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INTRODUCTION |
-LA1 represents one
of the most extensively investigated models for understanding the
mechanism of protein stability, folding, and unfolding. Under a variety
of conditions,
-LA adopts a partially structured conformation termed
as "molten globule" (1-9), which has been observed along both the
unfolding (1, 2, 5, 10-12) and folding pathways (13-18) of
-LA and
is considered one of the best characterized folding/unfolding
intermediates of globular proteins. At low pH (1, 19, 20), elevated
temperature (19, 21), or mild concentration of denaturant
(e.g. GdmCl; Refs. 22, 23), the native
-LA unfolds to the
state of a molten globule. During the refolding, the fully denatured
-LA undergoes a rapid collapse to the state of molten globule (13,
14, 18) as intermediate, which is followed by consolidation and search of side chain interactions to attain the native protein (14). The
structure of
-LA molten globule is characterized by a high degree of
native-like secondary structure and a fluctuated tertiary fold (1-4,
19). It is stabilized mainly by the core hydrophobic amino acids within
the
-helical domain (24-28) and may form even in the absence of its
four disulfide bonds (15). The structure of the molten globule of
-LA is also highly heterogeneous (3, 27, 29). Like most denatured or
partially denatured state of proteins (30, 31), it comprises a large
number of conformational isomers. So far, characterization of the
structure of denatured
-LA and the molten globule state of
-LA
have been achieved by measuring the average property of these
collective isomers using a wide range of spectroscopic and
physiochemical methods, including circular dichroism (1, 2, 19,
32), fluorescence (12, 32, 33), NMR (5, 14, 15, 20, 29, 34), disulfide replacement (35-37), limited proteolysis (38, 39), light scattering (40, 41), and calorimetric techniques (42). Further understanding of
the denatured state of
-LA and the molten globule state of
-LA
will require fractionation of diverse populations of conformational isomers that constitute the denatured
-LA.
In this report, the structure of denatured
-LA has been analyzed by
the technique of disulfide scrambling (43), which permits fractionation
and quantitative analysis of the denatured isomers. In the presence of
denaturant and thiol initiator, the native
-LA denatures by
shuffling its native disulfide bonds and converts to a mixture of fully
oxidized scrambled isomers that are trapped by non-native disulfide
bonds.
-LA contains four disulfide bonds and may adopt 104 possible
scrambled isomers. The technique of disulfide scrambling (43) presents
numerous advantages for characterizing the structure of denatured
-LA. 1) Scrambled isomers of
-LA are not interconvertible in the
absence of thiol catalyst or acidic pH. Because of their stability and
diverse physicochemical properties, scrambled isomers of
-LA can be
separated and purified by liquid chromatography and structurally
characterized (43-46). This permits a more detailed description of the
structure of denatured
-LA that is not attainable with conventional
methods. 2) Scrambled isomers of
-LA contain different sizes of
disulfide loops and adopt varied degree of unfolding. Compositional
analysis of scrambled isomers allows evaluation of the extent of
unfolding of the denatured
-LA (43, 44, 46). 3) The process of
denaturation of
-LA can be monitored in a time-course manner by acid
trapping of unfolding intermediates. This permits identification and
isolation of structurally defined intermediates present along the
unfolding pathway of
-LA. Specifically, it will allow us to examine
the structure of scrambled isomers that constitute the molten globule
state of
-LA.
Our specific aims are as follows: 1) to analyze the structure and
heterogeneity of denatured
-LA, 2) to elucidate the unfolding pathway of
-LA denatured under increasing concentrations of selected denaturants, and 3) to characterize the molecular structure of the
molten globule state of
-LA.
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EXPERIMENTAL PROCEDURES |
Materials--
Calcium-depleted bovine
-LA (L-6010) was used
throughout this study and was obtained from Sigma. The protein was
further purified by HPLC and was shown to be more than 97% pure.
GdmCl, GdmSCN, urea, acetonitrile, and 2-mercaptoethanol were also
purchased from Sigma with a grade purity of greater than 99%.
Denaturation of the Native
-LA--
The native
-LA (0.5 mg/ml) was dissolved in Tris-HCl buffer (0.1 M), pH 8.4, containing 2-mercaptoethanol (0.02-0.25 mM) and selected
concentrations of denaturants (urea, GdmCl, GdmSCN, or acetonitrile).
Denaturation was typically performed at 23 °C for 24-48 h. In the
case of thermal denaturation, the sample (in the presence of 0.1 mM 2-mercaptoethanol) was subjected to elevated temperature
(45-65 °C) for a time period of up to 60 min. To monitor the
kinetics and intermediates of unfolding, aliquots of the sample were
removed in a time-course manner, quenched with an equal volume of 4%
aqueous trifluoroacetic acid, and analyzed by reversed-phase HPLC. The
denatured sample was subsequently acidified with 4% trifluoroacetic
acid and stored at
20 °C.
Criteria for the State of Equilibrium and the End Point of
Denaturation--
The end point of denaturation, under a given
denaturing condition, implies that conversion of the native species to
the scrambled isomers has reached a state of equilibrium. This state of
equilibrium also includes those among scrambled isomers. The major
criteria to ensure that denaturation has reached equilibrium is
time-course kinetics analysis, which indicates that conversion of the
native
-LA to the scrambled species has reached a plateau, and the
ratio of scrambled to the native species remains constant during
prolonged sample incubation. Another method to verify the state of
equilibrium of denaturation is to renature (refold) the extensively
unfolded
-LA under the same conditions that denature the native
-LA, for instance, by allowing the sample denatured with 8 M GdmCl to refold after dilution of the denaturant. 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 native species (49). A systematic study has shown that
denaturation of
-LA by denaturants (in the presence of 0.1-0.25
mM 2-mercaptoethanol) completed generally within 2 h
(see Fig. 2). Nonetheless, the reaction has been routinely allowed to
proceed overnight (24 h). On the other hand, completion of denaturation
of
-LA by acetonitrile is relative slow and requires about 15 h. In the case of thermal denaturation, there are inherent risks of
prolonged sample incubation because of the heat-induced decomposition
of disulfide bonds. Experiments of thermal denaturation of
-LA were
therefore performed only in a time-course manner within 60 min.
Denaturation Is Distinguished from Unfolding--
The denatured
-LA may adopt varied degrees of unfolding. Denaturation and
unfolding are therefore two distinct terms. Using the method of
disulfide scrambling (43), it is possible to observe and follow
simultaneously the process of denaturation and unfolding of
-LA. The
extent of denaturation of
-LA is defined by the simple conversion of
the native structure to non-native structures (scrambled isomers).
There are 104 scrambled isomers of denatured
-LA versus
one isomer of the native
-LA. Unfolding of
-LA is defined by the
state of denatured
-LA and is structurally characterized by the
composition (relative concentration) among the 104 scrambled isomers.
Plotting of the Kinetic and Thermodynamic Denaturation Curves of
-LA--
The denaturation curve of
-LA was determined by the
fraction (%) of the native
-LA converted to the scrambled isomers.
Quantitative analysis of the relative yield of scrambled and the native
isomer was based on the integration of HPLC peak areas. There are two types of denaturation curves: 1) kinetic denaturation curve was calculated and derived from samples denatured in a time-course manner
under fixed denaturing conditions, and 2) thermodynamic denaturation
curve was derived from those that have attained the end point
(equilibrium) of denaturation under increasing concentrations of a
selected denaturant.
Plotting of the Kinetic and Thermodynamic Unfolding Curves of
-LA--
The unfolding curve of
-LA was determined by the ebb
and flow of different scrambled isomers as fractions of the total
denatured (scrambled) protein along the pathway of unfolding. Similar
to the denaturation curve, the unfolding curve can be established as
follows: 1) from samples denatured in a time-course manner by a defined
denaturing condition (kinetic unfolding curve) or 2) from samples that
have attained the end point of denaturation under increasing
concentrations of a selected denaturant (thermodynamic unfolding
curve). Calculation of the yield of scrambled isomers was based on the
peak area integration. Because of the complexity of minor isomers, the
data have a S.D. of ± 5%.
Structural Analysis of Scrambled Isomers of
-LA--
Fractions of scrambled
-LA (~10 µg) were isolated
and treated with 1 µg of thermolysin in 30 µl of
N-ethylmorpholine/acetate buffer (50 mM), pH
6.4. Digestion was carried out at 37 °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.
Amino Acid Sequencing and Mass Spectrometry--
Amino acid
sequence of disulfide-containing peptides were analyzed by automatic
Edman degradation using a PerkinElmer Procise sequencer (Model 494)
equipped with an online PTH-derivative amino acid analyzer. The
molecular mass of peptides was determined by MALDI-TOF mass
spectrometry (Perkin-Elmer Voyager-DE STR).
Nomenclature of Scrambled Isomers of
-LA--
Scrambled
species of
-LA are designated by the following formula:
X-
-LA-(species assigned on HPLC), where X stands for scrambled. For
instance, X-
-LA-a represents species "a" of scrambled
-LA.
 |
RESULTS |
Disulfide Structures of the Predominant Isomers of Denatured
-LA--
Denatured
-LA was shown to consist of at least 45 fractions of fully oxidized scrambled isomers. Their relative
concentrations vary substantially under different denaturing conditions
(see the following sections). Among them, six major fractions of
scrambled
-LA were isolated and structurally characterized. They are
X-
-LA-a, X-
-LA-b, X-
-LA-c, X-
-LA-d, X-
-LA-e, X-
-LA-h.
After digestion with thermolysin, peptides were isolated by HPLC and
were characterized by Edman sequencing and MALDI mass spectrometry to
identify the peptides that contain disulfides (data available upon
request). The disulfide structures are given in Fig.
1. X-
-LA-a and X-
-LA-d contain,
among the 104 possible isomers, the smallest sizes of disulfide loops
and represent the most extensively unfolded structures of
-LA. These
two isomers become well populated under strong denaturing conditions.
Specifically, X-
-LA-a adopts the beads-form structure with
disulfides formed by four consecutive pairs of neighboring cysteines.
X-
-LA-b and X-
-LA-c are partially unfolded isomers, with
X-
-LA-b possessing two native disulfide bonds within the
-sheet
region and X-
-LA-c retaining two intact native disulfide bonds at
the
-helical domain.

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Fig. 1.
The structures of native and denatured
-LA. Top, three-dimensional
structure of the native -LA and schematic presentation of four
denatured scrambled isomers (a, b, c,
and d). The dark gray and light gray
indicate the sections of -helical and -sheet structures,
respectively. Bottom, disulfide structures of the native
-LA and six scrambled isomers.
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Structures of
-LA Denatured by Urea, GdmCl, and
GdmSCN--
Time-dependent kinetics denaturation of the native
-LA was first analyzed using 6 M urea and 4 M GdmCl in the presence of varying concentrations of thiol
initiator (0.02-0.25 mM 2-mercaptoethanol). These
experiments were designed to establish conditions that would permit
denaturation of
-LA to reach a state of equilibrium. The results are
shown in Fig. 2. Both urea and GdmCl
denature the native
-LA at similar rates (Fig. 2, right
column). In the presence of 0.1-0.25 mM
2-mercaptoethanol, denaturation of
-LA is completed within 2 h.
The structure of
-LA denatured by 6 M urea comprises 4 major scrambled isomers (Fig. 2, left column).

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Fig. 2.
Kinetic denaturation of
-LA by urea (6 M) and GdmCl
(4 M). Denaturation was carried out at
23 °C in Tris-HCl buffer, pH 8.4 containing varying concentrations
of 2-mercaptoethanol (0.02-0.25 mM). Intermediates of
denaturation were trapped by acidification (4% trifluoroacetic acid)
and analyzed by HPLC, using the conditions described in the legend to
Fig. 3. Left, chromatograms of time course-trapped
intermediates denatured by 6 M urea in the presence of 0.1 mM 2-mercaptoethanol. Right, effect of the
concentration of 2-mercaptoethanol (inset) on the kinetic
denaturation curves of -LA. Fractions denatured indicate the
fraction (%) of native -LA converted to scrambled -LA.
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Thermodynamic denaturation of
-LA was subsequently performed in the
presence of 2-mercaptoethanol (0.1 mM) and increasing concentrations of urea, GdmCl and GdmSCN. Chromatograms of the denatured
-LA are shown in Fig. 3. At
mild concentrations of GdmCl (1.75 M) and GdmSCN (0.75 M), denatured
-LA was shown to consist of at least 45 fractions of scrambled isomers. Under strong denaturing conditions, the
denatured
-LA comprises three predominant isomers (X-
-LA-a,
X-
-LA-b, and X-
-LA-d). The thermodynamic denaturation curves
(Fig. 4) reveal that GdmSCN is about 2- and 7-fold more potent than GdmCl and urea, respectively. In the
presence of CaCl2 (5 mM), the potency of GdmCl
and urea to denature the native
-LA was diminished by 2-fold. The
thermodynamic unfolding curves (Fig. 5)
further display the structures of denatured
-LA evolved under
increasing concentrations of selected denaturants. Characteristically,
the progressive unfolding of denatured
-LA is accompanied by an
increasing yield of X-
-LA-a. For instance, the recovery of
X-
-LA-a as a fraction of the total scrambled
-LA increases from 5 to 27% as the concentration of GdmSCN rises from 0.2 to 4 M (Fig. 5). Similar phenomenon was observed with the urea
and GdmCl denaturation of
-LA.

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Fig. 3.
Thermodynamic denaturation of
-LA by different concentrations of urea,
GdmCl, and GdmSCN. Denaturation was carried out
at 23 °C for 24 h in Tris-HCl buffer, pH 8.4 containing
2-mercaptoethanol (0.1 mM). Denatured samples were
acidified with 4% trifluoroacetic acid and analyzed by reversed-phase
HPLC using the following conditions. Solvent A for HPLC was water
containing 0.1% trifluoroacetic acid. Solvent B was acetonitrile/water
(9:1 v/v) containing 0.086% trifluoroacetic acid. The gradient was
22-37% B in 15 min and 37-56% B from 15 to 45 min. The flow rate
was 0.5 ml/min. Column was Zorbax 300SB C18 for peptides
and proteins, 4.6 mm × 5 µm. Column temperature was 23 °C.
The concentration of denaturant and predominant isomers
(a-h) of denatured -LA are marked.
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Fig. 4.
Thermodynamic denaturation curves of
-LA. These curves were derived from data
presented in Fig. 3. In the case of GdmCl and urea denaturation,
experiments were also performed in the presence of CaCl2 (5 mM). Fractions denatured indicates the fraction
(%) of native -LA converted to scrambled -LA.
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Fig. 5.
Thermodynamic unfolding curves of
-LA. These curves were derived from data
presented in Fig. 3. The recoveries of seven major scrambled isomers
were used to construct these curves.
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Structures of
-LA Denatured by Organic Solvent--
Kinetic
denaturation of
-LA by organic solvent was first carried out in
Tris-HCl buffer (0.1 M), pH 8.4, containing 40%
acetonitrile. The experiments were performed both in the absence and
presence of CaCl2 (5 mM, Fig.
6). The results show that the rate of
disulfide shuffling induced by acetonitrile is about 10-fold slower
than those promoted by urea and GdmCl (Fig. 2). The reaction reaches equilibrium after about 15 h of incubation. In the absence of CaCl2 (Fig. 6, left column), unfolding of
denatured
-LA proceeds through X-
-LA-c as a predominant
intermediate, and the denatured sample consists of two major scrambled
isomers, X-
-LA-a and X-
-LA-b. In the presence of
CaCl2 (Fig. 6, right column), the yield of X-
-LA-c as unfolding intermediate diminishes drastically, and the
denatured
-LA comprises one prevalent isomer, X-
-LA-b.

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Fig. 6.
Kinetic denaturation of
-LA by acetonitrile. Denaturation was carried
out at 23 °C in Tris-HCl buffer, pH 8.4 containing acetonitrile
(7.28 M or 40% by volume) and of 2-mercaptoethanol (0.1 mM). Intermediates of denaturation were trapped by an equal
volume of 4% trifluoroacetic acid and analyzed by HPLC using the
conditions described in the legend to Fig. 3. Left,
denaturation carried out in the absence of CaCl2.
Right, denaturation performed in the presence of
CaCl2 (5 mM).
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Thermodynamic denaturation of the native
-LA was then performed with
increasing concentrations of acetonitrile (10-50%, by volume). All
reactions were allowed for 48 h to ensure the state of
equilibrium. The experiments were also conducted in the absence and
presence of CaCl2, and the results are presented in Fig.
7. Abrupt denaturation of
-LA occurs
at between 20 and 30% (1.82-3.64 M) acetonitrile. The
thermodynamic unfolding curves demonstrate that in the absence of
CaCl2, unfolding of denatured
-LA is characterized by an
increasing yield of both X-
-LA-a and X-
-LA-b. (Fig.
7A). In the presence of CaCl2, the structure of
denatured
-LA is dominated by the isomer X-
-LA-b (Fig.
7B).

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Fig. 7.
Thermodynamic denaturation of
-LA by acetonitrile. Denaturation was carried
out at 23 °C in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol
(0.1 mM) and different concentrations of acetonitrile
(10-50% by volume, equivalent to 1.82-9.10 M in molar
concentration). Denaturation proceeded for 48 h. Denatured samples
were acidified with 4% trifluoroacetic acid and analyzed by HPLC.
Left, chromatograms obtained from denaturation carried out
in the absence of CaCl2. The corresponding thermodynamic
unfolding curves are given in A. Right,
chromatograms obtained from denaturation performed in the presence of
CaCl2 (5 mM). The corresponding unfolding
curves are given in B.
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Thermal Denaturation of
-LA--
Thermal denaturation of
disulfide-containing proteins can be practically performed only in a
kinetic fashion. This is because prolonged incubation at high
temperature in alkaline pH may lead to the decomposition of the
disulfide bonds of scrambled isomers. The native
-LA was therefore
denatured at 45-65 °C for up to 60 min. The rate of
-LA
denaturation increases by 3- and 5-fold as the temperature rises from
45 to 55 °C and 65 °C, respectively (Fig.
8). At 65 °C, the presence of
CaCl2 (5 mM) reduces the rate of denaturation
by a factor of about 3-fold (Fig. 8).

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Fig. 8.
Kinetic denaturation curves of
-LA acquired from the experiments of thermal
denaturation. Denaturation was carried out at 45, 55, and 65 °C
in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol (0.1 mM). Denaturation at 65 °C was also performed in the
presence of CaCl2 (5 mM). Fractions denatured
indicate the fraction (%) of native -LA converted to scrambled
-LA.
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Heat-denatured
-LA comprises many of the isomers found within the
structures of
-LA denatured by GdmCl and GdmSCN (Fig. 9). The most striking feature is the
prevalence of X-
-LA-c during the early stage of thermal
denaturation. Under selected denaturing conditions (e.g.
65 °C, 2 min), the yield of X-
-LA-c may account for as much as
40% of the total content of the denatured
-LA. This is clearly
demonstrated by the kinetic unfolding curves of thermal denaturation of
-LA (Fig. 9A). The inclusion of CaCl2, which
binds to the
-sheet domain of
-LA, inhibits not only the rate of
thermal denaturation (Fig. 8) but also the prevalence of X-
-LA-c as
the unfolding intermediate (Fig. 9, B and top
right HPLC chromatograms).

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Fig. 9.
Kinetic denaturation of
-LA by elevated temperature. Denaturation was
carried out at 65 °C in Tris-HCl buffer, pH 8.4 containing
2-mercaptoethanol (0.1 mM). The experiments were conducted
both in the absence (left chromatograms) and presence
(right chromatograms) of CaCl2. Unfolding
intermediates were trapped by acidification (4% trifluoroacetic acid)
and analyzed by HPLC using the conditions described in the legend of
Fig. 3. Predominant isomers of denatured -LA are marked. The
corresponding unfolding curves are presented in A and
B.
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 |
DISCUSSION |
The Structure of Denatured
-LA Is Highly
Heterogeneous--
Structural analysis of denatured proteins is
inherently difficult. Unlike the native protein, which usually adopts a
well defined structure, a denatured protein comprises highly
heterogeneous conformational isomers that generally exist in a state of
equilibrium (30, 31). It is a daunting task to attempt isolation and
analysis of denatured 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 isolation of
conformational isomers, the structural property of a denatured protein
has been typically concluded from the measurement of the collective
isomers using various spectroscopic techniques (47-51). This has been
the case for the analysis of most denatured proteins, including
-LA.
The technique of disulfide scrambling permits isomers of a denatured
protein to be trapped by non-native disulfide bonds. Such stabilized
isomers can be separated by liquid chromatography and structurally
characterized (43). For a protein that contains four disulfide bonds,
like
-LA, there exist 104 possible species of scrambled isomers.
Denatured
-LA was shown to comprise about 50% of these possible
isomers. The heterogeneity is dependent upon the denaturing conditions
and is most evident for samples denatured at elevated temperature (Fig.
9) or low concentrations of denaturants (Fig. 3). A similar extent of
structural heterogeneity was observed with several three-disulfide
models. In the case of hirudin (45), tick anticoagulant peptide (43),
and potato carboxypeptidase inhibitor (46), about 50-80% of the
possible scrambled isomers were demonstrated to constitute their
denatured structures.
Conformational Stability of
-LA Is Characterized by Its
Denaturation Curves--
Denaturation curves of
-LA have been
determined here using the technique of disulfide scrambling (Fig. 4).
They are comparable with those evaluated by the CD signal of
-LA
(1). Both techniques reveal that
-LA (calcium-depleted) denatures
precipitously at 1-2 M GdmCl. The signal for disulfide
shuffling is as sensitive as that of fluorescence or CD spectra (49) in
measuring the denaturation curves of proteins. This was demonstrated in
the case of RNase A (52) and potato carboxypeptidase inhibitor
(46).
Aside from
-LA, conformational stability of seven different
disulfide proteins were similarly investigated using the technique of
disulfide scrambling. Their relative stability is presented in Table
I. The comparison is defined by the
concentration of a denaturant required to achieve 50% of the protein
denaturation thermodynamically. Among them, calcium-depleted
-LA
represents the least stable protein. It is about 2-fold less stable
than RNase A, a protein also containing four disulfide bonds and having a size almost identical to that of
-LA. However, calcium-bound
-LA exhibits stability similar to that of RNase A (Table I). The
following can be concluded from evaluation of this data. 1) These
results demonstrate and confirm that GdmSCN is typically 2-3-fold more
potent than GdmCl, which in turn is an additional 2-3-fold more
effective than urea (49). 2) Ranking of protein stability depends upon
the nature of denaturant. For example, based on the GdmSCN denaturation
curves, IGF-1 is 1.5-fold more stable than tick anticoagulant peptide.
This order is reversed when comparison of their stability is based on
GdmCl denaturation curves (Table I). 3) Numerous proteins are barely
denatured by 8 M urea, a condition that is general
considered as being capable to fully denature proteins (31). The most
remarkable case is BPTI, which remains practically intact at 8 M urea (53). For hirudin and EGF, less than 15% is
denatured under the same condition (45).
The Mechanism of Denaturation of
-LA Is Illustrated by Its
Unfolding Curves--
The unfolding curves of
-LA are derived from
the structural analysis of denatured
-LA, which is characterized by
the composition of its scrambled species. The structure of denatured
-LA varies, depending on the kinetics of denaturation, on the nature
and strength of denaturant. Unfolding curves of
-LA thus can be
obtained as follows: 1) from structural analysis of samples denatured
by a fixed denaturing condition and quenched in a time-course manner (Fig. 9, kinetic unfolding curves) or 2) from samples that
have reached equilibrium of denaturation under increasing
concentrations of a selected denaturant (thermodynamic unfolding
curves, see Figs. 5 and 7). These unfolding curves of
-LA,
constructed from the relative recoveries of seven major scrambled
isomers, display two important features about the mechanism of
denaturation of
-LA.
First, it demonstrates a general pathway for the unfolding of denatured
-LA-a involving progressive expansion and relaxation of the protein
conformation toward the shape of a linear structure. Among the 104 possible scrambled isomers of
-LA, X-
-LA-a (beads-form isomer)
and X-
-LA-d contain the smallest disulfide loops and presumably
represent the most extensively unfolded structures. The yield of
X-
-LA-a and X-
-LA-d are relative to the strength of the
denaturing condition. This phenomenon is most evident with samples
denatured with urea, GdmCl, GdmSCN, and acetonitrile. It can be
measured quantitatively from thermodynamic unfolding curves presented
in Figs. 5 and 7A. As the concentration of urea increases
from 3 to 8 M, the recovery of X-
-LA-a as a fraction of
the total denatured protein grows from 9 to 32%. The same tendency was
found with samples denatured by GdmCl and GdmSCN. Between 1 and 6 M GdmSCN, the recovery of X-
-LA-d rises from 3 to 23%.
-LA is not alone in displaying this property. The predominance of
beads-form isomer under strong denaturing conditions was
similarly observed with the unfolding behaviors of tick anticoagulant
peptide (43), insulin-like growth factor (44), hirudin (45), potato carboxypeptidase inhibitor, (46) and bovine pancreatic trypsin inhibitor (53). For example, in the presence of 6 M GdmSCN, more than 63% of the denatured tick anticoagulant peptide and 55% of
denatured BPTI were found to be the beads-form isomer. In the case of
IGF-1, the content of the beads-form isomer also rises from 5 to 30%
as the concentration of GdmSCN increases from 1 to 6 M.
Second, it reveals the presence of a partially structured unfolding
intermediate, which exhibits structural characteristics of the molten
globule state of
-LA. One of the scrambled isomers, X-
-LA-c was
found to contain two native disulfide bonds
(Cys6-Cys120 and
Cys28-Cys111) within the
-helical domain
and two non-native disulfide bonds (Cys61-Cys73 and
Cys77-Cys91) within the
-sheet
(calcium-binding) domain (Fig. 1). The disulfide structure of
X-
-LA-c implies that it is a partially unfolded
-LA with a
largely intact
-helical domain and an unstructured, disordered
-sheet region. X-
-LA-c accumulates only under mild denaturing
conditions. At low concentrations of GdmCl (1 M) and GdmSCN
(0.2 M, see Fig. 5), X-
-LA-c constitutes ~10% of the
total denatured
-LA. However, prevalence of X-
-LA-c as an
unfolding intermediate is most obvious with thermal denaturation (Fig.
9) or acetonitrile as denaturant (Fig. 6). In both cases, X-
-LA-c may account for more than 40% of the denatured
-LA during the early
stages of kinetic denaturation.
The disulfide structure of X-
-LA-c is consistent with the properties
of well characterized molten globule of
-LA. Structural elements
that stabilize and characterize the structure of molten globule of
-LA have been investigated by many different laboratories (15-17,
24, 25, 29, 33, 37). NMR analysis showed that the most persistent
structure in the
-LA molten globule is localized at the helical
domain. Kim and co-workers (29) have demonstrated that the molten
globule properties of
-LA are mainly confined to one of its two
domains. The
-helical domain forms a helical structure with a
native-like tertiary fold, whereas the
-sheet domain is essentially
disordered (24). These new findings differ somehow from the common
definition of molten globule in which a fluctuated tertiary fold is
believed to encompass the entire polypeptide chain (1, 2). Our data
support the finding that the molten globule of
-LA is composed of a
structured and an unstructured domain.
Binding of Ca2+ Stabilizes the
-Sheet Domain of
-LA--
Binding of Ca2+ is known to stabilize the
conformation of
-LA (for review, see Ref. 54). Our data confirm this
phenomenon. The GdmCl denaturation curves show that the stability of
-LA increases by 2-fold in the presence of 5 mM
CaCl2 (Fig. 4). A similar outcome was observed when
-LA
was denatured by acetonitrile in a thermodynamic fashion (Fig. 7).
Binding of Ca2+ to
-LA also impedes the initial rate of
its thermal denaturation by a factor of 3 (Fig. 8). Surprisingly, the
binding of Ca2+ does not decelerate the kinetics of
-LA
denaturation by acetonitrile (40%) (Fig. 6).
Structural analysis of denatured
-LA reveals that binding of
Ca2+ protects the two native disulfide bonds
(Cys61-Cys77 and
Cys73-Cys91) located within the
-sheet
domain of
-LA. This is displayed by thermal denaturation of
-LA
(Fig. 9) in which the binding of Ca2+ inhibits disulfide
scrambling of Cys61-Cys77 and
Cys73-Cys91 and suppresses the recovery of
X-
-LA-c. However, this effect is most remarkable in the case of
denaturation by acetonitrile. For example,
-LA denatured in the
presence of 30% acetonitrile and CaCl2 (5 mM)
comprises a single predominant scrambled isomers (X-
-LA-b) that
retains two intact native disulfide bonds of the
-sheet domain (Fig.
7). These data are consistent with the defined calcium binding site of
-LA, which is located at the
-domain. It is formed by oxygen
ligands from carboxylic groups of Asp82, Asp87,
and Asp88, and two carbonyl groups of the peptide backbone
(residues 79 and 84; Refs. 54, 55).