryptophan residues
in E2-DBD is unique, in that the indole rings of Trp37
(monomer 1) and Trp39 (monomer 1) stack in a characteristic
antiparallel fashion; the same occurs with Trp37 (monomer
2) and Trp39 (monomer 2). All four base-stacking
interactions occur within the dimer interface, giving rise to a
favorable aromatic-aromatic interaction. This makes it possible to
follow dissociation and unfolding processes using fluorescence, with
substantial sensitivity. The third tryptophan residue,
Trp59, which is present in HPV-16 E2-DBD but absent in
HPV-31 E2-DBD faces the solvent and is located in the smaller
-helix
(Fig. 1).
Pressure Dissociation of the E2-DBD: Concentration Dependence,
Reversibility, and Thermodynamic Parameters--
To monitor
dissociation of E2-DBD under pressure, we followed the shift of the
fluorescence emission spectra by measuring changes in the center of
spectral mass (see Equation 1). This reveals changes in the environment
of the tryptophan residues at the interface, which will eventually
become exposed to the solvent. Fig.
2A compares the tryptophan
emission spectra of E2 at pH 5.5 and 22 °C in the native state and
under denaturing conditions (5 M urea; high hydrostatic
pressure). Under high pressure (2.86 kilobars), the tryptophan
fluorescence intensity decreased approximately 40%, whereas
denaturation induced by urea was not accompanied by significant changes
in the total fluorescence intensity. The inset of Fig.
2A shows that despite differences in tryptophan fluorescence
intensity in urea and under pressure, both conditions caused the same
displacement toward the red (
max = 10 nm). In both
cases, the final value of center of mass was around 28,600 cm
1, reflecting complete exposure of the tryptophan
residues to the aqueous environment upon dissociation by pressure or
denaturation by urea. Titration of the changes in center of spectral
mass as a function of pressure is shown in Fig. 2B.

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Fig. 2.
Effects of pressure on the intrinsic
fluorescence emission of E2-DBD. A, nonnormalized intrinsic
fluorescence spectra of E2 (1 µM) at atmospheric pressure
(a), at 2.9 kilobars (b), and at atmospheric
pressure in the presence of 5 M urea (c),
excited at 278 nm in 50 mM bis-Tris-HCl, 1 mM
dithiothreitol, pH 5.5. The inset shows normalized spectra.
B, pressure-induced dissociation of E2-DBD as followed by
the center of spectral mass change at pH 5.5 at 22 °C. Other
conditions were as in panel A.
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The equilibrium dissociation constant and the accompanying volume
change for the dimer
monomer equilibrium were calculated using the
equation for dissociation by pressure (Equation 4). Fig.
3A shows the degree of
dissociation at each pressure, based on the center-of-mass data at pH
5.5 from Fig. 2B (Equation 2). The values derived from
fluorescence anisotropy (squares) coincide with those
obtained from the changes in fluorescence spectra (circles). The intercept of the semilogarithmic plot of these data (Equation 4)
yields the dissociation constant (Kd); the slope
provides the volume change of association (
V) (Fig.
3A, inset). The Kd values
obtained in this way from both fluorescence spectra and anisotropy data
are in excellent agreement with that calculated by Mok et
al. (17) from urea unfolding experiments (Table
I), especially so considering the
difference in techniques and the extrapolations to atmospheric pressure
and absence of denaturant, respectively. These results show that the
previous use of an empirical approach (
Gu =
Go + m[urea]) agrees very well with
the thermodynamic relation (
Gp =
Go + p
V) at pH 5.5.

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Fig. 3.
A, degree of dissociation as a function
of pressure at pH 5.5. The extent of dissociation ( ) was calculated
as described under "Experimental Procedures" (Equation 2) by using
the center of mass values from Fig. 2B ( ). For
comparison, the degree of dissociation calculated from the anisotropy
changes as a function of pressure (Fig. 7A) is also shown
( ). Inset, a plot of ln( 2/(1 ))
versus pressure (Equation 4), where the slope gives the
volume change of association and the intercept is related to the
dissociation constant at atmospheric pressure (Table I). B,
concentration dependence of the pressure-induced dissociation of
E2-DBD. , 1 µM; , 5 µM; , 10 µM. The shift in the intrinsic fluorescence emission was
used to follow dissociation of E2-DBD. The decompression curve at 1 µM protein concentration is also shown ( ). All
experiments were performed at 22 °C in bis-Tris-HCl 50 mM containing 1 mM dithiothreitol at pH
5.5.
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Table I
Dissociation constant at atmospheric pressure (Kd) and the
volume change of association ( V) for the equilibrium E22
2 E2 at different pH conditions
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Measurements of changes in volume are inaccessible to most denaturation
techniques but are of great importance, since they measure the
differences in packing of the states involved, adding valuable
information to the thermodynamic characterization. The calculated
volume change for the dissociation of E2-DBD by high pressure at 1 µM and pH 5.5 is 76 ml/mol (slope of plot in Fig. 3A), which falls within the range found for other dimers
(23).
An independent assessment of dissociation can be obtained from the
concentration dependence of the process (Fig. 3B). An
increase in E2-DBD concentration promotes a displacement in the
p1/2 value (the pressure that promotes 50% change)
from 1300 bars at 1 µM to 2070 bars at 10 µM E2-DBD. The difference in p1/2
between these two experiments (
p1/2 = 770 bars)
allows us to calculate
Vc (23), the expected
volume change for the dissociation of E2-DBD (Equation 5). The
Vc value was 73.2 ml/mol, in very good agreement
with the value obtained for
V at a fixed protein concentration (76 ml/mol; Fig. 3). A ratio of
V/
Vc that is close to 1 indicates
that the dissociation process complies with the law of mass
action in the concentration range analyzed and that there is no
significant conformational heterogeneity as observed in multisubunit
complexes (34), tetramers (35), and more recently in a dimeric protein
(36).
The reversibility of the dissociation process induced by pressure was
confirmed by following the spectral change on decompression. The value
of center of spectral mass for the Trp emission returns to the initial
value (prior to pressure application), with negligible hysteresis (Fig.
3B, open circles).
The stability of E2-DBD toward pressure denaturation depends markedly
on the pH (Fig. 4A). At pH 6.0 or 7.0 the process is incomplete, with a decrease in the center of mass
of only 200-300 cm
1 at 2.86 kilobars, the highest
pressure applied. At pH 5.8, the center of mass shift was greater, but
only at pH 5.5 is the final value (28,600 cm
1) compatible
with the value obtained in urea, indicating complete exposure of
tryptophan residues to the solvent. The urea unfolding curves also
showed a strong dependence on pH (17). It is noteworthy that at
atmospheric pressure, the center of mass of E2-DBD increases from
29,050 cm
1 at pH 5.5 to 29,280 cm
1 at pH
7.0 (Fig. 4A). This result clearly indicates that pH causes a change in the environment of at least one of the Trp residues. The
changes in the center of spectral mass with an increase in pH from 5.5 to 7.0, although rather small (~200 cm
1), are not
significantly dependent on protein concentration (not shown). Besides,
as previously shown by analytical ultracentrifugation (17), E2-DBD is
still a dimer, with no detectable monomers at pH 5.5. These data
suggest that an isomerization reaction rather than a dissociation
reaction is taking place when the pH is lowered from 7.0 to 5.5. The
existence of a predissociated dimer (17, 37) and of a denatured dimer
(38) has been demonstrated for the Arc repressor. Fig. 4B
shows the change in volume change as a function of pH. At high pH
values, the changes induced by pressure are less steep, which results
in a smaller volume change (~43 ml/mol). Between pH 6.0 and 5.5 there
is an abrupt transition, and
V increases to ~80 ml/mol.
The presence of a predissociation transition explains the smaller
volume changes (less steep dependence on pressure) at high pH values,
where the presence of a dimeric intermediate stretches the dissociation
curve (16, 23).

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Fig. 4.
A, effects of pH on the pressure-induced
dissociation of E2-DBD at 22 °C. Dissociation of E2-DBD was
performed at pH 5.5 ( ); 5.8 ( ); 6.0 ( ), and 7.0 ( ).
Other conditions as in Fig. 2A. B, apparent
volume change of association as a function of the pH calculated from
the experiments in Fig. 4A using Equation 4 (see
"Experimental Procedures").
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Eight histidines out of 10 per dimer of HPV-16 E2-DBD lie at the
interface between the two subunits (histidines 8, 38, and 46 are
indicated in the E2-DBD structure; Fig. 1). The histidines of each
subunit face each other at the interface, especially histidines 8 and
38; if they are protonated, they could destabilize the dimer. This may
account for the greater tendency to dissociate at lower pH. Proton
dissociation of the histidines, with a pK around 5.8 from
Fig. 4B, would suppress the charge repulsion at the
interface and engender a tight dimer. These histidines may exert
a crucial regulatory effect on the stabilization with a potential
effect on sequence-specific DNA binding.
Since the pressure dissociation process was not fully complete at pH
6.0 or 7.0 at the maximum pressures that were attained in Fig.
4A, we repeated the experiment at pH 6.0 with subdenaturing concentrations of urea added to the buffer (Fig.
5). In the presence of 0.5-0.95
M urea, pressure induced a decrease in the center of
spectral mass to a value similar to that observed at high urea concentrations at atmospheric pressure or at high pressure at pH 5.5 in
Fig. 4A, i.e. complete exposure of the Trp side
chains to the solvent. From these curves, we calculate the
V of association and the Kd at pH 6.0 in the presence of different concentrations of urea (Table I). The
Kd values are in good agreement with the value
previously obtained from urea unfolding curves (17). Interestingly, the
data show that
V increases dramatically with the
addition of a small concentration of urea (0.5 M urea). The
increase in
V observed when small amounts of urea are
added could reflect an extra transition from "native-like" monomer
to an unfolded monomer.

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Fig. 5.
Effects of subdenaturing concentrations of
urea on the dissociation of E2-DBD by pressure at pH 6. The center
of mass shift was used to follow dissociation in the absence of urea
( ) or in the presence of 0.5 M ( ); 0.75 M
( ), and 0.95 M ( ) urea. Other conditions were as in
Fig. 2A. The isolated symbol at the bottom
left corresponds to the center of mass value of 1 µM
E2-DBD in the presence of 6 M urea at atmospheric pressure
at the same pH.
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Residual Structure in the High Pressure Monomeric State of
E2-DBD--
In Fig. 2A, it was shown that the urea-induced
denaturation of E2 shifts the spectrum to the red but does not promote
any decrease in intensity, whereas dissociation induced by high
pressure affects both parameters. Fig.
6A shows the changes in the
spectral area of the tryptophan fluorescence emission as a function of pressure or urea. As it is seen, over a broad range of concentrations, urea did not promote changes in tryptophan emission intensity, while
pressure provoked a gradual decrease in the fluorescence intensity. The
changes in tryptophan fluorescence intensity under high pressure
resemble those induced by acid pH (17).

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Fig. 6.
A, comparison between urea denaturation
and pressure-induced dissociation of E2-DBD, based on tryptophan
fluorescence intensity. The tryptophan spectral area ratio is shown as
a function of pressure increase ( ) or urea addition ( ). The
ratios were calculated by dividing the area at any given condition by
the spectral area at atmospheric pressure or by the area in the absence
of urea. Other conditions were as in Fig. 2A. B,
Lorentzian distribution of Trp fluorescence lifetimes for E2-DBD at
atmospheric pressure ( -), in the presence of 6 M urea
(····) and at 3 kilobars ( ). Each data point represents the
average of five measurements, and the error is smaller than 0.2 degree
for the phase and 0.004 for the modulation measurements. The pH was
adjusted to 5.5 in all measurements. Other conditions were as under
"Experimental Procedures." The protein concentration was 10 µM in all experiments.
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Time-resolved fluorescence measurements allow us to probe the
tryptophan environment in the nanosecond time frame, providing a
dynamic characterization of the conformational state of a protein (30,
31). Fluorescence lifetime measurements were performed to compare the
excited state of the Trp residues of E2-DBD at pH 5.5 in three
different states: native state (atmospheric pressure), pressure-dissociated state (2.8 kilobars), and urea-denatured state
(Fig. 6B). In the native state, the best fit for the data is
achieved by a Lorentzian distribution of lifetimes, rather than
discrete exponential decays, centered at 1.745 ns. For the pressure-dissociated state, the distribution of lifetimes shifts to
shorter lifetimes, centered at 0.727 ns (Fig. 6B and Table II). The decrease of 2.2-fold in the
lifetime value is consistent with the decrease in tryptophan emission
that takes place under these conditions (Fig. 6A). On the
other hand, the center of the distribution of lifetimes for the
urea-denatured state (1.951 ns) is very similar to the native state,
consistent with the identical intensities of the native and
urea-denatured states. However, for both pressure-dissociated and
urea-denatured states, the width of the distribution increases
dramatically in comparison with the control at atmospheric pressure and
in the absence of denaturant (Fig. 6B; Table II). The
dramatic differences between the distribution of lifetimes of the
pressure-dissociated and urea-denatured states indicate that the phase
space explored by the Trp residues are very different. The shorter
lifetimes experienced by the Trp residues in the pressure-dissociated
state suggest that a conformation-dependent quenching takes
place.
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Table II
Comparison of the rotational hydrodynamic properties of E2 in the
native, pressure-dissociated, and urea-denatured states
Measurements of anisotropy and lifetime are the average of five
measurements. The error for anisotropy was smaller than 0.0005, and the
2 values for the fittings of lifetime distribution were
typically lower than 5.0.
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Hydrodynamic Evidence for Residual Structure in the
Pressure-dissociated Monomer--
To compare the rotational
hydrodynamic properties of E2 in the pressure-dissociated and
urea-denatured states, fluorescence anisotropy measurements were
performed. The steady-state anisotropy value for the dimer at
atmospheric pressure was 0.12. The anisotropy decreases to 0.0548 at
2.5 kilobars, suggesting a decrease in the rotational hydrodynamic
radius (Fig. 7A). Fig.
7A also shows the decompression curve (open
circles), indicating complete reversibility of the dissociation
process. Fig. 7B shows the change in anisotropy induced by
the increase in urea concentration. At 5 M urea, where the
fluorescence spectrum was already shifted completely to the red, the
anisotropy was 0.078, significantly higher than the value observed for
the pressure-dissociated protein. This result indicates that the
urea-denatured form of E2 is more expanded than the pressure-induced monomer. From the changes in anisotropy, in combination with the lifetime measurements, the average rotational relaxation times (<
>) were determined according to Equation 7 (Table II). The value
of <
> at atmospheric pressure is consistent with the expected value for a combination of local motions with global rotation of the
Trp residues in a protein with the dimensions of E2-DBD dimer (7, 21).
The tryptophans of the pressure-dissociated monomer rotate faster,
indicating a more compact state than is seen with the urea-denatured
form (Table II). The
V and Kd values
were 78.5 ml/mol and 4.3 × 10
8 M,
respectively, very close to those obtained from the center of mass data
(Fig. 3A and Table I).

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Fig. 7.
Dissociation of E2-DBD based on tryptophan
fluorescence anisotropy at pH 5.5 under pressure (A) or
denaturation in the presence of increasing concentrations of urea
(B). In A, the open circles
represent the decompression curve. The excitation was set at 295 nm,
and the emission was collected through 7-54 and 0-52 cut-off filters.
The anisotropy values under pressure were corrected for distortions in
the bomb windows.
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Persistent Binding of bis-ANS in the Pressure-dissociated
State--
As part of the characterization of the high pressure state,
we made use of the hydrophobic dye bis-ANS, which binds to accessible hydrophobic patches in structured proteins, which translates into a
large increase of its fluorescence emission (15, 39, 40). At pH 7.0, the E2-DBD dimer appears to bind two molecules of bis-ANS (40). When
the E2-DBD was pressurized in the presence of bis-ANS, there is a 30%
decrease in the bis-ANS fluorescence (Fig.
8), whereas the change in the center
of spectral mass of the tryptophan residues (not shown) is the
same as before. Urea denaturation causes a much larger decrease in the
bis-ANS fluorescence, which is compatible with the lack of long
range interactions in an unfolded polypeptide (Fig. 8). These data
indicate that the protein under pressure retains some degree of long
range tertiary interactions, whereas high concentrations of urea (5 M) cause a more extensive unfolding.

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Fig. 8.
Decrease in binding of bis-ANS to E2-DBD as
dissociation/denaturation is induced by urea ( ) or pressure
( ). The area of the bis-ANS fluorescence spectrum is normalized
to A0, the area obtained at atmospheric pressure
in the absence of urea. The difference between the values at 3000 bars
and in 5 M urea indicates that different conformations are
attained. Protein concentration was 1 µM, and bis-ANS was
5 µM. Excitation was set at 360 nm, and emission was
collected in the range 400-600 nm.
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Probing the Stability of the Pressure-dissociated State to Urea
Unfolding--
To gain more insight on the pressure-dissociated state,
we analyzed its stability toward urea denaturation. We reasoned that adding increasing concentrations of urea to the state at high pressure,
in the presence of bis-ANS, would further decrease the amount of bound
bis-ANS consistent with a fully unfolded polypeptide. In this way, we
could obtain an estimate of the stability of the species trapped under
high pressure. Fig. 9A shows
typical traces of the time course of changes in bis-ANS fluorescence of
E2-DBD after pressure (2.86 kilobars) is applied, at increasing
concentrations of urea.

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Fig. 9.
Effects of subdenaturing concentrations of
urea on the intermediate species trapped under pressure. A,
kinetics of E2-DBD denaturation under pressure (2860 bars), based on
the decrease in bis-ANS binding in the absence of urea ( ) or in the
presence of 1 M urea ( ) at pH 5.5. The fluorescence of
bis-ANS was measured as in Fig. 8. B, urea-induced
denaturation of the species trapped under high pressure ( ) and of
the protein at atmospheric pressure ( ). The end point of each
kinetic experiment (see panel A) is plotted as a function of
urea concentration. Note that in the presence of 0.75-1.00
M urea, bis-ANS decreases to a value similar to that seen
in the presence of 4-5 M urea at atmospheric pressure
( ). Protein concentration was 1 µM, and bis-ANS
concentration was 5 µM.
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The bis-ANS fluorescence values obtained after reaching the equilibrium
were plotted against urea (Fig. 9B). The structure present
at high pressure is completely unfolded at 1 M urea, with an apparent [U]50% of ~0.25 M. In
contrast, the stability of the folded dimer at atmospheric pressure but
otherwise similar conditions is much higher ([U]50% of
~2.5 M, Fig. 9). The species present at 2.6 kilobars in
the presence of 1 or 2 M urea could be defined as a largely
unfolded state in which the tryptophan residues are completely exposed
to the solvent (center of mass equal to 28,600 cm
1)
and its bis-ANS binding capacity is almost abolished, as expected for a
completely unfolded polypeptide.
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DISCUSSION |
When studying the folding of dimeric proteins, it is important to
determine the hierarchy of stability of possible structures formed in
the reaction and to be able to distinguish, when possible, between
folding and association events (41). In the particular case of several
DNA-binding proteins with structures that are highly intertwined,
folding and association are highly coupled processes. A good example is
the Arc repressor, in which a highly concerted folding and association
process is observed, as determined by fluorescence spectroscopy and NMR
(13, 15, 16, 37, 38).
High pressure constitutes a noninvasive technique in which reversible
dissociation or denaturation processes can be analyzed on a
thermodynamic basis (24). In the present work, we describe the
reversible dissociation of E2-DBD by high pressure, and we characterize
the partially folded monomers. The Trp fluorescence changes that take
place under pressure allow us to determine the volume change of
association by applying the thermodynamic principles associated
to pressure effects. A substantial fraction of the increase in volume
on folding and association of proteins results from the formation of
solvent-excluded void volumes. The structural importance of the
volume change can be evaluated by comparison with other dimer-monomer
dissociation reactions. The volume change of association for E2-DBD (76 ml/mol) is within the same range of that obtained for other dimers
(55-170 ml/mol) (for a review, see Ref. 23). However, most of the
dimers previously studied were much larger than E2-DBD. Only the Arc
repressor was smaller (15). An appropriate way to express the volume
change is to normalize to the molecular weight of the dimer (15, 23),
which furnishes the specific volume change. The value obtained for Arc and E2-DBD is much higher than for other dimers. The specific volume
change of association is 4.2 µl/g for E2-DBD; 7.69 µl/g for Arc;
and 0.688, 1.25, 1.88, and 4.73 µl/g for enolase, hexokinase, tryptophan synthase
2 subunit, and R17 coat protein
dimer, respectively (23). The large change in volume per mass of
protein found in Arc, R17 coat protein, and E2-DBD dissociation can be
explained by a high degree of interaction of buried amino acid side
chains with the solvent under dissociation. The hydration of
charges that were involved in salt bridges or the hydration of polar
and nonpolar groups results in volume contraction. It is suggested that
the partially unfolded states of these three proteins in the monomeric
state favor a higher degree of hydration when compared with other
dissociation systems. However, the Arc repressor undergoes an almost 2 times greater volume change per mass of protein than does E2-DBD,
indicating that in the case of Arc the disruption of the structure
under dissociation is more drastic.
An estimate of the solvent-excluded surface can be made from the volume
change (42). The volume change obtained experimentally (76 ± 4 ml/mol) for E2-DBD at pH 5.5 corresponds to a decrease in volume of 126 Å3/dimer dissociated. Considering a nonpolar solvent as a
model for calculating linear compressibility, this value corresponds to
exposure of 2775 ± 256 Å2 when E2-DBD dissociates.
This value is compatible with the x-ray diffraction structure, which
led to the conclusion that 2,467 Å2 of solvent-accessible
area is buried on formation of the dimer (7). This remarkable
similarity (within experimental error) between the pressure-dissociated
state and that calculated from x-ray diffraction for the native
monomer presents further evidence that the monomer retains some
tertiary structure; although it is less stable, it still maintains a
substantial proportion of hydrophobic residues buried in its
interior.
Although the structure of E2-DBD from HPV-16 has not yet been solved, a
structure of an equivalent protein from a strain with 80% homology
(HPV-31) has been solved by NMR methods and shows the same topology as
in the crystal structure of the bovine virus E2-DBD (Fig. 1; Refs. 7
and 21). This confirms that one of the three tryptophan
residues/monomer is effectively located at the surface in the minor
-helix, facing the solvent, and the other two are located at the
dimeric interface, facing the center of the barrel. It is, therefore,
very likely that the buried tryptophan residues are responsible for the
change in the fluorescence spectral mass, as they become exposed to the
solvent at high pressure or high urea concentration. However, although
the tryptophan residues in the pressure-dissociated state undergo
solvent relaxation (resulting in a spectral shift as large as that
caused by urea denaturation), the lifetime distribution is different
from that in the urea-unfolded state. The width of the distribution is
broader than in the native state but narrower than in the completely
unfolded state. Four lines of evidence distinguish the states attained
by pressure and urea: Trp intensity, excited-state lifetime, average
rotational relaxation time, and bis-ANS binding. This clearly shows the
presence of a state with persistent structure at high pressure. The
conformation of E2 trapped under high pressure is more compact than the
urea-denatured state, and it retains the ability to bind bis-ANS,
features compatible with molten globule-like conformations (43, 44).
High pressure has been used to trap molten-globule conformations of
different protein systems (15, 24, 45, 46).
The urea-unfolded state virtually binds no bis-ANS, whereas the
pressure-dissociated state retains ~70% of its bis-ANS binding capacity. This strongly suggests that there are persistent long-range interactions that allow for the formation of sites where the dye can be
inserted. The nature of those sites is unclear. They might lie in an
accessible core or in the DNA binding site, since folded E2-DBD binds
both ANS and bis-ANS (22, 28). It can be argued that the bis-ANS
binding is in fact due in part to a residual native-like conformation
of the DNA binding site.
The structure of E2-DBD in the monomeric state under pressure is very
unstable: urea titration of the bis-ANS binding reveals a midpoint
around 0.25 M urea, in contrast with the 2.5 M
for the native state (Fig. 9B). This decreased stability to
urea explains why the dissociation constant obtained from extrapolation
to zero in the urea-unfolding curves (atmospheric pressure) is similar to that obtained from the pressure-dissociation experiment. The lack of
resistance to unfolding of the high pressure state at low urea
concentrations can be interpreted as high sensitivity of the global
structure to the chemical denaturant or noncooperative unfolding of the
local structure that binds bis-ANS. Although we cannot discern between
these two explanations due to experimental limitations, the lack of
cooperativity is a defining characteristic in molten globule-like
conformations (43, 44).
Altogether, our data suggest the following pathway for the reversible
dissociation and unfolding of E2-DBD at pH 5.5.
Equilibrium 1 encompasses the process of dissociation of the dimer
to partially folded monomers (A) at pH 5.5. Unfolding by
high concentrations of urea involves both equilibria. Equilibrium 2 per se was monitored by the experiment of Fig. 9, where the pressure-dissociated state was denatured by urea to a completely unfolded species (U). The free energy change for reaction 2 (
G2) is less than 2.0 kcal/mol, indicating
that A is relatively unstable.
With the evidence accumulated in the present work, it is tempting to
suggest that the monomeric high pressure-dissociated state is related
to the nonnative monomeric intermediate species found in kinetic
refolding studies (22). The subsequent association of E2-DBD from the
monomeric intermediate is slow, consistent with a rearrangement that
must take place before the final association/folding step leading to
the native dimer, which must involve the acquisition of precise
geometric interactions and tight side chain packing (22). Further
experiments will be needed to test this hypothesis.
The overall characteristics of the pressure-dissociated state strongly
suggest a molten globule-like conformation. The implications of a
"structured" monomer in the absence of chemical denaturants or
extreme pH conditions contribute to the understanding of the equilibrium between folded and unfolded states in conditions that are
more compatible with the cellular environment, since pressure is a
thermodynamic variable that strictly affects the position of that
equilibrium. Further detailed characterization of the structure at high
pressure will require the use of high pressure NMR experiments (16).
Provided that enough structural detail can be obtained from NMR
experiments, future prospects will include the possibility of
developing synthetic compounds capable of trapping the monomeric state,
of potential therapeutic value against papillomaviruses.
We thank Emerson R. Gonçalves for
competent technical assistance, Y.-K. Mok and M. Bycroft for the
expressing E2-DBD clone; Martha Sorenson for critical reading of the
manuscript; and Fabio Almeida for the kind assistance with molecular
modeling.
Dedicated to the memory of Gregorio Weber.