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INTRODUCTION |
At the molecular level, much more is known about the state of
native proteins than that of their denatured counterparts. The three-dimensional structures of more than 4200 proteins have now been
determined, and the number is rising rapidly (1). Nevertheless, the
first case of molecular structure of a denatured protein has yet to be
solved. The striking disparity of our knowledge about the native and
denatured proteins is hardly surprising. Unlike the native protein,
which usually adopts a well defined conformation, a denatured protein
consists of a collection of highly heterogeneous conformational isomers
that exist in a state of equilibrium (2-4). Experimentally, it will be
a daunting task to isolate and analyze pure conformational isomers, not
only because of the exceedingly large number of isomers that may exist
but also because of their instability and rapid interconversion.
Without the isolation of conformational isomers, the average of their
chemical properties has been measured by most conventional methods,
including gel filtration, intrinsic viscosity, light scattering, and
various spectroscopic techniques (5-10). This is the case even with
high resolution NMR (11). Therefore, development of methods that may group and fractionate denatured species will represent a crucial step in the study of denatured states of proteins.
In addition, there are two important issues relating to the denatured
proteins that still need to be further clarified. One is the extent of
unfolding of denatured proteins. There is a distinction between
denaturation and unfolding (or between denatured and unfolded) (2).
Traditionally, denaturation is defined simply as conversion of the
native structure to non-native (or active to inactive) structures.
Measurement of denaturation curves does not really tell us the extent
of unfolding of denatured proteins (7, 10). Early studies by Tanford
and colleagues (5, 12) showed that proteins denatured in the presence
of 6 M GdmCl1
assume a near random coil conformation (13). However, mounting evidence
now indicates that denatured proteins do not always unfold as
extensively as was once thought, even in the presence of strong denaturing conditions (14-16). The extent of denaturation and
unfolding also varies from protein to protein under the same set of
denaturing conditions. The situation can be further complicated with
disulfide-containing proteins. The extent of unfolding of such proteins
hinges upon whether the native disulfide bonds become reduced or remain
intact (17, 18). Another issue of long standing interest is the
difference between denatured states of a protein induced by heating (2, 3, 19) and denaturants (urea, GdmCl, or organic solvents, etc.). These
differences may underline mechanisms and various forces that stabilize
native proteins.
In this report, I have attempted to address these issues by application
of a novel method for analyzing reversible denaturation of
disulfide-containing proteins (20, 21). In the presence of denaturant
and thiol catalyst, the native protein denatures by shuffling its
native disulfides to form a mixture of scrambled isomers. Scrambled
species are denatured structures that contain at least two non-native
disulfide bonds. For proteins that contain 3 disulfide bonds, there are
14 possible denatured states of scrambled isomers. In some cases, they
could be completely separated by reversed phase HPLC (21, 22). A major
advantage of this technique is that the scrambled isomers can be
trapped by acidification, purified by HPLC, and structurally
characterized. This allows quantitative analysis of the composition of
various denatured species and permits a more detailed identification
and description of the state of unfolding that has not been afforded
with conventional methods. This novel system has been used here to
examine and compare the denatured states of the tick anticoagulant
peptide (TAP) generated by urea, GdmCl, GdmSCN, organic solvents, and
elevated temperature. TAP is a factor Xa-specific inhibitor (23). It
includes 3 disulfides (24) and has a size (60 amino acids) similar to
that of bovine pancreatic trypsin inhibitor. Both TAP and bovine
pancreatic trypsin inhibitor belong to the Kunitz-type inhibitor and
share close structural homology in terms of disulfide patterns and
three-dimensional conformation (25, 26).
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EXPERIMENTAL PROCEDURES |
Materials--
TAP (CGP-55099) is a recombinant protein produced
by Novartis. The protein is more than 95% pure as judged by HPLC and
N-terminal sequence analysis. GdmCl, GdmSCN, urea, acetonitrile,
ethanol, and 2-mercaptoethanol were products of Merck (Darmstadt,
Germany) with purity of greater than 99%.
A Standard Protocol of Denaturation--
The protein (0.5 mg/ml)
was dissolved in the Tris-HCl buffer (0.1 M, pH 8.4)
containing 0.25 mM 2-mercaptoethanol and selected concentrations of denaturants (urea, GdmCl, GdmSCN, or organic solvents) or subjected to selected denaturing conditions (elevated temperature). When using denaturants, the reaction was typically performed at 23 °C for 20 h. In the case of heat denaturation, the reaction was carried out within 90 min (55 °C) and 45 min (69 °C). To monitor the kinetics and intermediates of unfolding, aliquots of the sample were removed in a time course manner, quenched with 4% trifluoroacetic acid, and analyzed by HPLC. The denatured sample was subsequently acidified with an equal volume of 4%
trifluoroacetic acid and stored at
20 °C.
Criteria for the Completion of Denaturation and Construction of
Denaturation Curves--
The completion of denaturation, under a given
denaturing condition, implies that conversion of the native species to
the scrambled species has reached a state of equilibrium. This state of
equilibrium also includes those among scrambled species. Two major
criteria are applied here to ensure that denaturation has reached
equilibrium. One is time course kinetics analysis, which indicates that
conversion of the native TAP to a scrambled species has reached a
plateau and the ratio of scrambled to native species remains constant during prolonged incubation. These results have revealed that denaturation of TAP by denaturants (in the presence of 0.25 mM 2-mercaptoethanol) was completed generally within 4-6
h. Nonetheless, the reaction has been routinely allowed to proceed
overnight (20 h). In the case of thermal denaturation, there are
inherent difficulties in performing prolonged kinetic analysis because
of the heat-induced decomposition of disulfide bonds. Another method to
verify the completion of denaturation is to renature (refold) fully
reduced/denatured TAP under the same conditions that denature the
native TAP. Both renaturation and denaturation are expected to reach
the same state of equilibrium and generate end products that consist of
the same ratio of scrambled to the native species (20).
The extent of denaturation at every given condition was simply
calculated from the fraction of TAP (using HPLC peak area integration) that is converted to the scrambled species. These data were then used
to construct the denaturation curves.
Structural Analysis of Scrambled TAP--
Isolated fractions of
scrambled TAP (~10 µg) were treated with 1 µg of thermolysin in
30 µl of N-ethylmorpholine/acetate buffer (50 mM, pH 6.4). Digestion was carried out at 23 °C for
16 h. Peptides were then isolated by HPLC and analyzed by amino
acid sequencing and mass spectrometry to identify the
disulfide-containing peptides.
Protein Analytical Methods--
The disulfide content of
scrambled proteins was determined by the dabsyl chloride precolumn
derivatization method (27), which permits direct quantification of the
disulfide bonds of proteins. Amino acid sequence analysis was performed
with a Hewlett-Packard G-1000A sequencer. The matrix assisted laser
desorption ionization mass spectrometer was a home-built time of flight
instrument with a nitrogen laser of 337-nm wavelength and 3-ns pulse
width. The calibration was performed either externally or internally,
by using standard proteins (hypertensin, Mr
1031.19 and calcitonin, Mr 3418.91).
Nomenclature of Scrambled Species--
To simplify the
description of scrambled species of TAP, they are designated by the
following: X-TAP-(species assigned on HPLC), where X stands for
scrambled. For instance, X-TAP-a represents species
a of scrambled tick anticoagulant peptide.
Clarification of Terminology--
Denaturation and unfolding
(denatured and unfolded) are not synonymous terms. In this report,
denaturation defines only the conversion of the native structure to the
non-native structure (scrambled species), whereas unfolding describes
the state of the denatured protein. For instance, the potency of a
denaturant to denature TAP is determined by the fraction of TAP that is
converted to scrambled species after the reaction has reached
equilibrium. On the other hand, the potency of a denaturant to unfold
TAP is determined by the composition of scrambled species and
specifically by the recovery of the most extensively unfolded scrambled
species (X-TAP-a). In addition, potency and efficiency have
been used alternatively to compare GdmCl and urea. Potency of a
denaturant is defined by the extent of protein denaturation (and
unfolding) as the reaction has reached a state of equilibrium.
Efficiency is related to the kinetics (rate constant) of unfolding.
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RESULTS |
The Composition and Disulfide Structures of X-TAP--
Along the
reversible pathways of unfolding and refolding of TAP (21, 28), 11 fractions of X-TAP have been detected as intermediates. Among them,
there exist seven major fractions that constitute more than 90% of the
total concentration of X-TAP. Four of them were structurally
characterized (28). An additional three fractions, e,
h, and i, have been isolated here. They were
digested by thermolysin at pH 6.5. Peptides were then isolated by HPLC
and characterized by amino acid sequencing and mass spectrometry. Each
was shown to consist of a single species. Their disulfide structures,
presented in Fig. 1, are the basis for
the examination of denatured states of TAP.

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Fig. 1.
Disulfide structures of scrambled TAP.
The structures of three species of X-TAP (e, h,
and i) are presented for the first time. Their structures
were derived from the sequence and mass analysis of
disulfide-containing peptides of thermolysin-digested samples.
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Denaturation of TAP in the Presence of Urea, GdmCl, and
GdmSCN--
Denaturation of the native TAP was analyzed in the
presence of increasing concentrations of urea, GdmCl, and GdmSCN. The
resultant denaturation curves are shown in Fig.
2. GdmSCN is more potent than GdmCl and
urea. Based on the concentration that is required to achieve the same
extent of denaturation, GdmSCN is about 2.5-fold more potent than
GdmCl, whereas urea and GdmCl display almost indistinguishable potency
in denaturing native TAP. Indeed, at a low concentration of denaturant
(3 M), urea is actually more potent than GdmCl.

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Fig. 2.
The denaturation curves of TAP in the
presence of different denaturants. Fractions denatured are
percentages of TAP that are converted to the X-TAP. The denaturants are
GdmSCN ( ), GdmCl ( ), urea ( ), acetonitrile ( ), and ethanol
( ). Denaturation was carried out at 23 °C for 20 h in
Tris-HCl buffer (0.1 M, pH 8.4) containing
2-mercaptoethanol (0.25 M) and increasing concentration of
the indicated denaturant. The contents of acetonitrile and ethanol are
also expressed in molar concentrations.
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The comparable potency of urea and GdmCl in denaturing native TAP
provides a useful case for further analysis of the kinetics of
denaturation. TAP can be almost fully denatured (>94%) by both urea
and GdmCl at concentrations ranging from 6 to 8 M (Fig. 2). However, the kinetics of its denaturation are dependent upon the concentration of the denaturant. At 8 M urea, the rate
constant of denaturation, which was measured to be 0.0137 min
1, is about 1.5- and 3-fold greater than that
performed at 7 and 6 M urea. With GdmCl, the rate constants
differ only slightly: 0.0138 min
1, 8 M GdmCl;
0.0163 min
1, 7 M GdmCl; and 0.0115 min
1, 6 M GdmCl. Surprisingly, the rate
constant of TAP denaturation at 7 M GdmCl is somewhat
greater than that performed at 8 M GdmCl. This marginal
difference is well reproducible.
Examination of the composition of denatured TAP reveals the
differential potency and distinct mode of actions of urea and GdmCl
(Fig. 3). Using GdmCl and GdmSCN as the
agents, denatured TAP consists of four well populated scrambled
species, a, d, g, and e. In
contrast, only two of them, d and g, predominate
in the urea solution. In comparing these two sets of denatured
structures (urea versus GdmCl), one notices that the
composition of urea-denatured TAP resembles that of the folding
intermediates of GdmCl-denatured TAP (Fig.
4). When X-TAP produced by 6 M GdmCl was allowed to refold, X-TAP-a and
X-TAP-e decreased rapidly. This suggests that TAP adopts a
more advanced state of unfolding in the GdmCl and GdmSCN solutions and
that X-TAP-a and X-TAP-e are highly unfolded species that become well populated only under these conditions. Furthermore, the pattern of X-TAP does not always remain constant under
the same denaturant. The relative yield of
X-TAP-a/X-TAP-g increases from 0.16 to 0.4 as the
concentration of urea doubles from 4 to 8 M. At the same
time, the concentration of X-TAP-a as a fraction of the
total X-TAP increases almost linearly from 9 to 16.5% (Fig.
5). The increase of X-TAP-a is
even more dramatic in the cases of GdmCl and GdmSCN denaturation (Fig.
5). The results thus indicate that the recovery of X-TAP-a is related to the strength of denaturing conditions and can be used to
plot the unfolding curves of TAP (Fig. 5). These unfolding curves are
distinguished from the denaturation curves shown in Fig. 2.

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Fig. 3.
Denatured states of TAP under different
concentrations of urea, GdmCl, and GdmSCN. Native TAP was allowed
to denature in Tris-HCl buffer (0.1 M, pH 8.4) containing
2-mercaptoethanol (0.25 mM) and indicated concentrations of
the denaturant. Denaturation was performed at 23 °C for 20 h.
The denatured sample was quenched with 4% trifluoroacetic acid and
analyzed by HPLC using the following conditions. Solvent A was water
containing 0.1% trifluoroacetic acid. Solvent B was acetonitrile/water
(9:1, by volume) containing 0.1% trifluoroacetic acid. The gradient
was 28-45% solvent B linear in 40 min. The column was Vydac C-18 for
peptides and proteins, 4.6 mm, 10 µm. Column temperature was
23 °C. N indicates the elution position of the native
species. Scrambled 3-disulfide species are marked alphabetically
(a to i).
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Fig. 4.
Renaturation (refolding) of X-TAP that are
generated by 6 M GdmCl (top panel) and heating
(55 oC, l h) (bottom panel). The
denatured samples were acidified with 4% trifluoroacetic acid,
desalted, and freeze-dried. To initiate folding, the samples were
reconstituted in Tris-HCl buffer (0.1 M, pH 8.4) containing
2-mercaptoethanol (0.25 mM). The folding intermediates were
trapped in a time course manner with 4% trifluoroacetic acid and were
analyzed by HPLC using the conditions described in the legend of Fig.
3.
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Fig. 5.
Recoveries of X-TAP-a as a fraction of the
total denatured TAP under different denaturing conditions. The
denaturing conditions are described in the text. GdmSCN has a maximum
solubility of around 6-6.5 M in the Tris-HCl buffer. The
data should be allowed a standard deviation of ±5%.
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Denaturation of TAP in Organic Solvents--
Organic solvents
promote denaturation of proteins presumably through association with
the hydrophobic residues of the protein, thereby disrupting hydrophobic
bonding. As for urea and GdmCl, the potency of organic solvents in
denaturing proteins depends on their concentration. Therefore,
denaturation of TAP was also characterized in the presence of
increasing concentrations of acetonitrile and ethanol. The denaturation
curves obtained with both organic solvents are included in Fig. 2. The
denaturation potency of acetonitrile is about 2-3-fold that of
ethanol. Although the maximum extent of TAP denaturation in the
presence of a high concentration of acetonitrile (9.6 M,
50% by volume) reaches 90%, it is only close to 60% in the buffer
containing 8.6 M (50% by volume) ethanol. Beyond 50%
content of acetonitrile or ethanol (by volume) in the buffer, TAP will
increasingly precipitate.
Several important features can be concluded from the composition of
X-TAP generated by organic solvents (Fig.
6). 1) The denatured TAP comprises only
two well populated species of scrambled isomers, X-TAP-d and
X-TAP-g. 2) The recovery of X-TAP-a, similar to
the cases of urea and GdmCl unfolding, is dependent upon the
concentration of the organic solvent. The content of X-TAP-a
as a percentage of the total X-TAP increases from 2 to 14% as the
concentration of acetonitrile rises from 4 to 8 M (Fig. 6).
3) In the case of ethanol or at low concentration of acetonitrile,
small but significant amounts of X-TAP-h and
X-TAP-i are detected (Fig. 6). These two species contain disulfide loops that are twice the size of that of X-TAP-d and X-TAP-g (Fig. 1). X-TAP-h and
X-TAP-i accumulate during the pathway of refolding of
GdmCl-denatured TAP (Fig. 4). Apparently, they adopt structures more
compact than that of X-TAP-d, X-TAP-g, or
X-TAP-a. 4) The denaturation curve and the
composition of X-TAP generated by acetonitrile resemble those produced
by urea.

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Fig. 6.
Right, compositions of X-TAP generated
in the presence of acetonitrile and ethanol. In the Tris-HCl buffer
containing 40% (6.9 M) ethanol, about 60% of the native
TAP was denatured to the state of X-TAP. 25, 30, and 40% acetonitrile
(by volume) correspond to the molar concentrations of 4.8, 5.7, and 7.7 M, respectively. Left, a time course thermal
denaturation of the native TAP is shown. Denaturation was carried out
at 55 or 69 °C. HPLC chromatograms of denaturation profiles obtained
at 55 °C are presented here.
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Heat Denaturation of TAP--
A time course denaturation of the
native TAP at 55 °C is shown in the left panel of Fig. 6.
The extent of TAP denaturation reaches a plateau after about 1 h.
Prolonged incubation leads to the decomposition of scrambled species,
as judged by their HPLC patterns. The rate of denaturation increases by
3-fold as the temperature rises from 55 to 69 °C (Fig. 6). The
process of thermal denaturation is reversible. As the sample was
removed from the heating block and placed at room temperature, X-TAP
renatured spontaneously to form the native structure (Fig. 4).
The composition of heat-induced X-TAP is different from those generated
by denaturants. It comprises all seven identified X-TAPs. Aside from
the presence of X-TAP-h and X-TAP-i, the recovery
of X-TAP-a is surprisingly high. For the samples that are
denatured at 55 and 69 °C, the content of X-TAP-a accounts for 17 and 21% of the total X-TAP, respectively. These are 60 and 90% higher than the content of X-TAP-a found in the
sample denatured by 7.7 M acetonitrile. Another
characteristic of heat-induced X-TAP is the existence of approximately
4% X-TAP-e, which populates well only in the presence of
GdmCl and GdmSCN but is barely detectable in solutions containing urea
or acetonitrile.
Furthermore, a systematic study has been performed to investigate the
combined effect of denaturant and temperature. In every case, the
addition of high temperature has resulted in a dramatic increase of the
yield of X-TAP-a to the extent that a sequential denaturation by 6 M GdmSCN (7 h at 23 °C) and elevated
temperature (69 °C, 15 min) denatured more than 99.5% of the native
TAP with 85% of the denatured species adopting the disulfide structure of X-TAP-a.
Inhibition of TAP Denaturation by Protein Stabilizers--
Protein
stabilizers are compounds (salts, sugars, amino acids, etc.) used to
preserve the native conformation of proteins in solution (29). Their
presence is known to shift the equilibrium constant of N (native)/D
(denatured) in favor of the native structure. Two commonly used protein
stabilizers, NaCl and lysine, were examined here. They were compared
specifically for their ability to inhibit the denaturation of TAP by
urea (8 M), GdmCl (6 M), acetonitrile (7.7 M), and high temperature (55 or 69 °C).
The results show that NaCl and lysine are able to effectively inhibit
all denaturing conditions, with the exception of GdmCl (Fig.
7). The extent of inhibition is, in
general, inversely related to the potency of denaturing conditions. For
instance, in the presence of 1 M NaCl, the extent of TAP
denaturation by 8 M urea and 7.7 M acetonitrile
is reduced by 42 and 81%, respectively, whereas the denaturation by 6 M GdmCl remains practically unaffected (Fig. 7). Indeed,
even at 4 M GdmCl that denatures only 50% of TAP, the
inhibitory effect of NaCl (1 M) is still undetectable. The
patterns of X-TAP denatured by urea and acetonitrile are not affected
by the presence of NaCl. In both cases, X-TAP-d and
X-TAP-g remain as predominant species.

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Fig. 7.
Inhibition of the TAP denaturation by NaCl
and lysine. The reaction was performed in the Tris-HCl buffer (0.1 M, pH 8.4) containing 2-mercaptoethanol (0.25 mM), selected denaturing conditions, plus increasing
concentrations of NaCl or lysine as inhibitors. The selected
denaturants are 6 M GdmCl ( ), 8 M urea
( ), and 7.7 M acetonitrile ( ). The reaction was
carried out at 23 °C for 20 h. The selected high temperatures
are 55 °C, 1 h ( ) and 69 °C, 15 min ( ). The fraction
of denatured TAP decreases with increasing concentrations of NaCl and
lysine. This was observed with all denaturaing conditions except for 6 M GdmCl. The data should be allowed a standard deviation of
±6%.
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Further experiments demonstrate that NaCl not only inhibits but also
reverses the denaturation of TAP. For instance, X-TAP renatures
spontaneously in the presence of 6 M urea and thiol catalyst to form the native structure as NaCl (1 M) is
added in the solution (data not shown).
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DISCUSSION |
The Structure of Denatured TAP Consists of Conformational Isomers
with Varied Degrees of Unfolding--
Denatured TAP consists of
disulfide isomers that exist in equilibrium and adopt varied degrees of
unfolding. There is a remarkable variation in the size of disulfide
loops among the seven species of X-TAP that have been isolated and
structurally characterized (Fig. 1). The order of the combined size of
the three disulfide loops (calculated by the number of amino acid
residues separating the two cysteines that form the disulfide) is as
follows: N-TAP (100 amino acids) = X-TAP-i (100 amino acids) = X-TAP-h (100 amino acids) > X-TAP-f (88 amino
acids) > X-TAP-e (56 amino acids) = X-TAP-g (56 amino acids) > X-TAP-d (52 amino acids) > X-TAP-a (20 amino acids). Those with large disulfide loops
(X-TAP-i, -h, and -f) presumably
assume a more compact conformation and thermodynamically might be
stabilized by additional noncovalent interactions (either native or
non-native like). This proposition is supported by two key
observations. 1) The concentrations of X-TAP-i,
-h, and -f become significant only under mild
denaturing conditions (Figs. 6). 2) X-TAP-i, h,
and f are the three major species that accumulate along the
pathway of the refolding of GdmCl-denatured TAP (Fig. 4). In contrast,
X-TAP-a, which contains the smallest disulfide loops,
represents the most extensively unfolded form of scrambled TAP. When
GdmCl is removed from the sample, the concentration of
X-TAP-a decreases precipitously along the pathway of
refolding (Fig. 4). Most strikingly, the recovery of
X-TAP-a, as a fraction of the total X-TAP, is invariably
determined by the strength and concentration of the denaturant. This
has been observed with all denaturants investigated here (Fig. 5).
Thus, recovery of X-TAP-a signals the extent of unfolding of
the denatured TAP. This work therefore provides a useful method for
measuring the extent of unfolding of the denatured protein. However, it
is important to point out that the property observed with TAP may not
necessarily apply to other disulfide-containing proteins. Proper
spacing of cysteines in the sequence of a protein will probably be a
major prerequisite.
Different Denaturants Generate Different Structures of Denatured
TAP--
Another important finding of this study is the demonstration
that different denaturants each produce a unique pattern of the structure of denatured TAP (see Figs. 3 and 6). The differences reflect
both their relative potency to denature the native TAP, as well as
their relative ability to unfold the denatured species. The results are
best illustrated by the comparison of urea and GdmCl. 1) Both GdmCl and
urea denature the native TAP not only with similar potency (Fig. 2) but
also with nearly indistinguishable kinetics. This property is somehow
unexpected because of the general experience that GdmCl is more potent
than urea (7). Indeed, some disulfide-containing proteins, such as
hirudin and epidermal growth factor, can be practically denatured only
with GdmCl. For hirudin, GdmCl is 9-fold more potent than urea (data
not shown). In the widely investigated case of ribonuclease A, the
potency of GdmCl as a denaturant exceeds that of urea by 2.5-3-fold.
These results, therefore, demonstrate that the relative potency of
GdmCl and urea in denaturing the native structures can vary
substantially from protein to protein. 2) Despite their comparable
potency to denature native TAP, GdmCl is definitely more potent than
urea in unfolding TAP. This is illustrated by the distinctive
compositions of X-TAP generated by both denaturants (Fig. 3) and by the
significantly higher recovery of X-TAP-a with GdmCl
denaturation (Fig. 5). 3) Urea and GdmCl also differ in their
interactions with protein stabilizers (co-solvents). Whereas NaCl (1 M) can drastically inhibit the denaturation of TAP by 8 M urea, it does not interfere with the TAP denaturation by
6 M GdmCl at all. These results thus indicate that NaCl is
able to counteract the destabilizing effect of urea but not that of GdmCl.
Urea and GdmCl are the two commonly used denaturants in protein
chemistry (3, 7, 10, 13). Although the chemical mechanism is not fully
understood, it is known that GdmCl and urea act by disrupting
noncovalent interactions that stabilize the native protein (2). The
existing evidence suggests that they cause water to become a better
solvent for nonpolar amino acids and thus weaken the hydrophobic
interaction, a dominant force of protein folding and stability (30).
GdmCl, being a salt, also suppresses electrostatic interactions among
charged groups of proteins. Pace and co-workers (31) have further shown that the unfolded states of proteins in urea and GdmCl solutions differ
significantly in the extent of their interaction with denaturants. Liepinsh and Otting (32) recently demonstrated that urea binds preferentially to the pockets and grooves on the surfaces of proteins. The data presented here further indicate that urea and GdmCl act with
two distinct modes of mechanism. These differences most likely reflect
a differential capacity in neutralizing various noncovalent forces
(hydrogen bonding, ion pairing, van der Waals, and hydrophobic interactions) that stabilize native proteins.
Thermal Denaturation--
Thermal unfolding has not been widely
applied in the experiments of reversible denaturation of proteins
because of the risk of disrupting covalent structures that often leads
to the irreversible denaturation of proteins (33). For
disulfide-containing proteins, it has been shown that the combination
of high temperature and alkaline pH can induce base-catalyzed
-elimination of disulfide bonds (34). This side reaction, which may
occur even at mild alkaline pH of 8-9, causes native proteins to form
a mixture of highly heterogeneous polymers that are intra- and
intermolecularly cross-linked by lanthionine and lysinoalanine (35). In
the case of TAP, denaturation at 55 and 69 °C is preferably carried
out within 20 and 90 min, respectively, to avoid possible destruction of disulfide bonds. Conditions selected here allow the reversible renaturation of heat-denatured TAP.
The molecular composition of heat-denatured X-TAP is distinguished from
that produced by various denaturants. The most intriguing aspect about
the structure of heat-denatured TAP is the simultaneous presence of
extensively unfolded isomers (X-TAP-a and
X-TAP-e) and compact isomers (X-TAP-h and
X-TAP-i). In solutions containing strong denaturants (GdmSCN
and GdmCl), the denatured TAP attains a more advanced stage of
unfolding. Therefore, X-TAP-a and X-TAP-e
populate well , but X-TAP-h and X-TAP-i are
absent (Fig. 3). In solutions with mild denaturants (e.g. 6.9 M ethanol), X-TAP-h and X-TAP-i
appear, and the concentrations of X-TAP-a and
X-TAP-e decrease accordingly. Coexistence of these four
X-TAPs as found in heat-denatured TAP is somehow unique. Again, these
data imply the complexity of noncovalent interactions that stabilize
native TAP and indicate that the mechanism of thermal denaturation
clearly differs from that of denaturants.
A Note of Caution--
The conclusion of this study is based on
the premise that the relative distribution of scrambled isomers of TAP
reflects merely the relative degree of unfolding of the conformational
ensemble present in the selected denaturing condition. It is important to mention that the chemistry of the disulfide interchange is thermodynamically linked to the conformational ensemble, and this thermodynamic linkage could potentially alter the preferred
conformational ensemble that exists in the absence of a disulfide interchange.
It is also relevant to point out that disulfide bonding signals only a
small fraction of the total conformational states of denatured
proteins. A given disulfide isomer, such as X-TAP-g, may
still consist of many conformational isomers. Nonetheless, the ability
to fractionate unfolded disulfide isomers, as demonstrated here with
the case of TAP, provides a very useful tool to differentiate the state
of denatured proteins and permits quantitative analysis of the
denaturation curve and unfolding curve separately.