From the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia and the § Department of Biochemistry, School of Biological Sciences, University of Southampton, Hampshire SO16 7PX, United Kingdom
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ABSTRACT |
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Protein misfolding plays a role in the
pathogenesis of many diseases. Protein folding is the process by which the primary sequence is
translated into tertiary structure. Modification of this sequence via
in vivo mutation or in vitro mutagenesis often
alters the ability of the protein to fold correctly. Misfolding of
proteins during their synthesis within the cell can lead to loss of
protein function (1), an example being The unfolding pathway of In order to study the folding of Materials--
1,1'-Bis-4-anilino-5-napthalinsulfonic acid
(dipotassium salt) and
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) were purchased from Molecular Probes Inc. (Eugene, OR). Ultrapure grade guanidine hydrochloride (Gdn HCl) was obtained from ICN Biochemicals.
Production of Proteins--
The recombinant Spectroscopic Methods--
Fluorescence emission spectra were
recorded on a Perkin-Elmer LS50B spectrofluorometer, using a
thermostatted cuvette holder at 25 °C in a 1-cm path length quartz
cell. Excitation and emission slits were set at 2.5 nm for all spectra,
and a scan speed of 10 nm/min was used. The absorbance at the
excitation wavelengths was monitored in all experiments and remained
below 0.05. Circular dichroism spectra were measured on a Jasco 720s
spectropolarimeter at 25 °C. Far UV spectra from 190 to 250 nm were
collected with 5 s/point signal averaging; Denaturation Transitions--
Unfolding curves were determined
by either measuring the fluorescence emission spectra of a 200 nM protein solution as a function of denaturant
concentration or the change in signal at 222 nm in the far UV as a
function of denaturant concentration. Attempts at measuring the near UV
CD unfolding curve proved impractical due to protein precipitation at
the concentrations required. Previous studies (31) and our own
preliminary data have shown that unfolding in Gdn HCl reaches
equilibrium within a matter of minutes at all concentrations of Gdn HCl
used. Therefore, the samples were allowed to equilibrate at 25 °C
for 2 h before measurement. For both the labeled and unlabeled
proteins, the reversibility of the unfolding process was established by
adding buffer to samples of unfolded protein, allowing each to
re-equilibrate at a lower Gdn HCl concentration. The fluorescence
emission spectra acquired for these samples, when corrected for
dilution, were identical with corresponding samples that had reached
equilibrium from the folded state, thus confirming the reversibility of
the systems. The inhibitory activity of these refolded proteins was
also analyzed, and they were found to be fully active.
Unfolding Data Analysis--
The Gdn HCl denaturation curves
were fitted to either a two-state or three-state unfolding model, using
a nonlinear least-squares fitting algorithm. Raw equilibrium data were
fitted directly to a two-state model of denaturation in which the
baselines are fitted simultaneously with the unfolding transition (32).
A three-state model was also employed for analysis of the denaturation
curves based upon the mechanism F Fluorescence Quenching Experiments--
Fluorescence quenching
measurements were performed in 50 mM Tris, pH 8.0, at 0 and
1 M Gdn HCl. Aliquots of KI (2 M stock) containing 1 mM Na2S2O3
were added to protein solutions (200 nM), and the change in
fluorescence emission intensity of the tryptophan residues
( Fluorescence Properties of
The addition of the IANBD label affords the protein new
fluorescence properties. The fluorescence emission spectra of the three
labeled proteins show distinct differences depending upon the location
of the probe within the Unfolding Studies--
The fluorescence characteristic of these
novel
Fig. 4 shows the normalized changes in
The labeled variants and unlabeled
The probe fluorescence changes are nonsuperimposable (Fig.
4B), implying that the probes are reporting site specific
conformational changes. The data in Fig. 4B were analyzed by
either a two-state or three-state mechanism as described previously
(25, 35). It was found that the data for
It is possible that the differences in probe monitored unfolding
profiles are due to local destabilizing effects of the probe. To assess
this, the unfolding curves for the labeled protein were measured using
far UV CD (Fig. 5). Far UV CD measures
secondary structure; therefore, deviation in the unfolding profile from native will indicate that the probes are affecting the unfolding process. However, it can be seen in Fig. 5 that the unfolding profiles
for all the labeled proteins are essentially the same as the unlabeled
native structure. The far UV CD data show two transitions, with average
midpoints of 0.77 and 2.6 M Gdn HCl (Table II). This is
similar to the fluorescence changes observed for
The nonsuperimposable transitions observed with different spectroscopic
probes and the biphasic nature of the CD unfolding denaturation curve
are consistent with the existence of a stable intermediate at 1 M Gdn HCl. The intermediate in 1 M Gdn HCl
possesses approximately 20% of the native CD spectral amplitude at 222 nm (Fig. 5), and the fluorescence data suggest that the environment around P3'Cys, S313C, and the two tryptophan residues has been altered
(Fig. 4). The structure of this equilibrium intermediate state was
further characterized by fluorescence quenching experiments. This
technique measures the accessibility of a nonfluorescent quenching
agent, such as iodide, to the fluorescence group, providing another
means of characterizing the nature of the intermediate. Various amounts
of iodide were added to the native (0 M Gdn HCl) and
intermediate (formed in 1 M Gdn HCl) states of
Unfolding Pathway of
Two-state unfolding has been observed in only a few small monomeric
proteins (38). It is often characterized by the coincidence of
unfolding curves monitored using different spectral probes. Fig.
4B shows that the fluorescence changes are nonsuperimposable when the different fluorescent probes are used to follow the unfolding pathway. This implies that the probes placed in different positions within
Examination of the x-ray crystal structure of native
An increase in the denaturant concentration causes further structural
changes in the protein that leads to complete unfolding. As a result,
the probes at P3'Cys and Cys-313 become fully exposed to solvent, and
the major portion of the tryptophan residue fluorescence transition
occurs. The final conformational change observed using these probes is
the disruption of the area around the B
Implications for Serpin Mechanism--
Specific parts of the
structure of a serpin, such as the reactive center loop and hinge
region, are extremely flexible; this mobility is required for their
inhibitory function (16, 20, 28). In recent years, the conformational
changes that occur within the serpin structure when bound to a
proteinase have been intensively studied (40, 41). Recent studies
suggest that the proteinase, once docked to the exposed reactive center
loop, attempts to cleave the scissile bond, which causes the reactive center loop to insert into the A Implications for Serpin Polymerization--
The fluorescence data
in this paper showed that the top of the A 1-Antitrypsin
misfolding leads to the accumulation of long chain polymers within the
hepatocyte, reducing its plasma concentration and predisposing the
patient to emphysema and liver disease. In order to understand the
misfolding process, it is necessary to examine the folding of
1-antitrypsin through the different structures involved
in this process. In this study we have used a novel technique in which
unique cysteine residues were introduced at various positions into
1-antitrypsin and fluorescently labeled with
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that
1-antitrypsin unfolded
through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular
modeling, we show that unfolding of
1-antitrypsin occurs via disruption of the A and C
-sheets followed by the B
-sheet. The implications of these data on both
1-antitrypsin
function and polymerization are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin
(
1-AT)1
deficiency (2-4). Many well characterized
1-AT variants
are associated with an
1-AT plasma deficiency; in these
cases, the variants aggregate at their site of synthesis within the
liver cell (5, 6). This aggregation is via an ordered polymerization process that is initiated by protein misfolding. In the case of the
1-AT Z mutation (Glu-342
Lys), the rate of protein
folding is much slower than in the native state, leading to the
accumulation of an intermediate, which then polymerizes (4). It has
recently been shown that the heat-induced polymerization of
1-AT also involves the formation of an unfolding
intermediate (7). The actual process of polymerization has been well
studied, and two mechanisms have been proposed: the loop-A-sheet and
loop-C-sheet mechanisms (8-11). Both processes involve the insertion
of the reactive center loop residues of the donating
1-AT molecule into either the A
-sheet (loop-A-sheet)
or the C
-sheet (loop-C-sheet) of the acceptor molecule (11). There
is evidence for the occurrence of both of these mechanisms; indeed,
in vitro
1-AT has been shown to undergo both
depending upon the buffer used (12-14).
1-AT is a member of the serine proteinase inhibitor
(serpin) superfamily and is composed of 394 amino acid residues
arranged into three
-sheets (A, B, and C) and nine
-helices
(A-I). X-ray crystallographic and biochemical data have shown that
1-AT can adopt a number of different structures
(15-17). These data demonstrate that areas such as the reactive center
loop and the A
-sheet of
1-AT are extremely mobile,
which is an essential requirement for inhibitory activity. Mutations
that affect the mobility of
1-AT cause it to become
either a proteinase substrate or to undergo polymerization (16,
18-20). Although many studies have examined the structural changes of
a serpin in relation to inhibitory mechanism, the conformational
changes, which occur during folding and misfolding, have not yet been characterized.
1-AT has been investigated
using size exclusion chromatography, intrinsic fluorescence, circular dichroism, and urea gradient gel electrophoresis (21-23). These studies revealed the presence of multiple intermediates, the
conformations of which have never been examined. A number of methods
have been employed for other proteins to study the conformational
changes during folding. The formation of disulfide bonds has been
widely used to trap folding intermediates (24), but
1-AT
contains only a single cysteine residue. NMR methodology has been used to examine the rate of exchange between backbone amide protons of the
folding protein and the solvent (25); however, due to the large size of
1-AT, this method is not applicable. Another commonly
adopted technique involves the insertion of unique tryptophan residues
(26, 27). The changes in tryptophan residue fluorescence upon folding
are then followed spectrophotometrically, yielding information about
the events occurring at specific locations within the protein
structure.
1-AT possesses two tryptophan residues, one
of which (Trp-194) is highly conserved and essential for activity (3).
Therefore, the insertion of additional tryptophan residues would lead
to a mixed population.
1-AT, we propose a
novel approach in which we have fluorescently labeled unique cysteine residues that have been inserted at positions throughout the tertiary structure of the molecule. The fluorescence properties of these unique
groups were then examined as a function of denaturant
concentration. The results demonstrate that the equilibrium unfolding
pathway of
1-AT consists of at least one stable
intermediate structure, which we have begun to characterize.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-AT
proteins were expressed in E. coli strain BL21 (DE3) as
described previously (28). The proteins were purified from cell
inclusion bodies using Gdn HCl denaturation and a continual dilution
procedure as recently described (29). In this study recombinant
1-AT(P1=Arg) was used as the control and the
construction of
1-AT(S313C) and
1-AT(P3'Cys) have been described (12). The
protein concentration was determined by Bradford assay against a
standard of purified recombinant
1-AT, the concentration
of which had been determined by amino acid analysis. The association
rate constant (kass) and stoichiometry of
inhibition were measured using thrombin for both the unlabeled and
labeled
1-AT forms, as described previously (30).
Fluorescent labeling of the proteins with IANBD was performed as
described previously (12). The extent of labeling was determined using
the extinction coefficient of IANBD (
= 25,000 M
1 cm
1).
222
measurements were made with the signal averaged over 60 s. The
concentration of
1-AT used was 0.1 mg/ml with a 0.1-cm
path length cell.
I
U, where F and U are the
fully folded and unfolded forms of the protein, respectively, and I is
an intermediate that is populated at equilibrium (33).
ex = 290 nm) and covalently bound IANBD
(
ex = 480 nm) were measured. Measurements in the
presence of 1 M Gdn HCl were made on individual samples, so
there was no dilution of the Gdn HCl. The data were analyzed as
described previously by Lehrer (34). All data were corrected for inner
filter effects where necessary.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-AT and Its
Variants--
The aim of this study was to monitor the structural
changes of
1-AT as it unfolds. To achieve this, we
generated
1-AT variants, which were fluorescently
labeled at specific sites on the protein. The structural changes
occurring in the unfolding process were then characterized using the
site-specific fluorescence changes.
1-AT possesses a
unique cysteine residue (Cys-232), which is situated on strand s2B
(Fig. 1). Two further cysteine residues were introduced into
1-AT at positions
P3'2 and Ser-313 (Fig. 1).
The substitutions were made using an
1-AT variant, in
which Cys-232 had been replaced by a serine residue. This variant has
previously been shown to have the same activity as the native protein
(12, 28). The native and mutant constructs were efficiently expressed
as inclusion bodies and purified as described previously (16). The two
mutations (P3'Cys and S313C) had no effect upon the inhibitory
properties of the proteins (Table I).
These cysteine residues were then selectively labeled with the
fluorescent probe IANBD. The extent of modification was measured using
the extinction coefficient of the probe and was found to be 1:1 in all
cases; this also confirmed the insertion of one unique cysteine residue
per monomer. The presence of the fluorescent label had no effect upon
the inhibitory properties of the protein (Table I).
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Fig. 1.
A schematic of native
1-AT in two orientations.
A, front view of antitrypsin, with Trp-194, Trp-238, Pro-361
(P3'), Cys-232, and Ser-313 in Van der Waals spheres and labeled.
B, side view, highlighting the position of Cys-232. In both
A and B, the P1/P1' is shown in ball and stick
form. This figure was produced using Molscript (47).
Association rate constants, stoichiometry of inhibition and
Stern-Volmer constants for the 1-AT variants
1-AT states. Each experiment was performed five
times, and the KSV values determined for all the
proteins had errors of less than 0.05. The KSV
determined for
1-AT(P1=Arg) was for the exposure of
the tryptophan residues and for the exposure of the fluorescence probe
in all the other cases.
1-AT structure (Fig.
2). The fluorescently labeled wild type
protein (
1-ATCys-232-IANBD)) had an emission
maximum at 531 nm, whereas the two other proteins exhibited
emission maxima at 527 nm
(
1-AT(S313C-IANBD)) and 532 nm
(
1-AT(P3'Cys-IANBD)) (Fig. 2). These spectra
indicate that the environment of the fluorescence probes at Cys-232 and P3'Cys are more solvent-exposed than the probe at Cys-313. Although the
labeled proteins were fully active (Table I), there was the possibility
that the presence of the IANBD altered the structure of the native
state. To examine this, we carefully measured the tryptophan emission
spectra and far UV CD spectra for all labeled and unlabeled proteins.
Both the tryptophan emission spectra and far UV CD spectra for the
three labeled proteins were identical to the unlabeled fluorescence and
CD spectra (data not shown). These data, in conjunction with the full
inhibitory activity of the proteins, demonstrate that the mutations and
the covalent modification had no significant effect upon the native
structure of the proteins.
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Fig. 2.
Normalized fluorescence emission spectra of
the three fluorescently labeled proteins. The emission spectra
( ex = 480 nm) of 200 nM
1-AT(P3'Cys-IANBD) (
),
1-AT(Cys-232-IANBD) (- - - - -), and
1-AT(Cys-313-IANBD) (
) were
recorded using a bandpass of 2.5 nm at a temperature of 25 °C.
1-AT variants allows the solvent induced
denaturation of
1-AT to be followed spectrofluorometrically. The fluorescence properties of both the tryptophan residues and the covalently attached probe were monitored following treatment with various concentrations of Gdn HCl. Incubation of the proteins in 6 M Gdn HCl resulted in the emission
maximum of the tryptophan residues (for all three proteins) becoming
red shifted from 337 to 352 nm, concomitant with an increase in
fluorescence emission intensity (Fig.
3A). This demonstrates that
the tryptophan residue fluorescence is heavily quenched in the native
structure. The emission spectra of the probes under the same conditions
showed a red shift to 538 nm and a decrease in fluorescence intensity (Fig. 3B).
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Fig. 3.
Native and denatured fluorescence emission
spectra of
1-AT(Cys-232-IANBD).
A, Trp emission spectra (
ex = 290 nm) of
native (
) and denatured (- - - - -) (6 M Gdn HCl)
1-AT(Cys-232-IANBD). B, probe
emission spectra
ex = 480 nm of the native (
) and
denatured (- - - - -)
1-AT(Cys-232-IANBD). The bandpass was 2.5 nm, and the temperature was 25 °C for both sets of spectra.
max as a function of denaturant concentration for both
the tryptophan residues and the probes. There was little change in the
max from the tryptophan residues below 1 M
Gdn HCl for all the variants. Between 1 and 3.5 M Gdn HCl,
max increased (Fig. 4A), indicating an
increase in solvent exposure of the tryptophan residues as the protein unfolds. The spectral changes associated with denaturation of the
modified proteins were unique to the probe position (Fig. 4B).
1-AT(Cys-232-IANBD) showed
no change in emission maximum (
max = 531 nm) until the
concentration of Gdn HCl reached 2 M. This is a 2-fold
increase in Gdn HCl concentration compared with the concentration
required to initiate changes in the tryptophan residue fluorescence.
The fluorescence changes of
1-AT(P3'Cys-IANBD) and
1-AT(S313C-IANBD) occurred at much lower Gdn
HCl concentrations (< 0.5 M).
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Fig. 4.
The apparent fraction of unfolded
protein as a function of Gdn HCl concentration. The fraction of
unfolded protein was determined as a function of Gdn HCl for
1-AT(P1=Arg) (
),
1-AT(Cys-232-IANBD) (
),
1-AT(P3'Cys-IANBD) (×), and
1-AT(S313C-IANBD) (
). The raw
fluorescence emission data were converted to plots of Fapp
versus denaturant concentration as described previously
(33). A, unfolding was monitored by changes in the Trp
residue fluorescence emission maximum (
ex = 290 nm).
B, unfolding was monitored by changes in the probe
fluorescence emission maximum (
ex = 480 nm). The
lines show the two-state and three-state curves fitted as
described under "Experimental Procedures." A two-state model was
used for all fits except for the probe fluorescence change of
1-AT(P3'Cys-IANBD), where a three-state
model was employed.
1-AT exhibited the
same tryptophan fluorescence change as a function of Gdn HCl
concentration, with a midpoint of 2.5 M Gdn HCl (Fig.
4A, Table II). Analysis of
these data using a two-state folding model allows calculation of
GF
U, (the free energy of unfolding in water) and
m (a constant that is proportional to the increase in degree
of exposure of the protein on denaturation) (Table II). It can be seen
that
GF
U is the same for both the unlabeled and
labeled proteins, with an average value of 2.22 ± 0.03 kcal/mol.
In addition, there is no significant difference in m with an
average value of 0.885 ± 0.02 kcal/mol/M. The
coincidence of these labeled and unlabeled data suggests that the
fluorescent probes do not perturb the equilibrium-unfolding pathway.
Parameters for the two- and three-state fits to the denaturation
transition curves
GF
U,
GF
I, and
GI
U
represent the free energy change for the unfolding of the protein in
water and the free energy change for the unfolding of the protein in
water and the free energy change for the F
I and I
U
transitions, respectively. mF
U, mF
I, and
mI
U represent the m values for the F
U, F
I, and I
U
transitions, respectively. D50% F
U,
D50% F
I, and D50%
I
U represent the midpoint of denaturation for the F
U,
F
I, and I
U transitions respectively. Presented are the two- and
three-state analyses of the fluorescence probe data (Fig.
4B) and the two-state analysis of the tryptophan
fluorescence data (Fig 4A) and the three-state analysis of
the far-UV CD data (Fig. 5).
1-AT(S313C-IANBD) and
1-AT(Cys-232-IANBD) could be adequately
described by the two-state analysis, whereas
1-AT(P3'Cys-IANBD) could only be described
using a three-state analysis. The data in Table II and Fig.
4B show that
1-AT(Cys-232-IANBD) has the highest denaturation midpoint (2.8 M Gdn HCl),
compared with the tryptophan observed transition (2.5 M Gdn
HCl). This indicates that the regions around the tryptophan residues
and Cys-232 are significantly more resistant to denaturation than the
regions around P3'Cys and Cys-313. The denaturation midpoint for
1-AT(Cys-313-IANBD) is 1.7 M,
over 1 M lower than the value obtained for
1-AT(Cys-232-IANBD). The midpoint for the
first transition of
1-AT(P3'Cys-IANBD) is
0.4 M Gdn HCl, whereas the midpoint of the second
transition is 2.5 M Gdn HCl. This is similar to the values
obtained from the tryptophan residue and
1-AT(Cys-232-IANBD) analysis, suggesting
that they may be monitoring the same transition. These data also imply
that the regions around both P3'Cys and Cys-313 are relatively
unstable. In contrast, the region around Cys-232 and the tryptophan
residues unfold at higher Gdn HCl concentrations, demonstrating an
increased stability compared with the other regions. The sequential
spectral changes demonstrate that
1-AT unfolding is not
a simple two-state process, in which only the fully folded and unfolded
states exist. In this case, there appears to be at least one
intermediate state, and the protein only becomes fully unfolded at
concentrations above 5 M Gdn HCl.
1-AT(P3'Cys-IANBD); however, the midpoints
of the transitions were approximately 0.2 M higher when
measured by far UV CD. We cannot discount the possibility that minor
local perturbations of the
1-AT structure due to
labeling may have occurred. However, the coincidence of 1) tryptophan
unfolding data (Fig. 4A), 2) far UV CD unfolding data (Fig.
5), 3) tryptophan emission spectra (data not shown), and 4) far UV CD
spectra (data not shown) of the labeled proteins compared with native
1-AT strongly suggests that the probes do not interfere
with the equilibrium unfolding process.
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Fig. 5.
The far UV CD unfolding profile of labeled
and unlabeled 1-AT. The
changes in far UV signal at 222 nm were measured as a function of
Gdn HCl concentration for
1-AT(P1=Arg)
(
),
1-AT(Cys-232-IANBD) (
),
1-AT(P3'Cys-IANBD) (×), and
1-AT(S313C-IANBD) (
). A three-state
unfolding model was used for the analysis of the data.
1-AT, and then the changes in the tryptophan and probe
emission intensity were monitored. The Stern-Volmer plot for both the
tryptophan residues and IANBD labeled proteins were linear over the
concentration range studied (Fig. 6.) At
1 M Gdn HCl, the tryptophan residues became far more
accessible to iodide than in the native state, with an almost 2-fold
increase in Ksv (Table I). Similar increases in
Ksv were also seen for
1-AT(Cys-232-IANBD) and
1-AT(P3'Cys-IANBD) in 1 M Gdn
HCl. Interestingly, although the spectral properties of
1-AT(S313C-IANBD) in 1 M Gdn HCl
changed, there was no change in Ksv (Table
I).
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Fig. 6.
Stern-Volmer plot for iodide quenching of the
tryptophan fluorescence of
1-AT(P3'Cys-IANBD).
1-AT(P3'Cys-IANBD) (200 nM) was
titrated with KI, and the fluorescence emission intensity
(
ex = 290 nm;
em = 337 nm) was recorded
after each addition. In the presence of 1 M Gdn HCl, KI was
added to individual samples so that the Gdn HCl was not diluted.
represents the states of native
1-AT(P3'Cys-IANBD), and
represents
1-AT(P3'Cys-IANBD) in the presence of 1 M Gdn HCl. The lines represent a least-squares
fit of the experimental data as described previously (34)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-Antitrypsin--
Investigations into the pathways by
which globular proteins fold are essential to understanding their
structural organization. This information is also critical if we are to
understand the molecular basis for protein misfolding diseases. In this
study, we have characterized the unfolding pathway of
1-AT using a combination of spectroscopic techniques. We
have placed the fluorescent probe, IANBD, in unique positions within
1-AT to obtain information about the movement of
specific structural motifs as the protein unfolds. This was achieved by
inserting cysteine residues at selected sites and then covalently
modifying them with the cysteine reactive probe IANBD. The positions of
the fluorescent probes used in this study are shown in Fig. 1. P3'Cys
is situated on the first strand of the C
-sheet (s1C) (Fig. 1).
Chang et al. (11) immobilized s1C using an engineered
disulfide bond and found that polymerization did not occur, indicating
that mobility of s1C is essential for this process. Cys-232 is the
native cysteine residue and is exposed at the rear of the molecule on
the B
-sheet (s2B) (Fig. 1). Cys-313 is located at the base of the
serpin molecule on a loop connecting strands 5A and 6A of the A
-sheet (Fig. 1). A detailed structural comparison between native and
cleaved
1-AT showed that this region moves upon reactive
center loop insertion into the A
-sheet (36); therefore, this probe
will monitor changes in the A
-sheet and in the surrounding regions.
The fluorescent properties of the native tryptophan residues (194 and
238) were also used in this study. Trp-194 is at the top of s3A and is
sandwiched between the A and B
-sheets, whereas Trp-238 on s3B is
partially exposed to the solvent. Previous studies have shown that
Trp-194 is the major contributor to the fluorescence properties of
1-AT (3, 37).
1-AT are reporting local structural changes.
Therefore,
1-AT unfolds via a series of structures. It
can be argued that the presence of the fluorescence probes themselves
affects the
1-AT unfolding pathway. We cannot entirely
discount this; however, we have shown that the tryptophan residue and
far UV CD spectral changes for the labeled proteins are
indistinguishable from the native protein during unfolding.
Furthermore, the labeled proteins are fully active as inhibitors.
Although small perturbations due to the presence of the probes cannot
be ruled out, we believe that the probes are reporting genuine
structural changes.
1-AT (17) in conjunction with the fluorescence and far
UV CD data allows insight into the structural changes as
1-AT unfolds. It is evident that the probes placed at
P3'Cys and Cys-313 are in regions of structure that are significantly
less resistant to denaturation than the structural areas around Cys-232
and the tryptophan residues (Fig. 4, A and B).
The midpoint of unfolding for both
1-AT(P3'3C-IANBD) (first transition) and
1-AT(S313C-IANBD) occurs at significantly
lower Gdn HCl concentrations than the corresponding values for
1-AT(P1=Arg) and
1-AT(C232S-IANBD). It has previously been
shown that conformational changes in the reactive center loop and s1C
cause a movement at the base of the serpin (36). Therefore, the initial
unfolding event, which occurs at low Gdn HCl concentrations, may
represent a movement in the reactive center loop and an opening of the
A
-sheet. This would alter the spectral properties of the
fluorescence probes at positions P3'Cys and Cys-313. Conformational
changes in these areas would be predicted not to affect the region
around Cys-232, and indeed the emission maximum of Cys-232 was
unaffected. The circular dichroism of the structure at 1 M
Gdn HCl in the far UV (222 nm) is decreased approximately 20% (Fig.
5), suggesting partial unfolding and disruption of some secondary
structure. This decrease may represent a local loss of secondary
structure as indicated by the changes in the fluorescence of the probes
at positions P3'Cys and Cys-313. There is a small change (< 8%) in
the environment of the tryptophan residues at 1 M Gdn HCl.
Kwon et al. (37) predicted that the opening of the A
-sheet would make Trp-194 more accessible to solvent and hence alter
its fluorescence properties. This was confirmed by the 2-fold increase
in the Stern-Volmer constant of the tryptophan residues (Table I).
Complete disruption of the A
-sheet has been shown to account for
the change in tryptophan fluorescence (37); therefore, the small change
we observed at low denaturant concentrations may represent a prelude to
this conformational change.
-sheet (Cys-232). These data
demonstrate that the B
-sheet region is the most resistant to
denaturant. The pathway of conformational change suggested by these
fluorescence data begins with an opening at the top of the A
-sheet
and a movement in s1C to form a stable intermediate. This is then
followed by the unfolding of the A and C
-sheet regions, which in
turn leads to loss of the B
-sheet and formation of the unfolded structure.
1-AT and other serpins can undergo a conformational
switch termed the stressed (native) to relaxed (latent) transition.
This transition is crucial to the inhibitory properties of the serpin family (5, 39). The area that holds the molecule in this trapped,
stressed state has been located to a region within the hydrophobic core
of the molecule (37, 39). Through side chain locking, these core
residues prevent the rearrangement of native
1-AT into a
more stable but inactive state (39). Our study suggests that the
residues that make up this core around the B
-sheet are unaffected
by low denaturant concentrations. Therefore, this arrangement is intact
in the intermediate structure formed in 1 M Gdn HCl. Once
the denaturant concentration is increased, the residues within the core
region become disordered and the molecule unfolds completely. These
data suggest that regions within the serpin molecule have evolved that
are significantly more resistant to denaturant than others; this has
implications for both the inhibitory mechanism and polymerization process.
-sheet. The extent of this
insertion is unknown; however, biophysical studies have shown that the
effect is to move the proteinase from the top of the molecule to a
position probably on the face of the A
-sheet (40, 41). The
unfolding results presented above explain how
1-AT is
designed to undergo these conformational changes. We show here that the
regions around the top and bottom of the A
-sheet are less stable
than the other parts of the molecule, unfolding at lower Gdn HCl
concentrations. Comparison of x-ray crystallographic structures of
native and cleaved serpins show that the area around Ser-313 moves upon
either full (complete A
-sheet opening) or partial (partial A
-sheet opening) reactive center loop insertion (36). Furthermore,
the x-ray crystal structures of latent antithrombin (10) and
plasminogen activator inhibitor-1(42) demonstrate the mobility of s1C.
The B
-sheet represents a stable platform for these conformational changes, and our study shows that this is a far more stable region in
the protein (unfolding with a midpoint at 2.8 M Gdn HCl).
The data presented herein show that the region around the top of the A
-sheet of
1-AT is more flexible and facilitates
movement of the reactive center loop into the A
-sheet once the
proteinase is bound.
-sheet is more flexible
than the rest of the molecule, and this is required for correct serpin
function. However, if this instability is enhanced due to mutations,
this can induce the unwanted effect of polymerization.
1-AT polymerization in vivo leads to a plasma
deficiency of the inhibitor and eventually to lung and liver damage (6,
43). A number of mutations have been identified that cause the A
-sheet movement to be disturbed (9, 44, 45). An alternative
explanation may be that these mutations alter further the
conformational instability of this region, which would lead to an open
A
-sheet, and therefore increased chances of polymerization and disease.
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ACKNOWLEDGEMENTS |
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We thank Drs. Carla Chinni and Robert Pike for critical reading of the manuscript and James Irving for producing Fig. 1.
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FOOTNOTES |
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* This work was supported by Australian Research Council Grant A0972079 and a Monash Medicine Faculty Research Initiatives grant (to S. P. B.).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.
Supported by the Monash Research Fund.
¶ To whom correspondence should be addressed. Tel.: 61-3-99053703; Fax: 61-3-99054699; E-mail: Steve.Bottomley{at}med.monash.edu.au.
2 The nomenclature for residues within the reactive center loop is based on that described by Schechter and Berger (46) for the substrates of proteases.
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ABBREVIATIONS |
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The abbreviations used are:
1-AT,
1-antitrypsin;
IANBD, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
ethylene diamine;
Gdn HCl, guanidine hydrochloride;
ex, excitation wavelength;
em, emission wavelength;
max, emission maximum wavelength;
Ksv, Stern-Volmer constant;
F, folded form;
U, unfolded form;
I, intermediate form.
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REFERENCES |
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