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J Biol Chem, Vol. 275, Issue 12, 8287-8289, March 24, 2000
From the Research Center for Protein Chemistry, Institute of
Molecular Medicine, University of Texas, Houston, Texas 77030
The disulfide folding pathway of bovine
pancreatic trypsin inhibitor (BPTI) is characterized by the
predominance of folding intermediates with native-like structures. Our
laboratory has recently analyzed the folding pathway(s) of four
3-disulfide-containing proteins, including hirudin, potato
carboxypeptidase inhibitor, epidermal growth factor, and tick
anticoagulant peptide. Their folding mechanism(s) differ from that of
BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide
intermediates and 2) the presence of 3-disulfide scrambled isomers as
folding intermediates. To search for the underlying causes of these
diversities, we conducted kinetic analyses of the reductive unfolding
of these five proteins. The experiment of reductive unfolding was
designed to evaluate the relative stability and interdependence of
disulfide bonds in the native protein. It is demonstrated here that
among these five proteins, there exists a striking correlation between
the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of
heterogeneity of folding intermediates and the accumulation of
scrambled isomers along the folding pathway. A sequential reduction of
the native disulfide bonds is associated with the presence of
predominant intermediates with native- like structures.
Bovine pancreatic trypsin inhibitor
(BPTI)1 is a compact
single-domain protein stabilized by three disulfide bonds
(Cys30-Cys51,
Cys5-Cys55,
Cys14-Cys38) (1). Unfolded and fully reduced
BPTI refolds spontaneously to form the native structure under selected
redox conditions. The mechanism of BPTI unfolding and refolding has
been a subject of intensive investigation and represents one of the
best and most extensively characterized models (2-7). The folding
pathway of BPTI was elucidated by trapping and structural
characterization of disulfide bond intermediates. The original model of
Creighton (2-5) identified seven predominant intermediates, two
1-disulfide species and five 2-disulfide species, with 75% of the
disulfide bonds being native. The native disulfide of BPTI
(Cys30-Cys51) was found to be a major
component of both 1- and 2-disulfide intermediates. In the revised
model proposed by Weissman and Kim (6, 7), five well populated
intermediates, two 1-disulfide and three 2-disulfide species, were
described, and all of them were shown to contain only native disulfide
bonds. Another important feature of the BPTI folding pathway is the
kinetic role of a 2-disulfide intermediates that contains two of
disulfide bonds of the native BPTI
(Cys30-Cys51,
Cys5-Cys55)
(NSHSH) (3, 5, 6, 8).
NSHSH is the immediate precursor of the
native BPTI. Formation of the third native disulfide, Cys14-Cys38,
completes the folding and accounts for the final step of the BPTI
folding pathway. Prevalence of the native disulfide bonds along the
folding pathway has major implications. It implies that non-covalent
specific interactions that stabilize the native BPTI and local
structures of BPTI play a crucial role in guiding the folding in its
early stages and hence dictate the formation of a limited number of
well populated intermediates that admit only native disulfides.
Our laboratory has analyzed the folding pathway(s) of four
single-domain, 3-disulfide-containing proteins that have sizes similar
to that of BPTI. These four proteins are hirudin (9), potato
carboxypeptidase inhibitor (PCI) (10), epidermal growth factor (EGF)
(11), and tick anticoagulant peptide (TAP) (12). Their folding
mechanism(s) have been shown to differ from that of BPTI in two crucial
aspects. Their folding intermediates, including 1- and 2-disulfide
species, are far more heterogeneous than those described for BPTI.
Aside from EGF, there is also no evidence for the accumulation of
predominant intermediates. The most noticeable difference, however, is
the presence of scrambled 3-disulfide isomers as folding intermediates,
which has not been observed with BPTI. Scrambled isomers are fully
oxidized species that contain at least two non-native disulfide bonds
(13). They exist in high concentration along the folding pathways of
hirudin, PCI, and TAP, as well as EGF. With the exception of EGF,
accumulation of scrambled isomers as folding intermediates can be
greatly enhanced by allowing the folding in the buffer containing
oxidized glutathione or cystine (14). For instance, when folding of PCI
was performed in the presence of 0.5 mM cystine, more than
98% of the total protein was trapped as scrambled species before trace
amounts of the native PCI even appeared (10).
These discrepancies suggest that the folding pathway of small
disulfide-containing proteins is indeed more versatile than what has
been learned from the BPTI model alone. To further understand the
mechanism of protein folding, it is essential to identify and
characterize the underlying cause that generates such diversity of
folding pathway(s).
Materials--
BPTI was obtained from Roche Molecular
Biochemicals. Hirudin core domain (residue 1-49) was derived from the
recombinant hirudin variant 1 (HV1) by selective removal of its
disordered C-terminal section using Reductive Unfolding--
The native protein (0.5 mg/ml) was
dissolved in Tris-HCl buffer (0.1 M, pH 8.4) containing
varying concentrations of dithiothreitol (0.5-100 mM).
Reduction was carried out at 23 °C. To monitor the kinetics of
unfolding, aliquots of the sample were removed at various time
intervals, quenched with an equal volume of 4% aqueous trifluoroacetic
acid, and analyzed by HPLC. The samples were stored at Structural Analysis of Partially Reduced EGF and BPTI--
The
unfolding intermediates of EGF and BPTI (EGF-II and BPTI-II, both
2-disulfide species) were purified from HPLC and freeze-dried. The
samples (20 µg) were derivatized with 50 µl of vinylpyridine (0.1 M) in Tris-HCl buffer (0.1 M, pH 7.5) at
23 °C for 45 min. Vinylpyridine-derivatized EGF-II and BPTI-II
(~20 µg) were further purified by HPLC and treated with 2 µg of
thermolysin (Sigma, P-1512) in 65 µl of
N-ethylmorpholine/acetate buffer (50 mM, pH 6.4). Digestion was carried out at 37 °C for 16 h. Peptides
were then purified 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
sequences of disulfide-containing peptides were analyzed by automatic
Edman degradation using a Perkin-Elmer Procise sequencer (model 494)
equipped with an on-line PTH-amino acid analyzer. The molecular mass
values of disulfide-containing peptides were determined by MALDI
time-of-flight mass spectrometry (Perkin-Elmer Voyager-DE STR).
The mechanisms of reductive unfolding for five different proteins,
including hirudin, PCI, TAP, EGF, and BPTI were analyzed here.
Unfolding experiments were performed at pH 8.4 using various concentrations of dithiothreitol (DTT) as the reducing agent. The
unfolding intermediates were trapped in a time course manner by mixing
aliquots of samples with an equal volume of aqueous trifluoroacetic
acid (4%) and were subsequently analyzed by reverse phase HPLC. The
experimental data were analyzed and plotted using MS Excel and Gepasi
software (15). The rate constants for the sequential irreversible
transformations were calculated using Gepasi software and nonlinear
regression. The results show that the mechanisms of reductive unfolding
of these five proteins can be divided into three groups.
For hirudin, PCI, and TAP, reduction leads to the direct conversion of
the native structure (N) to the fully reduced species (R) without
accumulation of 1- and 2-disulfide intermediates (Fig. 1). This phenomenon of concurrent
reduction was observed with the concentration of the reducing agent
ranging from 0.5 to 100 mM. The rate constant
(kN The mechanism of EGF unfolding is different from that of hirudin, PCI
and TAP. Reduction of the native EGF undergoes a stable 2-disulfide
intermediate (II). This 2-disulfide intermediate subsequently converts
to the fully reduced EGF (R) with no significant buildup of a
1-disulfide intermediate along the pathway (Fig. 1). The calculated
rate constants of N to II (kN
ACCELERATED PUBLICATION
The Underlying Mechanism for the Diversity of Disulfide
Folding Pathways*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chymotrypsin (9). Tick
anticoagulant peptide (TAP, CGP-55099) is a recombinant protein
produced by Novartis (Basel, Switzerland). Potato carboxypeptidase
inhibitor was a gift from Dr. F. X. Aviles (University of
Barcelona, Spain). Recombinant human EGF was supplied by the Protein
Institute Inc. (Broomall, PA). The purity of all five proteins was
greater than 96% as judged by HPLC and N-terminal sequence analysis.
Dithiothreitol was a product of Sigma with a purity grade of more than
99%.
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R) displays a linear dependence upon the
concentration of DTT in all three cases (Fig.
2). Based on the observed rate constants
of reduction, hirudin is about 11-fold more stable than PCI and
TAP.
View larger version (21K):
[in a new window]
Fig. 1.
Mechanism(s) of the reductive unfolding of
hirudin, PCI, TAP, EGF, and BPTI. The native protein (0.5 mg/ml)
of hirudin, TAP, and EGF were treated with indicated concentration of
DTT. Time course intermediates were trapped with acid (4%
trifluoroacetic acid) and analyzed by HPLC. The native proteins of PCI
and BPTI were treated with various concentrations of DTT for the
indicated time intervals. The end products were similarly trapped with
acid prior to HPLC analysis. N, II, and
R indicate the elution positions of the 3-disulfide native
species, the 2-disulfide species, and the fully reduced species,
respectively. Solvent A for the HPLC was water containing 0.05%
trifluoroacetic acid. Solvent B was acetonitrile/water (9:1, by volume)
containing 0.042% trifluoroacetic acid. The flow rate was 0.3 ml/min.
Column was Zorbax C-18 for peptides and proteins, 4.6 mm, 10 µm.
Column temperature was 23 °C. The gradient varied for each protein:
for hirudin, the gradient was 14-36% B in 60 min; for TAP, 28-45%
solvent B linear in 40 min; PCI, 14-42% solvent B linear in 50 min;
EGF, 14-56% B in 50 min; BPTI, 10-60% B in 60 min.
View larger version (20K):
[in a new window]
Fig. 2.
Rate constant for the direct conversion of
the native species to the fully reduced species. The three
disulfide bonds of hirudin, TAP, and PCI were shown to be reduced
simultaneously and collectively by DTT. For all three proteins, the
observed rate constants (kN R) display a
linear dependence upon the concentration of DTT. Quantitative analysis
was based on the peak integration of HPLC data.
II) and II to R (kIIR) are plotted in Fig.
3. kNII also
exhibits a linear dependence on the DTT concentration, but
kIIR reaches a plateau at of DTT
concentration of more than 10 mM. Therefore, accumulation
of the 2-disulfide intermediate was most evident when a high
concentration of the reducing agent was applied. The structure of this
stable 2-disulfide intermediate was characterized through analysis of
the thermolytic peptides by both Edman sequencing and MALDI mass
spectrometry (results not shown). The data revealed that this
intermediate contains two free cysteines (Cys6 and
Cys20) and two native disulfide bonds of EGF
(Cys14-Cys31,
Cys33-Cys42).
View larger version (20K):
[in a new window]
Fig. 3.
Rate constant for the sequential conversion
of N to II (kN II) and II to R
(kIIR). The disulfide bonds of EGF
and BPTI were shown to undergo sequential reduction. The HPLC-generated
data were analyzed and plotted by MS Excel. The rate constants for the
sequential irreversible transformations were calculated using Gepasi
software.
The unfolding mechanism of BPTI bears resemblance to that of EGF.
Reduction of the native BPTI (N) also goes through a 2-disulfide intermediate (II) that eventually converts to the fully reduced BPTI
(R) without an accumulation of 1-disulfide intermediates (Fig. 1). The
structure of this 2-disulfide intermediate has been analyzed by us as
well, and it was found to contain two free cysteines (Cys14
and Cys38) and two native disulfide bonds of BPTI
(Cys30-Cys51,
Cys5-Cys55) (data not shown). These results
are consistent with those reported earlier (8, 16). Unlike with EGF,
there is a huge disparity between the two rate constants that
characterize the conversion of N to II (kNII)
and II to R (kIIR) (Fig. 3). The transformation of N to II is extremely rapid and requires only mild a
concentration of DTT. For instance, in the presence of 2 mM
DTT, the conversion is completed within 1.5 min. However, the
subsequent conversion of II to R is exceedingly slow (8, 17). In the
presence of 100 mM DTT, only 7% of the fully reduced BPTI
was recovered after 90 min of incubation, and the observed rate
constant (kIIR) was found to be 0.75 × 10
3 min1. By comparison, the rate constant
kNII is greater than kIIR by a factor of 250,000. The vast
difference of these two rate constants reflects both the weak nature of
the Cys14-Cys38 bond and the outstanding
stability of the structures surrounding Cys30-Cys51 and
Cys5-Cys55. It is relevant to point out that
the native disulfide of BPTI, Cys14-Cys38, is
about 10-fold more susceptible to DTT reduction than non-native disulfides of scrambled hirudin and TAP.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Our data demonstrate the distinctive unfolding mechanisms of BPTI, EGF, TAP, PCI, and hirudin. These differences in the mechanisms reflect how their native disulfide bonds are stabilized and may well account for the diversity of their folding pathways (2-7, 9-12). The correlation is elaborated as follows. 1) Hirudin, PCI, and TAP share the same all-or-none mechanism of reductive unfolding, which suggests that their native disulfide bonds are stabilized in a concerted and interdependent manner. These three proteins also display common characteristics in their folding pathways that include high heterogeneity of 1- and 2-disulfide intermediates and the presence of scrambled 3-disulfide isomers as folding intermediates (9, 10, 12). 2) BPTI unfolds and refolds through a unique mechanism. Unlike in hirudin, PCI, and TAP, the three native disulfide bonds of BPTI are not stabilized in a concerted fashion. Native-like stable structures containing 2- and 1-disulfide bonds exist along the folding and unfolding pathways of BPTI (18-21). These properties govern the formation of limited numbers of predominant intermediates that admit mainly native disulfide bonds and preclude the formation of scrambled 3-disulfide species, such as those observed in the cases of PCI and hirudin. 3) The unfolding and refolding mechanisms of EGF appear to rest in between those of BPTI and hirudin and exhibit characteristics of both BPTI and hirudin. Similar to BPTI, there is a stable unfolding intermediate that contains two native disulfides (Cys14-Cys31, Cys33-Cys42). This native-like 2-disulfide species was shown to accumulate rapidly along the folding pathway of EGF (11). But unlike BPTI, the major 1-disulfide intermediates of EGF were shown to contain both non-native and native disulfide bonds (11, 22). Moreover, scrambled 3-disulfide species were found along the folding pathway of EGF (11), which is characteristic of the folding of hirudin and PCI.
Among these diversities, the one observed between TAP and BPTI is most
intriguing. Both TAP and BPTI belong to the Kunitz-type inhibitor and
share close structural homology in terms of disulfide pattern and
three-dimensional structure (Fig. 4)
(23). Despite the structural similarity, their native disulfide bonds
are apparently stabilized in very different ways (Fig. 1). Our results
also demonstrate that the folding intermediates of TAP are more
heterogeneous than those of BPTI (12). At least 18 fractions of the 1- and 2-disulfide intermediates of TAP were detected. In addition,
scrambled (3-disulfide) TAP were shown to exist along the folding
pathway and act as folding intermediates (12). However, among the
heterogeneous folding intermediates of TAP, some major species have
been shown to contain the native disulfide bonds, as was observed in
the case of BPTI.
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All these data, taken together, clearly show that the folding mechanism
of small 3-disulfide-containing proteins is more complex that what has
been understood previously. The major characteristics of the disulfide
folding pathway, including the extent of heterogeneity of folding
intermediates, their disulfide structures, and the accumulation of
scrambled species, are closely associated with the manner in which
native disulfide bonds are stabilized and vary among different proteins.
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FOOTNOTES |
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* This work was supported, in part, by an endowment from the Robert Welch foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of Molecular
Medicine, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-500-2458; Fax: 713-500-2424; E-mail: rchang@imm2.imm.uth. tmc.edu.
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ABBREVIATIONS |
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The abbreviations used are: BPTI, bovine pancreatic trypsin inhibitor; PCI, potato carboxypeptidase inhibitor; EGF, epidermal growth factor; TAP, tick anticoagulant peptide; HPLC, high performance liquid chromatography; DTT, dithiothreitol; MALDI, matrix-assisted laser desorption ionization.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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-sheet) and adopt comparable
three-dimensional conformations. The three corresponding native
disulfides are numbered.