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INTRODUCTION |
Dsb family proteins, comprising at least six members (DsbA, DsbB,
DsbC, DsbD, DsbE, and DsbG), have been characterized to be thiol
oxidoreductases in the periplasm of prokaryotic cells and responsible
for the formation of disulfide bonds in newly synthesized proteins
(1-3). Among the Dsb family, DsbC, a homodimer, is homologous, to the
highest extent, with eukaryotic protein-disulfide isomerase
(PDI)1 in terms of biological
properties and functions (4-8). PDI has been characterized to be not
only an isomerase but also a chaperone (9, 10). Recently we have
reported that DsbC shows even more pronounced chaperone activity than
PDI in promoting the in vitro reactivation and suppressing
aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding (11), whereas DsbA shows a
weaker chaperone activity than that of PDI (12). DsbG, strongly homologous to DsbC, has also been characterized to have chaperone activity (13). The crystal structure of DsbC reveals that it is a
V-shaped molecule with each arm of the V, a monomer consisting of a
C-terminal thioredoxin-domain and a N-terminal association domain
connected by a hinged linker (14). DsbC has four cysteines in each
23-kDa subunit. Two in the active site
(Cys98-Gly-Tyr-Cys101) with Cys98
protruded out of the molecule are responsible for the oxidoreductase activity (6). Single replacements of Cys98 with different
residues all result in complete loss of enzyme activities; however, the
contributions of Cys101 to the biological activities were
reported diversely (5, 15). The nonactive site cysteines,
Cys141 and Cys163, were reported to form a
stable disulfide bond somehow buried and disrupted only in the presence
of large excess of dithiothreitol (DTT) (6, 16). Among the members of
the thioredoxin superfamily characterized so far, only DsbC has a
nonactive site disulfide bond, Cys141-Cys163,
which was suggested to play a purely structural role (6, 17). Recent
crystal structure analysis shows that this unique disulfide bond
located in a helical insert within the C-terminal catalytic domain is
partially solvent exposed (14).
DsbC, like other oxidoreductases, exists in both oxidized and reduced
forms. Reduced DsbC is the functional and dominating form (8, 15, 16)
and is regenerated from oxidized form by membrane-bound DsbD (8, 15,
18, 19). Overexpression of DsbC results in the accumulation of oxidized
form due to exceeding the reductive capacity of DsbD (15). Unlike DsbA
(20), reduced and oxidized DsbC show the same intrinsic fluorescence
and CD spectra (6).
In this paper we report that five DsbC mutants, with Cys replaced by
Ser, overproduced in Escherichia coli using a His-tagged signal to direct export as described in our previous work (21), show
different properties in terms of folding, export, solubility, stability, and biological activities compared with wild-type DsbC.
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EXPERIMENTAL PROCEDURES |
DNA Construct--
Primers designed for mutations of Cys at the
positions of 98, 101, 141, and 163 of DsbC into Ser are listed in Table
I. The plasmid BS-DsbC was constructed by insertion of the coding
sequence of DsbC precursor amplified from the plasmid pDsbC, a generous gift from Dr. Rudi Glockshuber (Eidenössische Technische
Hochschule, Hönggerberg, Switzerland), into Bluescript
II/SK(+) (Stratagene) as described previously (21). The preparation of
single-stranded DNA of BS-DsbC and oligonucleotide-directed mutagenesis
in vitro were performed essentially according to Carter
(22). The full-length coding sequences of the corresponding precursors
of the five mutants were confirmed with both strands, and then
subcloned into pQE-30 vector (Qiagen) via BamHI site to
create corresponding expression plasmids pQE-C98S, pQE-C98S/C101S,
pQE-C101S/C163S, pQE-C141S, and pQE-C163S according to Liu et
al. (21), resulting in modified precursors with a
MRGSH6GS-fused signal sequence at the N termini (Table
I).
Expression and Purification--
Transformed M15[REP4] cells
(Qiagen) were grown in 2× YT media with 100 µg/ml ampicillin and 25 µg/ml kanamycin. The overnight culture was diluted 100-fold and
incubated at 37 °C for 2 h followed by induction with isopropyl
-D-thiogalactopyranoside (IPTG) of different
concentrations from 0.001 mM to 1 mM. Osmotic
shock was used to release soluble periplasmic proteins, and the shocked cells were sonicated and centrifuged at 35000 rpm for 90 min at 4 °C. The pellets were treated with 1% Triton X-100 in 50 mM Tris buffer (pH 8.0) to release membrane-bound proteins.
Proteins obtained at each step were analyzed by SDS-PAGE (12%).
-Lactamase (23, 24), NADH oxidase (24), and glucose 6-phosphate
dehydrogenase (25) were used as markers to assign
subcellular-compartments of periplasm, inner membrane, and cytoplasm of
M15[REP4] cells transformed with plasmid pQE-30, respectively.
Processed species of wild-type DsbC prepared from the cells harboring
pQE-DsbC (21) showed two peaks on a Q-Sepharose Fast Flow column
(Amersham Pharmacia Biotech). According to free thiol determination
(26) and native-PAGE analysis (6) of the S-carboxymethylated products, the first eluted peak has been characterized to be oxidized DsbC dimer and the second to be a mixture of oxidized dimer,
heterodimer of oxidized and reduced subunit and reduced dimer. The
oxidized DsbC dimer was used as a control in all experiments if not
specified otherwise.
Induction by 0.01 mM IPTG for 6 h at 37 °C was used
for preparation of processed species. Osmotic shock was used to release periplasmic soluble C98S. To prepare C141S, C163S, and C101S/C163S from
aggregates, the osmotically shocked cells were washed with 1% Triton
X-100 followed by treatment with 8 M urea and 0.5% SDS in
50 mM Tris buffer (pH 8.0) at room temperature for 4 h, and the denatured proteins were renatured by thorough dialysis
against the same Tris buffer. For extracellular C98S/C101S, the
overnight culture was diluted only 1-fold and induced with 0.01 mM IPTG at 37 °C overnight. The cell-free culture was
fractionated with 25% and then 100% saturated
(NH4)2SO4. Proteins obtained as
indicated above were further purified by successive chromatography on
Q-Sepharose Fast Flow and Superose 12 HR 10/30 columns (Amersham
Pharmacia Biotech). All the purified proteins were examined by SDS-PAGE (12%) followed by Western blot. Antiserum was prepared from a New
Zealand rabbit immunized with a mixture of DsbC and the five mutants
extracted from SDS-PAGE gels.
Proteinase K Accessibility--
Cells harboring pQE-C98S,
pQE-C101S/C163S, pQE-C141S, or pQE-C163S were induced with 0.01 mM IPTG at 37 °C for 6 h. The osmotically shocked
cells prepared from 1.5 ml of cell culture was suspended in 0.4 ml 10 mM Tris-HCl (pH 7.5) with 5 mM
CaCl2 and incubated with 0.02 mg/ml proteinase K (Merck) at
25 °C for 1 h. The reaction was stopped by 0.174 mg/ml
phenylmethylsulfonyl fluoride and analyzed by SDS-PAGE (15%) and
Western blot.
In Vitro Unfolding and Refolding--
Proteins were denatured in
0.1 M Tris buffer (pH 7.5) containing different
concentration of guanidine hydrochloride (GdnHCl) overnight at room
temperature and the values of ellipticity at 222 nm
([
]222) were determined.
Refolding of fully denatured proteins prepared by incubation with 6 M GdnHCl overnight at room temperature in the presence or
absence of 0.2 M DTT was initiated by dilution 100-fold
into 0.1 M Tris buffer (pH 7.5) and monitored immediately
at 25 °C by measuring the fluorescence emission intensity at 312 nm
with an excitation at 280 nm.
Other Determinations--
The N-terminal amino acid sequences of
purified mutant proteins were determined on a MilliGen/Biosearch model
660 ProSequencer linked to a Waters HPLC system (Franklin) with
simultaneous detection at 269 and 313 nm.
Protein concentrations were determined by the method of Bradford (27).
DsbC, S-carboxymethylated DsbC, and DsbC mutants were all
considered as protomers in the calculations of molar ratios. Enzymatic
activities were assayed according to oxidative refolding of reduced and
denatured RNase A (28) and reduction of insulin (5, 29). Chaperone
activity was determined according to the assisted-reactivation of
denatured GAPDH upon dilution (11).
To examine the redox state of DsbC overexpressed in the cells cultured
in the media complemented with or without reductants, proteins in two
eluted peaks of Q-Sepharose Fast Flow column were mixed and then
S-carboxymethylated by iodoacetic acid (6). The reaction
mixture after desalting was analyzed by 6% native PAGE (6). A
hybridized product of DsbC and S-carboxymethylated DsbC was
used to mark the net negative charge of 0, 2, and 4 (6).
CD spectra from 200 to 250 nm were determined with a Jasco 720 spectropolarimeter at 25 °C. Intrinsic and
8-anilino-1-naphthalenesulfonic acid fluorescence spectra were
measured in a Shimadzu RF-5301PC spectrofluorometer at 25 °C with
280 and 373 nm for excitation, respectively.
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RESULTS |
Expression and Processing of Wild-type and Mutant DsbC
Proteins--
As shown in Fig. 1,
precursors and processed species of wild-type DsbC and mutants move
with significantly different mobility in SDS-PAGE (12%). Induced with
IPTG at low concentrations of 0.001 and 0.01 mM, all
proteins were efficiently processed, and higher concentrations of IPTG
(
0.1 mM) resulted in the accumulation of unprocessed
species. Wild-type DsbC was overexpressed in the periplasm as a soluble
protein, consisting 20-30% of the total cellular proteins.
C98S/C101S, similar to wild-type DsbC, appeared in the periplasm as a
soluble protein when induced for 4 h, but all secreted or leaked
into the medium if induced overnight (data not shown). More than 180 mg
of processed C98S/C101S was obtained from 1 liter of cell-free culture.
Processed species of C98S was partially released by osmotic shock while
those of C141S, C163S, and C101S/C163S totally aggregated. Decreasing
the yield of synthesis by using 0.001 mM IPTG did not
improve the solubility. All five mutants show the same N-terminal
sequence of Asp-Asp-Ala as that of wild-type DsbC, confirming that the
signal sequences in all mutants have been processed correctly.

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Fig. 1.
Expression and processing of wild-type and
mutant DsbC proteins. Transformed M15[REP4] cells were induced
with IPTG of different concentrations at 37 °C for 6 h except
4 h for C98S/C101S. Proteins were examined by SDS-PAGE (12%).
Panel shows low range molecular weight markers (M); oxidized
DsbC (C), total cellular proteins before (B) and
after induction with IPTG of 1.0, 0.1, 0.01 and 0.001 mM,
respectively (lanes 1-4); proteins in
supernatant (lanes 5-8) and in osmotically
shocked cells (lanes 9-12) after osmotic shock
corresponding to lanes 1-4, respectively. Arrows indicate
the positions of precursors.
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Subcellular Location of Processed Mutants--
As shown in
Table II, the supernatant (S1) after
osmotic shock showed almost all cellular
-lactamase activity,
indicating that the proteins in the supernatant fraction prepared by
the method of osmotic shock are indeed located in the periplasm. The activities of glucose-6-phosphate dehydrogenase and NADH oxidase mainly
appeared in the fractions of sonicated supernatant (S2) and Triton
X-100 extract (S3), respectively, indicating that S2 and S3 were mainly
from cytoplasm and membranes, respectively. Therefore, as shown in Fig.
2 with the optimal induction of 0.01 mM IPTG for 6 h at 37 °C, C98S was expressed partly
as a periplasmic soluble protein, partly as a membrane-bound protein,
and partly as inclusion bodies in the periplasm. Although C163S
and C101S/C163S totally formed inclusion bodies in the periplasm as
they appeared only in the pellet of each preparation step, C141S mainly
formed inclusion bodies in the periplasm with a small part as a
membrane-bound protein.
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Table II
Subcellular compartment marker enzyme activities
M15[REP4] cells transformed with plasmid pQE-30 were fractionated as
described in Fig. 2, and the activities of marker enzymes in the
fractions of S1, S2, and S3 were determined.
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Fig. 2.
Subcellular location of processed DsbC
mutants. Transformed cells were induced with 0.01 mM
IPTG at 37 °C for 6 h. Osmotically shocked cells were sonicated
and centrifuged, and the pellets were treated with 1% Triton X-100 and
centrifuged again as described in the text. Panel shows oxidized DsbC
(C) and total cellular proteins before (B) and
after (A) induction, respectively; supernatant
(S1) and osmotically shocked cells (P1) after
osmotic shock, respectively; supernatant (S2) and pellet
(P2) after sonication, respectively; supernatant
(S3) and pellet (P3) after treatment with Triton
X-100, respectively, on SDS-PAGE (12%).
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Proteinase K Accessibility of the Osmotically Shocked
Cells--
As shown in Fig. 3, after
digestion with proteinase K, the bands of the mutants C98S,
C101S/C163S, C141S, and C163S in the osmotically shocked cells all
disappeared and no new bands with lower molecular weight appeared even
detected with Western blot, providing further evidence that the
insoluble mutants were indeed located in the periplasm and the
membrane-bound C98S and C141S were not imbedded inside the membranes
but likely peripheral inner membrane proteins toward the periplasm.
Many proteins sequestered in aggregates become highly resistant to
proteolysis, but this is not the case for C98S, C141S, C163S, and
C101S/C163S, suggesting that they are in a protease-accessible
conformation.

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Fig. 3.
Proteinase K accessibility of osmotically
shocked cells. Osmotically shocked cells were digested with (+) or
without ( ) proteinase K as described in the text and analyzed by
SDS-PAGE (15%) (panel A) and Western blot
(panel B). Panel shows low range molecular weight
marker (M), oxidized DsbC (C), C98S
(1), C101S/C163S (2), C141S (3), C163S
(4).
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Effect of Reductants on the Formation of
Cys141-Cys163 Disulfide--
The presence of
5 mM DTT or 10 mM N-acetylcysteine
in the culture does not affect the processing, export, solubility, and activity of DsbC (Fig. 4A and
Table III). Under nondenaturing
condition, S-carboxymethylated products of DsbC from the
strains grown in the media with DTT or N-acetylcysteine
showed three bands with net negative charge of 0, 1, and 2, corresponding to the oxidized DsbC, heterodimer of oxidized and reduced
subunits, and reduced DsbC, respectively (Fig. 4B), and
showed about 30% of reductase activity of DsbC (Table III). The
activity might be contributed by the Cys98 originally in
the oxidized form and not alkylated in both oxidized DsbC and oxidized
subunit of the heterodimer. The S-carboxymethylated products
prepared in the presence of 6 M GdnHCl showed three bands with net negative charge of 0, 2, and 4, respectively, because of the
alkylation of Cys101, which was not attacked by iodoacetic
acid under nondenaturing condition. The above indicates that the
nonactive site cysteines, Cys141 and Cys163,
form a disulfide bond even with the cells grown in a reducing culture.

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Fig. 4.
Effects of reductants in the media on the
formation of Cys141-Cys163 disulfide.
Overnight-cultured cells harboring pQE-DsbC were diluted 100-fold in
the absence (N) or presence of 5 mM DTT
(D) or 10 mM N-acetylcysteine
(C), followed by incubation for 2 h, and then induced
with 0.01 mM IPTG at 37 °C for 6 h.
Panel A (12% SDS-PAGE), DsbC (lanes
1-3) from the cells cultured in different media as
indicated was a combination of two peaks from the Q-Sepharose Fast
Flow; total cellular proteins before (lane 4) and
after induction (lanes 5-7); supernatants after
osmotic shock (lanes 8-10); osmotically shocked
cells (lanes 11-13). Panel
B (6% native-PAGE), DsbC from the cells cultured in the
presence of different reductant as indicated was
S-carboxymethylated in 0.1 M Tris-HCl (pH 8.8)
containing 5 mM EDTA with (+) or without ( ) 0.5 M iodoacetic acid (IAA) and 6 M
GdnHCl at room temperature for 30 min. The desalted
S-carboxymethylated products were examined on a native PAGE
as described by Zapun et al. (6). Lane
S is a net charge marker prepared according to Zapun
et al. (6). The digits indicate the net negative
charge of the corresponding bands.
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As shown in Fig. 5, under nondenaturing
condition, the Cys141-Cys163 disulfide bond of
C98S/C101S was only reduced by a large excess of the strong reductant
DTT but not by reduced glutathione (GSH), indicating that this
disulfide, although partially solvent-exposed (14), is stable in a
reductive environment.

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Fig. 5.
Reduction of
Cys141-Cys163 disulfide bond. C98S/C101S
of 2.1 µM was incubated in 0.1 M Tris buffer
(pH 7.5) without (dotted line) or with 0.2 M DTT (dashed line) or 0.15 M GSH (solid line) at 37 °C for
30-60 min and then analyzed on a PepPRCTM column (Amersham Pharmacia
Biotech) with a linear gradient of acetonitrile (35-45% (v/v) in 20 ml) in 0.1% trifluoroacetic acid.
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Enzyme Activities--
As shown in Table III, compared with
wild-type DsbC, C98S and C98S/C101S are devoid of enzyme activities and
C101S/C163S shows approximate 30% reductase activity and 60% activity
for catalyzing the oxidative refolding of RNase A. C141S and C163S show
full enzyme activities of DsbC.
Chaperone Activity--
As shown in Fig.
6, C98S and C98S/C101S, although inactive
as enzymes, show the same ability as that of wild-type DsbC to assist
the in vitro reactivation of denatured GAPDH to 30% at molar ratios larger than 15. C141S and C163S at high concentrations stimulate the reactivation of GAPDH to a lower extent of 15%, and
C101S/C163S to only 10%.

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Fig. 6.
Effect of wild-type and mutant DsbC proteins
on the reactivation of denatured GAPDH upon dilution. The
reactivation of GdnHCl-denatured GAPDH at 140 µM was
initiated by 50-fold dilution into 0.1 M phosphate buffer
(pH 7.5) containing 5 mM DTT, 2.5 mM EDTA, and
oxidized DsbC ( ), C98S ( ), C98S/C101S (×), C141S ( ), C163S
( ), or C101S/C163 ( ) at different molar ratios to GAPDH. The
reactivation mixture was first kept on ice for 30 min and then at
25 °C for an additional 3 h before an aliquot containing 2 µg
of GAPDH was taken for activity assay at 25 °C. The data were the
averages of two independent experiments.
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Unfolding and Refolding--
C98S, C98S/C101S, C141S, C163S, and
C101S/C163S all show similar CD spectra, the same intrinsic
fluorescence maximal emission wavelength of 312 nm and the same
retention time on a Superose 12 HR 10/30 column as that of wild-type
DsbC (data not shown), indicating that the mutations do not affect the
secondary structure in general and the dimerization of the subunits.
As shown in Fig. 7, the Cm
values, GdnHCl concentrations at the midpoints of unfolding curves
measured by the ellipticity at 222 nm, of DsbC, C98S, and C98S/C101S
are 2.19, 2.16, and 1.95 M, respectively, which are
apparently higher than 1.46, 1.47, and 1.08 M for C141S,
C163S, and C101S/C163S, respectively, suggesting that C98S and
C98S/C101S appear to be as stable to GdnHCl denaturation as DsbC, while
the proteins with the nonactive site Cys residues mutated are less
stable. The above suggest that the nonactive site disulfide
Cys141-Cys163 indeed plays an important
structural role in the stability of the DsbC molecule. Moreover, the
Cm values appear to be parallel to the solubility of the
mutants in the periplasm; the higher the Cm, the more stable
and soluble the proteins.

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Fig. 7.
Unfolding of wild-type and mutant DsbC
proteins by GdnHCl. GdnHCl-induced transitions of oxidized DsbC
( ), C98S ( ), C98S/C101S (×), C141S ( ), C163S ( ) and
C101S/C163S ( ) were measured by ellipticity at 222 nm
([ ]222) as described in the text. Data were normalized
using the relation fx = (Yx Yd)/(Yn Yd). Yx, determined
[ ]222; Yn and
Yd, the [ ]222 value of native
and fully denatured proteins, respectively.
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As shown in Fig. 8A, the
refolding rates of denatured C98S and C98S/C101S upon dilution as
monitored by the fluorescence emission intensity at 312 nm are the same
as that of wild-type DsbC, and much faster than that of C101S/C163S,
C141S, and C163S, suggesting that the formation of the nonactive site
disulfide bond may accelerate the folding of DsbC molecule. Denatured
and reduced DsbC, C98S, and C98S/C101S refolded markedly slower than
the corresponding nonreduced species (Fig. 8B), providing
further support to the above suggestion.

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Fig. 8.
Refolding of denatured wild-type and mutant
DsbC proteins upon dilution. Panel A,
proteins were fully denatured and refolded by 100-fold dilution to 4.25 µM as described in the text. The emission fluorescence
intensity at 312 nm with an excitement of 280 nm was monitored
immediately after dilution and normalized using ft
= (It Id)/(In Id): It, observed
fluorescence intensity at the time of t;
In and Id, fluorescence
intensity of native and denatured proteins, respectively.
Curves 1, 2, and 3 are for
oxidized DsbC, C98S, and C98S/C101S, respectively; curve
4 for C101S/C163S; curves 5 and
6 for C141S and C163S, respectively. Panel
B, the fully denatured oxidized DsbC, C98S, and C98S/C101S
were reduced by 0.2 M DTT for 30-60 min at 37 °C and
refolded as described in panel A
(curves 1, 2, and 3,
respectively).
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DISCUSSION |
The DsbC subunit consists of a C-terminal thioredoxin-like domain
with a CGYC- motif as the active site and a unique nonactive site
disulfide in a helical subdomain and N-terminal association domain for
dimerization (14). The present results have indicated that, as in
eukaryotic PDI (30), the -CXYC- motif of DsbC is necessary
for enzyme activities but not required for chaperone activity; thus,
the chaperone activity of DsbC is independent of the active site.
Mutants C98S and C98S/C101S with Cys98 mutated lose enzyme
activities completely but retain full chaperone activity, while
C101S/C163S with Cys101 mutated retains partial enzyme
activities, indicating that Cys98 is essential whereas
Cys101 is relatively less important for DsbC to function as
a thiol oxidoreductase. It was reported that a single mutation of
Cys101 to Ser resulted in decrease of in vivo
biological activities (16); however, a C101V mutant showed no activity
in catalyzing the reduction of insulin (5). Cys101 has been
suggested to facilitate rapid disruption of mixed disulfides with
substrate, allowing efficient scanning of several disulfide isomers and
preventing trapping of DsbC in disulfide-linked complexes as in the
case of PDI (31).
The nonactive site cysteines form a stable disulfide bond,
Cys141-Cys163, which is resistant to the
physiological reductant GSH at higher than in vivo
concentrations, and the formation of the disulfide bond is not affected
by the presence of strong reductant DTT in culture. This disulfide is
not required for enzymatic activity as C141S and C163S are fully active
as a thiol oxidoreductase; however, the lack of this disulfide bond
results in markedly decreased chaperone activity. Compared with both
the wild-type DsbC and the mutants with intact
Cys141-Cys163 disulfide, the mutants C141S,
C163S, and C101S/C163S, with no Cys141-Cys163
disulfide, show higher sensitivity to GdnHCl-denaturation with lower
Cm values and lower refolding rates. The present data have provided evidence that the disulfide,
Cys141-Cys163, does play a substantial role in
sustaining the stability of the molecule and accelerating the refolding
of denatured DsbC and contributes to the formation of a large uncharged
surface of the V-shaped cleft (14) for substrate binding. Lack of the Cys141-Cys163 disulfide, although it affects
neither the export nor the processing, does decrease the solubility of
the expressed mutants greatly so as to aggregate in the periplasm.
It is known that many overexpressed recombinant proteins are liable to
form inclusion bodies in the cytoplasm, but mostly soluble if targeted
in the periplasm (32, 33). There are a few reports on periplasmic
inclusion bodies (33, 34). Using the export pathway directed by a
His-tagged signal and optimal induction by IPTG at 0.01 mM
recently established in this laboratory (21), wild-type DsbC has been
overproduced as a properly processed and soluble periplasmic protein;
however, the mutants with Cys replaced by Ser, although processed
correctly, behave differently in terms of solubility and/or cellular
distribution. Lowering temperature to 25 °C increased the export and
processing efficiency but did not improve the solubility of
overexpressed proteins in the periplasm (data not shown). The
His-tagged signal sequence does not affect the correct processing of
the modified signal, since all the mutants have the authentic N
terminus. The aggregation of C98S, C141S, C163S, and C101S/C163S in the
periplasm does not appear to be resulted from the proteolysis
resistance, a driving force for the formation of inclusion bodies in
many cases, since all the insoluble mutants are digested by proteinase
K. The possibility that the His tag at the N terminus of the signals
affected the folding of mutated molecules was also excluded since
wild-type DsbC is expressed as an easily soluble protein in the
periplasm using the same expression system and the mutation sites are
distant from the signal in both sequence and three-dimensional
structure (14). Thus, it is suggested that the low stability of mutants of C141S, C163S and C101S/C163S with low Cm values may
contribute to the insolubility in the periplasm. It is also possible
that the solubility of a periplasmic protein may relate to its
conformation before it reaches the periplasm or during translocation.
From the facts that DsbC, C98S/C101S, and C98S containing the intact Cys141-Cys163 disulfide are soluble or
partially soluble at least in the periplasm, and that C141S, C163S, and
C101S/C163S with no Cys141-Cys163 disulfide
aggregate in the periplasm and refold in vitro apparently slower than the formers do, it is rational to speculate that the formation of the nonactive site disulfide bond
Cys141-Cys163 is a key factor to stimulate
further folding to a proper conformation. Failure of the formation of
this disulfide may allow the folding intermediates to have their
non-native hydrophobic surface exposed for a longer time and therefore
more chance for incorrect interactions to form aggregates.