 |
INTRODUCTION |
The analysis of the fundamental processes that govern protein
folding relies on the perturbation of the protein-solvent system to
attain a denatured polypeptide. For this purpose, modifications in the
solution are introduced such as temperature, pH, and particularly, chemical denaturants. The latter are generally more amenable for complete thermodynamic and kinetic analysis because changes in pH and
temperature are not always fully reversible, making the interpretation
more difficult. Other complications arise from changes either in
volume or in the thermal energy of the system when temperature is the
variable or from the fact that the action of chemical denaturants is
based on the binding of these molecules to proteins (1). Often,
temperature denaturation leads to aggregation, which further difficult
a full thermodynamic characterization.
Proteins are assembled and evolved to work in biological systems, in
defined ranges of pH and temperature, and in the absence of chemical
denaturants. Under these conditions, however, proteins are normally in
their folded conformation, and the population of unfolded molecules is
extremely low. Pressure constitutes an ideal noninvasive technique to
perturb the equilibrium between folded and unfolded states in protein
systems, because it does not introduce changes in the composition of
the solution, and its often fully reversible effect can be readily
interpreted in thermodynamic and structural terms (2-4). A great
advantage is that upon pressure release, it leaves no residue in the
solution and the change produced can be analyzed in real time. It has
been used extensively for the study of oligomeric systems and, to a lesser extent, in monomeric proteins in combination with other denaturing agents (5-9). This is because monomeric proteins are normally resistant to the mild effect of high pressure (10), compared
with other denaturing agents. On the other hand, small monomeric
proteins have been the object of intensive studies because their small
size allows detailed structural and theoretical analysis in combination
with experimental approaches to elucidate the molecular mechanisms of
protein folding.
Chymotrypsin inhibitor-2
(CI-2)1 is a small monomeric
protein (64 residues) that constitutes a paradigmatic example of a
simple two-state model for folding (11-13). CI-2 is a potent inhibitor of the proteases chymotrypsin and subtilisin and both x-ray crystal (14) and NMR solution structures (15) are available. It is, however, a
highly stable protein in terms of the generally used denaturing agents,
a property that, in principle, anticipates resistance to denaturation
with pressure as the sole perturbant. CI-2 was cleaved into two peptide
fragments of 40 and 24 residues, respectively, which were showed to
reconstitute readily to yield a native-like noncovalent complex (16).
Both the structure attained and its mechanism of folding are highly
similar to the refolding of the uncleaved protein, except for the
second order kinetic mechanism, which appears to differ only in the
concentration (17-20). Because the complex formed is much less stable
than the intact protein (16) but conserves its folding mechanism, we
decided to investigate possible effects of high hydrostatic pressure on this noncovalent complex. We hypothesize that the existing bimolecular equilibrium in the [CI-2(1-40)·(41-64)] complex will be the
target of the mild action of pressure, and this will allow a reversible denaturation reaction that we aim to analyze in detail as a model for
folding in the absence of chemical denaturants.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All chemicals used were of maximal purity
available. Bis-Tris buffer and guanidine chloride (GdnCl) were
purchased from Sigma. CI-2 was obtained recombinantly and its 1-40 and
41-64 fragments (abbreviated W and Y, respectively, for their contents
of tryptophan and tyrosine) were cleaved and purified as described
previously (16). Water was deionized and distilled twice prior to use.
Fluorescence Spectroscopy and High Pressure
Measurements--
Fluorescence spectroscopy was carried out either
with Hitachi F-4500 (Tokyo, Japan) or ISS-K2 (Champaign, IL) equipment.
Fluorescence measurements under pressure were made using a high
pressure bomb described in (21), fitted with sapphire windows, and
adapted to the ISS-K2 fluorimeter.
Tryptophan fluorescence was measured with excitation at either 278 or
295 nm, and the emission scanned from 300 to 400 nm or 315 to 415 nm,
respectively. We focused on changes in spectral area, fluorescence
intensity and center of spectral mass (CM), defined as
|
(Eq. 1)
|
where
is the center of mass in wave numbers,
Fi is the fluorescence emitted at wave number
i. Shifts in CM due to pressure were used to follow the
denaturation process by converting them into fraction of denatured
species at each pressure (
p) according to Weber (2).
|
(Eq. 2)
|
where
p is the CM at a given pressure,
N
and
U the CMs corresponding to native and unfolded species,
respectively. Thermodynamic parameters for the pressure unfolding for a
unimolecular transition were determined according to Equation 3.
|
(Eq. 3)
|
where
V is the volume change and
Ku is equilibrium constant for pressure unfolding
extrapolated to atmospheric pressure (4).
Prior to pressure unfolding-refolding experiments, equimolar
concentrations of fragments were incubated overnight at 25° C in 50 mM Bis-Tris buffer, pH 6.0, to form the complex. The
mixture was placed in a capped quartz bottle, which was not sealed
inside the pressure bomb, as previously described (21). Fluorescence spectra were recorded at atmospheric and different increasingly higher
pressures. The temperature was kept at 25 ± 0.1° C using a
circulating water bath connected to the high pressure bomb.
GdnCl denaturation experiments were carried out by incubating the
preformed [CI-2(1-40)·(41-64)] complex for 1 h with
increasing concentrations of the denaturant in 50 mM
Bis-Tris HCl, pH 6.0. Data for fluorescence intensity and CM were
recorded for each concentration, and the resulting curve was analyzed
using a two-state equation corresponding to a
concentration-dependent equilibrium, fully described in
Ref. 22.
Gel Filtration Chromatography--
Gel filtration experiments
were performed using a Superdex 75 column (Amersham Pharmacia Biotech)
attached to a Shimazdu high pressure liquid chromatography system
(Tokyo, Japan) with absorbance and fluorescence detectors. In GdnCl
denaturation experiments, the column was equilibrated in 100 mM sodium phosphate pH 6.0 buffer with increasing
denaturant concentration as in each of the individual samples that were
loaded. The flow rate was kept at 1 ml/min. Chromatographic experiments
that were carried out after pressure release were performed at the same
low temperature as in the corresponding pressure experiment
(10° C).
 |
RESULTS |
Resistance of Uncleaved CI-2 to Pressure
Denaturation--
Monomeric proteins are often too stable for
denaturation at the range of pressures normally attainable with
standard pressure equipment used in fluorescence studies (21). CI-2 is
not an exception. Being extremely stable to chemical denaturation, it cannot be fully unfolded by urea, and its thermal denaturation midpoint
is above 75° C, even in the presence of denaturants (23, 25). The
unfolding of CI-2 is accompanied by a maximum wavelength shift and a
large increase in fluorescence caused by a yet unidentified residue or
residues that quench the tryptophan side chain in the folded state
(11). We applied increasingly high pressures to uncleaved CI-2 and
followed changes in spectral area and in CM to assess solvent exposure
of its unique tryptophan residue. The protein is resistant to
denaturation at the maximum pressures tested (Fig.
1a). If we preincubate the
inhibitor with subdenaturing concentrations of GdnCl (Fig.
1a, inset), its unique buried tryptophan residue
is gradually exposed to the solvent as the change on its CM indicates.
Subdenaturing GdnCl concentrations of 2.25 M are required
to attain the maximum shift in the center of spectral mass of the
tryptophan at the highest pressures, compatible with a large solvent
accessibility. Despite this large shift (Fig. 1b), the area
increases 2.5-fold in the high pressure state (3450 bar, 2.25 GdnCl),
whereas the GdnCl-denatured state has its spectral area increased by
6-fold.

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Fig. 1.
Pressure denaturation of intact CI-2.
a, pressure titration experiment of 10 µM CI-2
in 50 mM Bis-Tris HCl buffer, pH 6.0 (closed
circles), buffer plus 2.0 M GdnCl (open
circles), and 2.25 M GdnCl (closed
triangles). The inset shows a GdnCl denaturation curve
of CI-2, followed by the change in the center of mass, in excellent
agreement with the same process followed by fluorescence intensity
(11). b, fluorescence spectra of intact CI-2 in 2.25 M GdnCl (and the same buffer as in a),
pressure-denatured CI-2 in the same conditions and GdnCl-denatured
protein at atmospheric pressure (6.0 M denaturant).
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|
We can calculate the thermodynamic parameters associated with this
transition using equation (3). In doing so, we obtain a
G
of 2.5 kcal mol
1 and a
V of 42.5 ml
mol
1.
Pressure Denaturation of the [CI-2(1-40)·(41-64)]
Complex--
The spectrum of the tryptophan in the free fragment that
contains the tryptophan residue, CI-2(1-40) displays a large quenching on association/folding to yield the noncovalent folded complex (16). A
ribbon diagram for the structure of CI-2 is shown in Fig.
2a, indicating the position of
the buried tryptophan. If we closely compare the normalized spectra of
the complex with that of the intact folded CI-2, we find that the
tryptophan fluorescence of the former is not completely shifted to the
blue as in the case of the intact protein (Fig. 2b). Both
crystal and solution structures are available for the complex (20), and
the apparent exposure of the chromophore to the solvent is not an
indication of major unfolding but probably of solvent accessibility and
conformational dynamics. Nevertheless, the chemical shift of the
tryptophan in the complex corresponds unequivocally to a folded
conformation (20). The change exerted by pressure on the complex (Fig.
2b) is only partial in terms of the release of the
fluorescence quenching of the Trp residue when we compare it with the
change observed either on fragment association or GdnCl unfolding of
the complex (Fig. 2b, inset).

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Fig. 2.
Spectral properties of the unique tryptophan
in [CI-2(1-40)·(41-64)]. a, ribbon representation
of CI-2, showing the position of the chromophore and the two regions
corresponding to the fragments. b, spectra of intact CI-2
(dashed line) and [CI-2(1-40)·(41-64)] (solid
line). Inset, GdnCl-denatured (dashed) and
folded (solid) [CI-2(1-40)·(41-64)] complex.
c, spectra of [CI-2(1-40)·(41-64)] complex at
atmospheric pressure (3450 bar) and at atmospheric pressure,
1 h after pressure release (dashed line).
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The [CI-2(1-40)·(41-64)] complex, which shows similar folding
behavior to the intact protein but with lower stability, was next
analyzed for its ability to unfold under high hydrostatic pressure
without the addition of chemical denaturants. The complex shows a large
red shift of the intrinsic fluorescence upon the application of
pressure due to the exposure of its unique tryptophan residue to the
solvent (Fig. 2c), indicative of substantial denaturation (23, 24). The spectral area changed concomitantly by only 2-fold, which
is considerably lower than what would be expected from the complete
unfolding/dissociation of intact CI-2 or from the association of
fragments in water (Fig. 2b, inset).
The effect of a gradual increase of pressure was followed by CM and
shows a transition that is over at the highest pressures attainable by
our equipment, with a value of 28500 cm
1 at 3.5 kilobar
(Fig. 3, top panel). The
process is thus accompanied by a CM of 650 cm
1 and a
2-fold increase in the spectral area or intensity. When the pressure
was gradually released, both the CM and the area returned to their
initial value, indicating a full reversibility of the effect (Fig.
2c).

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Fig. 3.
Reversible pressure denaturation of the
[CI-2(1-40)·(41-64)] complex. Top, pressure
titration of 10 µM complex (see "Experimental
Procedures") followed by the change in the center of fluorescence
spectral mass (closed circles) and the return to atmospheric
pressure (open circles). Bottom, gel filtration
chromatograms (Superdex 75) of the [CI-2(1-40)·(41-64)] complex
before (solid line) and 45 min after (dashed
line) pressure treatment. The peak eluting at 22 min corresponds
to Bis-Tris buffer and marks the V0.
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|
Although the CM attained by the complex at the highest pressures is
compatible with the tryptophan residue being fully exposed to the
solvent (28600 cm
1), we confirmed the end point of the
transition by unfolding [CI-2(1-40)·(41-64)] by pressure
titration in the presence of 0.7 M GdnCl, corresponding to
the midpoint at atmospheric pressure and gives an identical result (not
shown). As a control experiment, we also carried out a pressure
titration of the isolated CI-2(1-40) fragment, with no significant
changes observed.
To check for complete reversibility, we carried out gel filtration
chromatography of the complex before and after pressurization. Fig. 3
(bottom panel) shows that the chromatograms in both
conditions are superimposable; there is no evidence of dissociation
into fragments when the complex is renatured immediately after pressure release.
Comparative Unfolding of CI-2 and [CI-2(1-40)·(41-64)] by
GdnCl and Pressure--
Pressure appears to exert only partial changes
in the fluorescence quantum yield of the chromophore, and the changes
in spectral properties of the complex and intact CI-2 appear to be much
more extensive in response to GdnCl than to high pressure. In similar conditions to the pressure experiments, we analyzed the
denaturation/dissociation of [CI-2(1-40)·(41-64)] by GdnCl by
monitoring the change in CM (Fig.
4a). The latter changes 750 cm
1, coincident with the total change observed by
pressure, indicative of complete exposure of the tryptophan probe. On
the other hand, the change in spectral area is 6-fold, much larger than
the change observed at high pressures (Fig. 2c).

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Fig. 4.
Concentration dependence of the GdnCl
denaturation of the [CI-2(1-40)·(41-64)] complex.
a, GdnCl denaturation of the complex (5 µM)
followed by the change in CM. Inset, titration of
CI-2(1-40) by the complementary fragment CI-2(41-64), indicating a
similar overall change in CM. b, recovery of folded CM in
0.35 M GdnCl at increasing concentrations of the
complex.
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|
To further investigate these differences, we monitored the formation of
the complex upon mixing CI-2(1-40) with increasing concentrations of
CI-2(41-64) in the same manner as described previously (16) but
followed the change in CM. The change in this parameter is of similar
magnitude to those observed for pressure and GdnCl denaturation (Fig.
4a, inset). However, the spectral area changes
7-fold as in the case of GdnCl denaturation and larger than the change
exerted by high pressures (16).
Monitoring Dissociation during GdnCl Denaturation of
[CI-2(1-40)·(41-64)]--
The question arises whether the GdnCl
denaturation equilibrium is linked to protein concentration. As Fig.
4a shows, the transition of the CM is over at 1.2 M GdnCl, with a total change of 750 cm
1. A
protein concentration dependence would translate into a displacement of
the curve and may be followed by the change in the CM at a concentration near the [GdnCl]50 of the transition, with
increasing concentrations of the complex. For this purpose, we
incubated [CI-2(1-40)·(41-64)] in 0.35 M GdnCl at
concentrations ranging from 2 to 50 µM complex and
measured the CM. Fig. 4b shows the recovery of the CM as the
protein concentration is increased, shifting to values corresponding to
a fully folded conformation. The change in CM for the complex either in
the absence of denaturant or at 3.5 M GdnCl with increasing
complex concentrations is minimal (not shown). A CM of ~390
cm
1 is expected from near midpoint values to folded
complex in the absence of denaturant (Fig. 4a), and this
value is approached by the protein concentration-dependent
CM recovery (Fig. 4b). This is equivalent to a shift in the
GdnCl curve, which indicates an increase in the stability of the
complex to denaturation that depends on protein concentration.
Next, we followed changes in molecularity directly for the GdnCl
denaturation process, making use of gel filtration chromatography. The
[CI-2(1-40)·(41-64)] complex at 5 µM was incubated
with different concentrations of the denaturant and then injected into
a gel filtration column equilibrated in the same denaturant
concentration. At zero denaturant, only complex can be observed, but as
the GdnCl concentration increases a clear dissociation is observed that is completed at 2.0 M denaturant, coincident with the end
of the denaturation transition observed by fluorescence intensity data (Figs. 5 and
6).

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Fig. 5.
GdnCl denaturation/dissociation of the
complex by gel filtration. Top, control chromatogram
indicating the elution position of the complex and the two fragments,
CI-2(1-40) (W) and CI-2(41-64) (Y). The concentration of the complex
was 5 µM, and the denaturant concentration is indicated
in each chromatogram. Bottom, elution of the separated
fragments in 2.0 M GdnCl.
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Fig. 6.
GdnCl denaturation/dissociation of the
complex analyzed by a two-state concentration-dependent
model. Conditions were similar to those described for Fig. 5. The
fluorescence emission at 356 nm was plotted against denaturant
(closed circles) and analyzed according to the equation
described in the text. The arrow shows the accurate
measurement of the total change in fluorescence intensity expected for
a complete dissociation of the fragments. The open circles
correspond to the elution times of the peak corresponding to the
associated complex going to free (1-40) fragment in the gel filtration
experiment in Fig. 5 (major peak at 0 M denaturant).
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The GdnCl denaturation process is linked to the dissociation of the
fragments and can thus be analyzed using a
concentration-dependent two-state approach as in the case
of a dimer (22). Fluorescence intensity changes give the most accurate
data, which were used to calculate a free energy of unfolding of 8.4 kcal mol
1 and an m value (the cooperativity
unfolding parameter) of 1.8 kcal mol
1
M
(Fig. 6). The overall fluorescence change
observed in previous spectra is measured accurately as 6-fold, as the
arrow in the plot indicates. The elution times from the gel
filtration experiment (Fig. 5) change in parallel with fluorescence
intensity (Fig. 6), supporting a concerted unfolding/dissociation process.
Concentration Dependence of [CI-2(1-40)·(41-64)] Denaturation
by Pressure--
Although with structure and folding mechanisms
similar to those of the intact CI-2 protein, [CI-2(1-40)·(41-64)]
clearly differs in that its folding equilibrium is in principle linked
to an association-dissociation process and its kinetic mechanism of
association/folding in solution is indeed second order. To test for
concentration dependence, we carried out pressure titrations at
different concentrations of the complex, ranging from 5 to 100 µM (Fig. 7a).
Surprisingly, the process does not depend on protein concentration,
although in all the concentrations tested there is a similar
displacement of the spectral parameters corresponding to the exposure
of its unique tryptophan residue to the solvent. We cannot rule out a protein concentration dependence at lower concentrations, but the
7-fold change in the fluorescence of the single tryptophan residue
precludes a sensitive measurement below 5 µM because the folded conformation at atmospheric pressure is largely quenched, posing
limits to the sensitivity.

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Fig. 7.
Concentration independence of the pressure
denaturation of the [CI-2(1-40)·(41-64)] complex.
a, pressure titration of different concentrations of the
complex: 5 µM (closed circles), 50 µM (open circles), and 100 µM
(triangles). The data were transformed as described under
"Experimental Procedures" to obtain the thermodynamic parameters.
b, gel filtration chromatogram in a Superdex 75 column,
indicating the elution times for the separate fragments and the complex
at 10 µM concentration, followed by absorbance at 220 nm.
c, [CI-2(1-40)·(41-64)] complex after 2.5 h of
pressurization (solid line) and after 10 h of
pressurization in conditions similar to those described for
b (dashed line). d, same control as in
b but followed by tryptophan fluorescence. Large
dashes, CI-2(1-40); small dashes, CI-2(41-64);
solid lines, the complex. It should be noted that at the
same molar ratio, the area of the peak corresponding to CI-2(1-40),
containing the single tryptophan, is 5-fold larger than the complex,
and CI-2(41-64), with only a tyrosine residue, contributes little to
the fluorescence. e, same as in c but followed by
fluorescence. Experiments b-e were performed at 10° C,
as described under "Experimental Procedures."
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It is very important for us to confirm that the high pressure state
remains associated but the availability of techniques to measure
directly the molecularity at high pressure is limited to gel
electrophoresis (26), which is not able to operate at the 3.5 kilobar
required to denature the complex. Taking advantage of a slow
renaturation process after pressure release, we decided to use gel
filtration chromatography. Data from our laboratory indicate that the
overall refolding t1/2 at 25° C is approximately
150 s (not shown). To slow down the renaturation process and allow
a characterization of the state attained at high pressure, we decreased
the temperature to 10° C, and the t1/2 for the
renaturation after pressure release increases
5-fold.2 This leaves enough
time to release the pressure and inject the sample immediately in a gel
filtration column, equilibrated at the same temperature. Fig.
7b shows a control experiment indicating the eluting times
of the complex and the free fragments under these conditions, monitored
by absorbance at 220 nm. A very similar chromatographic behavior is
observed for a complex that has been pressurized for 2.5 h and for
10 h; both show only an incipient shoulder that could be
interpreted as a small degree of dissociation (Fig. 7c). If
we instead monitor tryptophan fluorescence, two peaks of identical
height are observed after release from 2.5 h of pressurization
(Fig. 7d). This cannot be interpreted as 50% dissociation,
because the tryptophan is 6-7-fold quenched in the folded complex.
This means that the actual dissociated fragment, at the lowest
concentrations we use in our studies (5 µM), is less than
10%. Nevertheless, it is something significant, and the fraction that
dissociates will probably become higher as the concentration of the
complex is lowered. After 10 h of pressurization, the peak
corresponding to the free fragments increases concomitantly with a
decrease of that corresponding to the complex, suggesting that an
extremely slow dissociation may take place.
Thermodynamic Parameters Associated to the Pressure Denaturation
Process--
From the pressure titration data at different
concentrations we can calculate the parameters associated with the
pressure denaturation process (5). The lack of protein concentration dependence and the chromatographic experiments led us to conclude that
the greater population of the pressure-denatured state is not
dissociated in the range of concentrations at which the sensitivity of
our equipment allows us to work in equilibrium conditions (5-100 µM). If we add the limited change in the quantum yield at
high pressure, which does not recover the values of the free
CI-2(1-40) fragment, we assume a process in which no changes in
molecularity take place in our present experimental conditions. These,
besides protein concentration, include pH, temperature, and the
experimental time frame.
Thus, for the analysis of the thermodynamic parameters, we do not need
to account for protein concentration, and we use the equation described
in the experimental section for a unimolecular unfolding transition. By
analyzing the linear transformation of the data of the pressure
titration curves at 5, 50, and 100 µM (Fig. 7a
and see "Experimental Procedures"), we obtain an average free
energy of 1.36 ± 0.03 kcal mol
1. The corresponding
value for the volume change upon pressure denaturation is 35 ± 0.7 ml mol
1.
 |
DISCUSSION |
Intact CI-2 proved to be resistant to denaturation by high
pressure, something not at all unexpected for small monomeric proteins. Subdenaturing concentrations of GdnCl allowed pressure denaturation, indicated by a shift in the CM of its tryptophan. This reversible qualitative spectral change was, however, not accompanied by a quantitative change of the fluorescence quantum yield as in the case of
GdnCl unfolding. Thus, at high pressure, there must be a structure
different from a fully extended denatured polypeptide. Nevertheless,
the structural basis for the large fluorescence quenching in the folded
state of both intact CI-2 and fragment complex remains unknown.
Without the addition of chemical denaturants, the complex
[CI-2(1-40)·(41-64)] undergoes a reversible denaturation
transition. The final value for the center of spectral mass is
coincident with the tryptophan residue in CI-2(1-40), which is
disordered and solvent exposed already at atmospheric pressure. The
fact that the fluorescence intensity changes only 2-fold compared with 7-fold in fragment association or complex unfolding by GdnCl strongly suggests the presence of residual structure. The lack of fluorescence quenching in the free CI-2(1-40) fragment at atmospheric pressure indicates that the basis for the quenching in the pressure-denatured state cannot be caused by interaction with neighboring residues and
must have a long range, i.e. tertiary nature.
The lack of protein concentration dependence in the pressure
denaturation equilibrium must be interpreted as the absence of dissociation. A deterministic behavior, found in multi-protein subunit
assemblies such as virus or large protein oligomers or aggregates, can
be ruled out for the small size and structural simplicity of the
complex (5). Gel filtration chromatographic experiments after pressure
release support the idea of a major population of undissociated species
at high pressures in our experimental conditions.
GdnCl denaturation of [CI-2(1-40)·(41-64)] is accompanied by a
concerted dissociation into fragments. A major difference is that the
state at high GdnCl (dissociated fragments), although with a similar CM
to the high pressure state, shows no quenching of the tryptophan. We
conclude that there must be a link between the dissociation and the
complete release of the quenching.
It is clear that the pressure-denatured state is different from that of
GdnCl. The latter is expected to be a better denaturant, for its
ability to solvate hydrophobic side chains and for its strong ionic
effect that would be more effective in releasing quenching of
chromophores by charged residues. It is difficult to imagine how the
[CI-2(1-40)·(41-64)] complex can be denatured and remain
associated. This puzzling observation can be explained by the existence
of a persistent residual structure in the high pressure state,
associated and still quenched. Because the tryptophan is located at the
center of the main hydrophobic core, which nucleates all the major
secondary structure elements into the tertiary structure, it is
tempting to suggest that any structure present at the high pressure
state should be non-native, compatible with a full exposure of the
chromophore to the solvent. However, fluorescence is extremely sensitive to the environment of the probe, and the CM shift could be
due to a large solvent accessibility of the residue but not to full
global unfolding. A molten globule-like conformation appears more
compatible with these results. This hypothesis is supported by previous
work with large CI-2 molten globule-like fragments, in which the
fluorescence indicated solvent accessibility to the single tryptophan
residue, but both its chemical shift and the overall backbone
conformation as judged by its far UV circular dichroism spectrum
corresponded unequivocally to a folded conformation (27-30).
Pressure does not affect all interactions as GdnCl does. Although
pressure weakens electrostatic interactions and hydrophobic contacts,
it is known to stabilize hydrogen bonds in macromolecules (31). An
explanation consistent with our results would be that intrafragment
interactions in the folded state are more affected by pressure, and a
more drastic treatment, i.e. GdnCl, is needed to perturb
interfragment interactions leading to dissociation. The natural
protease inhibitory function of CI-2 requires that it remains stable
for long enough after cleavage by the target enzyme, resulting in a
very slow dissociation. The nature of the interactions that hold
together the cleaved protein must be different from those of nature
designed dimers that evolved for eventually undergoing dissociation.
Table I shows the free energy values
calculated for the different reactions analyzed for this system in this
and previous work (16, 17). GdnCl denaturation and fragment
association/folding are in very good agreement, considering the
different approaches involved. Partly structured and undissociated
intermediate species were neither detected in the fragment
association/folding nor in the GdnCl denaturation reactions. Thus, the
complete transition that takes place upon either fragment
association/folding or GdnCl denaturation/dissociation of the complex
is accompanied by an absolute free energy change of 8.5 to 10 kcal
mol
1, whereas the pressure transition involves only 1.4 kcal mol
1 change.