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INTRODUCTION |
Protein-disulfide isomerase
(PDI)1 is a multifunctional
enzyme that both catalyzes the formation of disulfide bonds (1, 2) and
acts as a subunit of prolyl-4-hydroxylase (3). PDI has been proposed to
function as a molecular chaperone by binding to unfolded protein
species, thereby preventing aggregation and misfolding (4, 5). Despite
these interesting studies, the situation is complicated because PDI,
unlike other chaperones, catalyzes disulfide bond formation and reduction.
Several laboratories have attempted to show the site of binding of
small molecular weight polypeptides that compete with misfolded protein
substrates. Mutated PDI with the carboxyl terminus deleted shows
neither peptide binding nor chaperone activity in assisting the
refolding of denatured D-glyceraldehyde-3-phosphate
dehydrogenase (6). On the other hand, it has been reported that
deletion of the carboxyl-terminal domain (C domain) has no inhibitory
effect on the assembly of recombinant prolyl-4-hydroxylase (7). Other investigators have reported that small molecular weight peptides bind
to the b' domain of PDI (8).
The possibility exists that more than one site in the structure of PDI
is involved in recognition and refolding of protein substrates. The
binding and hydrolysis of ATP by PDI has been reported by Guthapfel
et al. (9); strikingly, the ATPase reaction is stimulated in
the presence of denatured polypeptides, whereas the disulfide oxidation
of PDI is not influenced by ATP. However, the functional role played by
ATP hydrolysis during the refolding of denatured proteins has not been
investigated in detail.
The aim of the present work is 2-fold: first, to study regions of the
primary structure of PDI involved in recognition of unfolded protein
substrates and, second, to investigate whether the free energy of
hydrolysis of ATP is required for unfolding of misfolded protein substrates.
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EXPERIMENTAL PROCEDURES |
Purification of Proteins--
PDI was purified by the method
described in Ref. 10 with small modifications. Fresh porcine livers
(600 g) were homogenized in 0.1 M phosphate (pH 7.5)
containing 1% Triton X-100 and 5 mM EDTA. After
centrifugation, the supernatant was treated with ammonium sulfate, and
the fractions obtained between 55-85% saturation were suspended in 25 mM citrate buffer (pH 5.3), dialyzed against the same
buffer (buffer A), applied to CM-Sephadex C-50 column and eluted with
the same buffer. Fractions displaying PDI activity were pooled,
dialyzed against 20 mM sodium phosphate (pH 6.3) (buffer
B), and applied to a DEAE-Sepharose fast flow column, which was eluted
using a linear gradient of 0-0.7 M NaCl in buffer B. Purified PDI was kept at 4 °C and used in subsequent studies.
The concentration of PDI was determined using absorbance at 280 nm = 1 for 1 mg of protein/ml (11). The activity of PDI was determined
using the insulin reduction assay as described in Ref. 11.
The Escherichia coli GroEL-GroESL gene (12), inserted in a
plasmid provided by Dr. F. Larimer (Oak Ridge Laboratories), was
expressed in E. coli strain BL 21 (DE 3) cells. The protein GroEL was purified by modifications of the procedure included in Ref.
13. After ammonium sulfate fractionation, the protein was purified by
means of three chromatographic steps: DEAE-Sephacel, gel filtration
through Sepharose CL-4B, and affinity chromatography through red
agarose (14). The last step removes contaminating proteins trapped by
GroEL. The protein concentration was calculated using absorbance at 280 nm = 0.15 for 2.5 mg of protein/ml.
Porcine heart mMDH was purchased from Roche Molecular Biochemicals. The
enzyme was dialyzed against 20 mM Tris/HCl buffer, pH 7.5, at 4 °C, applied to a DEAE-cellulose column, and eluted by means of
a linear gradient made with the equilibrium buffer (20 mM)
and the same volume of 100 mM Tris/HCl (pH 7.5). The active fractions were concentrated by ultrafiltration. The enzyme
concentration was calculated using A280 = 2.5 for a 1% solution. (15).
Denaturation of mMDH--
A solution of MDH (5 mg/ml) was
prepared in 100 mM Tris/HCl, pH 7.5, containing 3 M GdnHCl and allowed to denature at 25 °C for 1 h.
The protein solution was 5-fold diluted with 100 mM
Tris/HCl, pH 7.5, and then passed through a Sephadex G-25 column to
remove GdnHCl. Renaturation of MDH was initiated by diluting denatured protein (0.1 µM) in renaturation buffer and incubated for
3 h at 25 °C. The renaturation buffer consisted of 100 mM Tris/HCl, pH 7.5, dithiothreitol (1 mM), and
KCl (0.1 M) with and without chaperone proteins. Aliquots
of this renaturing reaction were withdrawn at specific times and
assayed for MDH activity. The assay buffer consisted of 100 mM Tris/HCl, pH 7.5, 0.5 mM oxaloacetate, and
0.2 mM NADH. The initial rate of conversion of NADH to NAD was determined by measuring changes in absorbance for 1 min at 25 °C.
Materials--
Porcine livers were obtained from a local
slaughter house. DEAE-Sephacel, DEAE-Sepharose, CM-cellulose,
DEAE-cellulose, and Sephadex-G-25 were purchased from Amersham
Pharmacia Biotech. Insulin, GSH, NADPH, DTT, gluthione reductase, and
scrambled RNase A were purchased from Sigma. The polypeptide mastoparan
was obtained from Sigma. The reagent iodoacetamide fluorescein was
purchased from Molecular Probes, Inc. (Eugene, OR).
Labeling of PDI--
PDI (1 mg/ml) was reacted with
iodoacetamide fluorescein (IAF) at a final concentration of 0.1 mM in 0.1 M potassium phosphate buffer (pH 7.4)
containing 0.5 M GdnHCl at 4 °C for 12 h. Under this set of experimental conditions, SH groups located in the central
domain of the protein are exposed to the alkylating reagent.
The labeled protein was dialyzed against 0.1 M potassium
phosphate (pH 7.4) to remove unreacted dye, followed by gel filtration chromatography on Sephadex-G-25. The labeled protein displayed catalytic activity when assayed using insulin as a substrate, suggesting that alkylation on the thioredoxin domain has not taken place. The degree of labeling (1.3 mol of IAF/mol of monomer) was
determined by using an extinction coefficient of 4.9 × 104 M
1 cm1 at 490 nm.
Purification of the Peptide Containing the Modified
Residue--
The modified protein was denatured in 0.8 ml of 6 M guanidinium chloride containing dithiothreitol (1 mM) for 1 h at 37 °C. A freshly prepared solution
of 20 mM iodoacetic acid was then added, and the mixture
was incubated in the dark at room temperature for 30 min. The mixture
was then dialyzed against 2 liters of 0.1 M ammonium bicarbonate.
The labeled protein (100 nmol) was suspended in 0.8 ml of 0.1 M ammonium bicarbonate, pH 8, and digested with trypsin for 24 h at 37 °C at a substrate/trypsin ratio of 40:1 (by mass). To 0.8 ml of tryptic digest, 50 µl of acetic acid was added, and the
precipitate was removed by centrifugation. The solution was then
lyophilized, and the peptides were separated by reverse-phase HPLC
(Vydac C18 column). The separation was performed with a
linear gradient of 10-80% B over 70 min at a flow rate of 0.5 ml/min. Eluant A was 0.1% trifluoroacetic acid, and eluant B was 0.1% trifluoroacetic acid in 80:20 acetonitrile/H2O. Absorbance
was monitored at 220 nm, and fluorescence was monitored at 535 nm for
the detection of labeled peptides. The fluorescent peptides were
further purified with a linear gradient of 5-60% B over 30 min at a
flow rate of 0.5 ml/min. The sequence of the isolated peptide, labeled
with fluorescein, was determined by Edman degradation using an Applied
Biosystems model sequencer (model 492 cLC).
Fluorescence Spectroscopy--
Emission spectra were recorded in
a Perkin-Elmer LS-50B spectrofluorimeter. For fluorescein-labeled
protein, the excitation was 480 nm, whereas for unlabeled proteins the
excitation was set at 295 nm. Excitation and emission slits were set at
2.5 nm. The results of the fluorescence titration experiments were
fitted to Equation 1 as follows,
![&agr;/1−&agr;=[<UP>mMDH</UP>]F/K<SUB>D</SUB>](/content/vol274/issue46/fulltext/32757/img001.gif)
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(Eq. 1)
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Where 
= (F 
Fo)/(FM Fo). F is the observed fluorescence,
Fo and FM the fluorescence
intensities of free and bound IAF-PDI, respectively. In the analysis of
the results, it was assumed that the stoichiometry of binding is 1 mol
of IAF-PDI/1 mol of denatured mMDH.
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RESULTS |
Reactivation of Partially Folded mMDH--
Since PDI is a
multifunctional enzyme endowed with ATPase and chaperone activities, it
was thought to be of interest to investigate whether both catalytic
activities are linked during the process of refolding of a protein
substrate. Is the ATPase activity enhanced during the refolding of the
protein substrate? As a protein substrate of PDI, we have chosen an
intermediate of denatured mMDH generated by GdnHCl denaturation of the
wild type protein. Partially folded mMDH, containing 12% -helix,
exhibits exposed hydrophobic amino acid residues and is devoid of
catalytic activity (15). Moreover, the unfolded conformations of mMDH
recognizes GroEL and regains a good deal of its catalytic activity
(90%) in the presence of Mg ATP (16-18).
The results included in Fig. 1 show the
time course of recovery of dehydrogenase activity in the presence of 1 mM DTT. Under this set of experimental conditions, the
recovery of catalytic activity is due to spontaneous refolding of the
protein after reduction of disulfide bonds generated during GdnHCl
treatment (15). A significant recovery of catalytic activity was
observed in the presence of increasing concentrations of PDI. Maximum
recovery of dehydrogenase activity, which amounts to approximately 50% of the wild-type protein, takes place when the concentration of PDI
(2.2 µM) was 20-fold higher than the concentration of the protein substrate. A further increase in PDI concentration has no
effect either on the rate or extent of recovery of catalytic activity.
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Fig. 1.
Reactivation of mMDH assisted by PDI.
mMDH was denatured in 3 M guanidinium chloride as described
under "Experimental Procedures." Refolding was initiated by
diluting partially folded mMDH to 0.1 µM in 0.1 M Tris/HCl (pH 7.5) containing DTT (1 mM), 0.1 M KCl, and various concentrations of PDI. The mixtures were
incubated at 25 °C, and aliquots were withdrawn at the indicated
times for enzymatic assays. Spontaneous refolding of partially folded
mMDH in the presence of DTT (1 mM) ( ) is shown. Results
were obtained in the presence of PDI (0.5 µM) ( ) and
PDI (2.2 µM) ( ). The effect of the addition of Mg-ATP
(1 mM) to the refolding mixture containing mMDH (0.1 µM), PDI (2.2 µM), and DTT (1 mM) is shown (+). Reactivation of denatured mMDH (treated
with guanidinium chloride) upon dilution with the renaturation buffer
containing DTT (1 mM) and PDI (2.2 µM) is
shown ( ).
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No antichaperone activity was detected at PDI concentrations above 2.0 µM.
When similar reactivation experiments were performed in the presence of
Mg-ATP (1 mM) at the optimal mixing molar ration of PDI/protein substrate of 20:1, the final recovery of catalytic activity
was practically identical to that observed in the absence of Mg-ATP
(Fig. 1).
To ascertain whether the binding of the protein substrate influences
ATPase activity displayed by PDI, the hydrolysis of ATP (1 mM) in the absence and presence of partially folded mMDH
was measured using a coupled enzymatic assay consisting of pyruvate kinase and lactate dehydrogenase.
As shown by the results included in Fig.
2, the ATPase activity
(kCAT = 0.26 min1) characteristic
of PDI remains practically invariant upon increasing the concentration
of partially folded mMDH from 2 to 8 µM. In marked
contrast to the lack of inhibition of ATPase activity caused by the
binding of mMDH, the glutathione-dependent reduction of insulin is strongly inhibited by small concentrations of partially folded mMDH as shown in Fig. 2. This finding suggests that insulin and
denatured mMDH might share common binding sites on PDI.
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Fig. 2.
Effect of partially folded mMDH on the
catalytic activities of PDI. ATPase activity of the enzyme in the
presence of increasing concentrations of partially folded mMDH. ().
Measurements were made using the pyruvate-kinase-lactate
dehydrogenase-coupled assay (9) in the presence of 0. 1 mM
ATP. , glutathione-dependent reduction of insulin
measured in the presence of increasing concentrations of partially
folded mMDH.
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Although partially folded mMDH has been used in most of the studies
reported in this work, it should be emphasized that the rate of
reactivation of malic dehydrogenase is not affected by the nature of
the initial unfolded species used in the refolding experiments. Indeed,
the kinetics of reactivation of partially folded mMDH, prepared as
described under "Experimental Procedures," is similar to that
observed when 3 M guanidinium chloride-treated enzyme is
immediately diluted into the renaturation buffer containing PDI (Fig.
1).
Recognition of PDI by Denatured mMDH--
PDI contains
tryptophanyl residues, which exhibit a structureless emission band
centered at around 340 nm when excited at 295 nm. The intensity of the
emission band is enhanced by the addition of equimolar amounts of
denatured mMDH (Fig. 3). mMDH does not
contain tryptophanyl groups; and under the experimental conditions
chosen for the luminescence experiments, the enzyme does not display
any emission band centered at 340 nm. A gradual increase in
tryptophanyl fluorescence is observed when the concentration of
denatured protein varies from 0 to 1.5 µM. As shown in
the inset of Fig. 3, the fluorescence intensity reaches a
maximum value at a mixing molar ratio of the proteins of approximately 1:1, taking the molecular masses of mMDH and PDI as 35 and 55 kDa,
respectively.
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Fig. 3.
Interaction between PDI and mMDH. Shown
are the emission spectra of PDI (0.96 µM) in the absence
(curve 1) and presence of denatured mMDH (1.36 µM) (curve 2) in 0.1 M
Tris/HCl buffer (pH 7). The spectrum of the mixture was recorded 2 min
after mixing. A sample of denatured mMDH (1.36 µM) does
not show any emission band upon excitation at 295 nm, the wavelength
selected for these experiments. Inset, titration of PDI (0. 96 µM) with increasing concentrations of denatured mMDH
(). The fluorescence intensity was recorded at 350 nm immediately
after the addition of mMDH. Excitation wavelength was 295 nm.
F/Fo is the fluorescence ratio in the
presence (F) and absence (Fo) of
denatured mMDH. Included is the effect of increasing concentrations of
native mMDH on the fluorescence emitted by PDI ().
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In order to determine the affinity of mMDH for PDI at a concentration
comparable with those used in the reactivation experiments, it was
desirable to label PDI with a fluorescent probe characterized by high
fluorescence yield at protein concentrations ranging from 0.05 to 0.1 µM. Derivatized IAF-PDI exhibits a maximum of
fluorescence at around 535 nm due to the presence of the fluorescence
chromophore. The fluorescence quantum yield of the probe
(Q = 0.35), excited in the spectral region (460-480
nm) located far away from the aromatic amino acid residues tyrosine and
tryptophan, can be used to detect the presence of protein-protein
complexes in solution. Moreover, IAF is bound to cysteinyl residues,
which can be localized on the polypeptide chain by sequencing the protein.
The emission of the chromophore excited at 480 nm is increased upon the
addition of increasing concentrations of denatured proteins. Fig.
4 shows the emission spectra of IAF-PDI
(0.96 µM) recorded after the addition of denatured mMDH
and scrambled RNase A. To measure the affinity of IAF-PDI for partially
folded mMDH, mixtures containing IAF-PDI (0.05 µM) and
increasing concentrations of denatured protein were allowed to
equilibrate for 2 min at 25 °C, and their spectra were immediately
recorded. As shown in Fig. 4, inset, the fluorescence
emitted by IAF-PDI is increased until it remains practically constant
when the concentration of partially folded mMDH is greater than 0.6 µM. Assuming a stoichiometry of binding of 1:1, the
dissociation constant of the protein-protein complex was estimated to
be 0.2 µM.
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Fig. 4.
Binding of IAF-PDI to mMDH. Samples of
IAF-PDI (0.96 µM) were mixed with increasing
concentrations of denatured proteins, and their spectra were
immediately recorded upon excitation at 480 nm in 0.1 M
Tris/HCl buffer (pH 7). From bottom to top,
spectra recorded in the presence of 0, 10, and 20 µM
scrambled RNase and 0.6, 0.8, and 1 µM partially folded
malate dehydrogenase. Inset, titration of IAF-PDI at a
concentration of 0.05 µM with increasing concentrations
of denatured mMDH (). The samples were mixed for 2 min before
recording their emission intensity at 535 nm upon excitation at 480 nm.
An apparent KD = 0. 2 µM is obtained
by fitting the fluorescence data to Equation 1. Included are the
results obtained when IAF-PDI is incubated with increasing
concentrations of native mMDH ().
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Sequence of the Labeled Peptide--
To ascertain whether labeling
of PDI by the extrinsic probe IAF has taken place in the central
domains of the protein, derivatized PDI was subjected to trypsin
digestion, and the peptides were separated by HPLC as outlined under
"Experimental Procedures." Upon separation of tryptic peptides by
HPLC using a reverse-phase column, one major labeled peptide was
identified (Fig. 5).
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Fig. 5.
Separation of tryptic peptides from modified
PDI by reverse-phase HPLC. Separation was performed with a linear
gradient of 10-80% B over 70 min at a flow rate of 0.5 ml/min.
Elution was monitored at 220 nm. Fractions of 0.5 ml were collected,
and each fraction was analyzed by fluorescence measurements using an
excitation wavelength of 480 nm and emission wavelength of 530 nm. The
fractions showing fluorescence (*) were pooled, lyophilized, and
purified by reverse-phase HPLC with a linear gradient of acetonitrile
5-60% B over 30 min at a flow rate of 0.5 ml/min. The purified
labeled peptide was used for amino acid sequencing.
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Rechromatography of the labeled peptide with the same column using a
different solvent composition resulted in further purification. The
fractions, characterized by a fluorescence maximum at 535 nm, due to
the presence of the extrinsic chromophore fluorescein, were pooled,
lyophilized, and sequenced. Edman degradation gave the following amino
acid sequence: Ile-Thr-Glu-Phe-X-His-Arg. Comparison of the
determined sequence of the modified peptide with the reported primary
structure of human PDI (19) indicated that the labeled peptide
corresponds to amino acid residues 322-328 of the human enzyme. The
position indicated as X in the peptide sequence corresponds
to cysteine 326 in the human enzyme. The absence of any labeled peptide
covering the sequence Trp-Cys-Gly-His-Cys-Lys pertaining to the active
site region of thioredoxin, strongly suggests that the reaction with
IAF is restricted to free cysteine groups in the central domain b' of
PDI.
Aggregation State of PDI--
It has been reported that monomeric
species of PDI are in dynamic equilibrium with dimeric and tetrameric
species at pH 7.4 in 0.3 M NaCl (9). The possibility that
high molecular "clusters" of PDI preferentially bind small
molecular weight peptides was investigated by gel filtration
chromatography using 0.1 M Tris/HCl, pH 7, as the mobile
phase. When pure PDI, stored at 4 °C, was submitted to gel
filtration, one major peak was detected. The retention time of PDI is
slightly different from bovine serum albumin (monomeric) characterized
by a molecular mass of 67 kDa (Fig. 6). The addition of the peptide
mastoparan (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu) does not cause any change in the monomeric state of PDI, even at
concentrations required for saturation of the enzyme (20). Hence, PDI
does not undergo a reversible process of association in the absence or
presence of low molecular weight peptides. When the same experiments
were performed with IAF-PDI mixed with mMDH and the elution profiles
were monitored by fluorescence measurements at 535 nm, we were unable
to detect macromolecular species corresponding to tetramers of PDI
(results not shown).
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Fig. 6.
HPLC separation of PDI. Elution profiles
are shown of PDI in the absence (solid line) and
presence of the peptide mastoparan (1 mM) (). 50 µl of
a solution of 2 mg/ml of PDI was applied to a TSK 3000 SW gel
filtration column equilibrated with 0.1 M Tris/HCl (pH 7).
Flow time was 0.3 ml/min. The elution profile of the standard bovine
serum albumin (dashed line) is shown. Two elution
bands are detected in bovine serum albumin corresponding to species of
134 and 67 kDa, respectively. One elution profile is detected in PDI
(55 kDa).
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Our results do not show dimerization or aggregation of PDI at neutral
pH. The discrepancy with other published data must be attributed to the
conditions of storage of purified PDI, since it has been reported that
a metastable dimer is induced by freezing the samples in phosphate
buffer (21).
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DISCUSSION |
Mitochondrial MDH, a dimeric protein, undergoes structural
transitions in solutions containing GdnHCl. In the presence of 3 M GdnHCl, the protein shows little residual secondary
structure; and dilution of the denatured protein with buffer restores a
fraction of the -helix content (15). The partially folded species
recover only 16% of the catalytic activity after the addition of 1 mM DTT. The rate of recovery of catalytic activity of the
dehydrogenase is influenced by the presence of PDI in the renaturation
buffer. Under optimal conditions of temperature (25 °C) and PDI
concentration (2.2 µM), the recovery of activity amounts
to 50% of the wild-type protein. Like other well characterized
chaperone proteins, i.e. GroEL, PDI functions primarily by
unfolding misfolded protein intermediates; but it appears that binding
of the protein substrate to the chaperon would provide the free energy
required for unfolding. Two lines of experimental evidence are
consistent with this hypothesis: first, the rate of reactivation of
partially folded mMDH is not influenced by the presence of Mg-ATP
together with PDI in the refolding buffer, and second, the intrinsic
ATPase activity of PDI is not perturbed by its interaction with the
protein substrate.
In agreement with the results published by other laboratories (16, 17),
it was found that unfolded mMDH recovers a good deal of its catalytic
activity (90%) when the denatured protein substrate interacts with
GroEL and Mg-ATP at equimolar concentrations of the proteins.
In view of these results, it is pertinent to ask why PDI is less
efficient than GroEL in assisting the refolding of partially folded
mMDH. Based on the studies reported by other laboratories on the
binding of small molecular weight peptides to PDI, it has been
suggested that weak affinity of PDI for denatured proteins might
explain low recovery of catalytic activity. Our studies on mMDH
indicate that derivatized IAF-PDI binds denatured mMDH with a
dissociation constant of 0.2 µM, which is 5-fold higher than the dissociation constant determined for denatured mMDH bound to
GroEL in the absence of Mg-ATP (17). Hence, a 20-fold molar excess of
PDI over the denatured protein substrate (0.1 µM) would be sufficient to ensure binding and reactivation of mMDH. However, the
reactivation experiments conducted in the presence of a 20-fold excess
of PDI have shown partial recovery of catalytic activity.
There are some aspects of the interaction of chaperone proteins with
unfolded substrates that should be considered in the analysis of the
behavior of PDI. GroEL interacts with denatured mMDH, and the binding
of Mg-ATP drives the chaperone complex through a functional state in
which refolding of the protein substrate occurs inside the cavity of
GroEL (18). Encapsulation of the protein substrate provides a folding
environment that facilitates conformational arrangements and prevents
no unspecific interactions with other nonnative proteins during the
chaperone cycle.
PDI does not possess a cavity for encapsulation of a protein substrate
of the size of mMDH. On the other hand, the binding of small molecular
weight polypeptides does not promote association of PDI into
"clusters" of large molecular weight that would prevent exposure of
the protein substrate to the surrounding solvent. Several laboratories
have reported that the interaction of misfolded proteins
(i.e. scrambled RNase A) with PDI competes with the binding of peptides (11, 20). Based on these observations, it has been
suggested that the peptide binding site corresponds to a site at which
PDI interacts with unfolded regions of proteins during its action in
the cell. Although the hypothesis is attractive, the domain associated
with peptide binding has not been completely elucidated. Initially it
was reported that deletion of 51 amino acid residues of the C-terminal
domain totally prevented peptide binding and chaperone activity (6).
Subsequent studies have shown that the deletion includes amino acid
residues pertaining to the C-terminal domain and a fraction of the a'
domain (7). More recently, it has been reported that the b' domain
provides the principal peptide binding site of PDI (8). In view of
these observations, it seems very likely that amino acid residues
outside the C domain might contribute to the interaction of PDI with
the protein substrate. Our fluorescence results have shown that binding of the protein substrate causes conformational changes in PDI propagated to different domains of its structure. Not only the fluorescent probe positioned in the b' domain, but also tryptophanyl residues distributed along the a and a domains, sense the
conformational changes induced by the protein substrate. It is
conceivable that the flexibility of the protein plays an important role
in reactions such as isomerization of disulfide bonds and refolding of
protein substrates.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Fax: 852-2766-6692;
E-mail: bcchurch@inet.polyu.edu.hk.
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