From the National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China
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ABSTRACT |
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Two
D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
folding intermediate subunits bind with chaperonin 60 (GroEL) to form a
stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the
two GAPDH subunits bind at one end of the GroEL molecule displaying a
"half of the sites" binding profile. For lysozyme, GroEL binds with
either one or two folding intermediates to form a stable 1:1 or 1:2
complex with one substrate on each end of the GroEL double ring for the
latter. The 1:1 complex of GroEL·GroES binds with one lysozyme or one
dimeric GAPDH folding intermediate to form a stable ternary complex.
Both complexes of GroEL·lysozyme1 and
GroEL·GAPDH2 bind with one GroES molecule only at the
other end of the GroEL molecule forming a trans ternary
complex. According to the stoichiometry of GroEL binding with the GAPDH
folding intermediate and the formation of ternary complexes containing
GroEL·GAPDH2, it is suggested that the folding
intermediate of GAPDH binds, very likely in the dimeric form, with
GroEL at one end only.
D-Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH,1 EC 1.2.1.12) is a
homotetrameric enzyme playing a key role in glycolysis. It has been
used as a model for studies on unfolding, refolding, dissociation and
association of oligomeric proteins (1-4), but little is known about
details of its folding and association. Kinetic analysis of activity
recovery of denatured GAPDH indicated that dimerization of the dimer is
the rate-limiting step in the process of reactivation of the denatured
enzyme (5). At low temperatures, tetrameric GAPDH can be induced by ATP
to dissociate into inactive but structurally compact monomers, useful
for the study of its reassociation and reactivation (6). A cold
intermediate of a hyperthermophilic GAPDH was characterized by
Jaenicke's group to represent a native-like "assembled molten
globule" displaying a reversible and highly cooperative
conformational transition to the unfolded state (7, 8). GAPDH has also
been used as a target protein to examine the chaperone activity of
eukaryotic protein disulfide isomerase (9, 10) and bacterial DsbA (11) and DsbC,2 independent from
their disulfide isomerase activity. Recently, a burst-phase
intermediate of denatured rabbit muscle GAPDH during refolding has been
characterized in this laboratory to be similar to the relatively stable
unfolding intermediate of the enzyme denatured in 0.5-1.0
M guanidine hydrochloride (GdnHCl). This intermediate binds
to GroEL with suppression of both its reactivation and aggregation. The
stable complex with GroEL can be dissociated in the presence of ATP
resulting in the reactivation of GAPDH to a level considerably higher
than that obtained by spontaneous reactivation of the denatured GAPDH
upon dilution and to a still higher level if GroES is also present
(13).
Molecular chaperones such as GroEL play an essential role in assisting
the folding of nascent peptides to form biologically functional
proteins by binding with folding intermediate and thereby preventing
reactions that lead to aggregation (14, 15). Stoichiometric analysis of
the suppression of GAPDH reactivation by binding with GroEL suggested
that the tetradecameric GroEL binds with one GAPDH dimer or two
monomers (13). In this paper we have examined the binding profiles of
GroEL itself and various GroEL complexes, such as GroEL·GroES,
GroEL·GAPDH, and GroEL·lysozyme with different ligands, and the
results are consistent with the suggestion that GroEL binds two
lysozyme intermediates symmetrically but with a dimeric species of the
GAPDH folding intermediate only at one end of the double ring.
Materials--
D-Glyceraldehyde-3-phosphate was
prepared from its diethyl acetal monobarium salt (Sigma) by the method
provided. Hen egg white lysozyme, ADP, and oxidized glutathione were
purchased from Serva. Bovine serum albumin (98-99% albumin, Fraction
V), Micrococcus lysodeikticus dried cells, and GdnHCl were
from Sigma. Dithiothreitol was from Promega. Reduced glutathione was
from Boehringer Mannheim. Tris was from Amresco. All other chemicals
were local products of analytical grade. In all experiments, unless
otherwise specified, 100 mM Tris-HCl buffer (pH 7.5)
containing 200 mM KCl, 5 mM MgCl2, and 2 mM EDTA was employed and referred to hereafter simply
as the Tris buffer.
Preparation and Determination of Proteins--
The multicopy
plasmid pGroESL containing the coding sequences of GroEL and GroES, a
gift from Dr. S. L. Yang of the Shanghai Research Center of
Biotechnology, Academia Sinica, was overexpressed in Escherichia
coli HB101. The expression product was purified basically
according to Landry and Gierash (16). Further purification of the GroES
fraction was carried out by hydroxyapatite chromatography using 5-200
mM phosphate gradient to remove the co-expressed
chloramphenicol acetylase, and the GroEL fraction was treated by ATP
according to Schmidt et al. (17) to remove the endogenously
bound polypeptides. GroEL and GroES thus purified both showed one band
on SDS-PAGE with the expected molecular mass (MM). The purified GroEL
preparation had very low intrinsic fluorescence, especially with an
excitation wavelength of 295 nm. Preparation and activity assay of
rabbit muscle GAPDH were as described by Liang et al.
(2).
Protein concentrations were determined by measuring the absorbance at
280 nm with the following absorption coefficients
(A1 cm0.1%): 1.00 for GAPDH (18), 0.66 for
bovine serum albumin, 2.63 for native lysozyme, and 2.37 for denatured
lysozyme (19). The concentrations of GroEL and GroES were determined by
the method of Bradford (20) with bovine serum albumin as a standard.
Unless otherwise specified, GroEL, GroES, and GAPDH were considered as tetradecamer, heptamer, and monomer, respectively, in the calculations of concentrations and molar ratios as denatured GAPDH is presumably fully dissociated.
Denaturation of Lysozyme and GAPDH--
Lysozyme at 20 mg/ml was
completely denatured and reduced in 100 mM sodium phosphate
(pH 8.0) containing 8 M GdnHCl and 150 mM
dithiothreitol at room temperature for 4 h. The reaction mixture was brought to pH 2.0 with 6 M HCl, dialyzed first against
10 mM HCl, and then against 100 mM acetic acid
at 4 °C thoroughly. The denatured and reduced lysozyme was aliquoted
at 200 µM and stored at Preparation of Complexes of GroEL with Different
Ligands--
The complex of GroEL·GroES was prepared according to
Azem et al. (21) by incubation of 2 µM GroEL,
2 µM GroES, and 1 mM ADP in the Tris buffer
at 37 °C for 30 min. The complexes of
GroEL·GAPDH2, GroEL·lysozyme1, and
GroEL·lysozyme2 were obtained by 50-fold dilution of 200 µM denatured GAPDH and 100- or 50-fold dilution of 200 µM denatured and reduced lysozyme, respectively, into the Tris buffer containing 2 µM GroEL and incubation at
25 °C for 30 min. The complexes of
GroEL·GroES·GAPDH2 and
GroEL·GroES·lysozyme1 were made by rapid dilution of
denatured GAPDH to 4 µM and denatured and reduced
lysozyme to 2 µM, respectively, in the Tris buffer containing 2 µM GroEL·GroES and 50 µM ADP
and incubation at 25 °C for 30 min. The complexes of
GroEL·GAPDH2·lysozyme1 and
GroEL·lysozyme1·GAPDH2 were prepared,
respectively, by dilution of denatured and reduced lysozyme to 2 µM in the Tris buffer containing 2 µM
GroEL·GAPDH2 and denatured GAPDH to 4 µM in
the Tris buffer containing 2 µM GroEL·lysozyme1 and incubation at 25 °C for 30 min.
The complexes of GroEL·GAPDH2·GroES and
GroEL·lysozyme1·GroES were obtained by incubating 2 µM GroES and 1 mM ADP with 2 µM
GroEL·GAPDH2 and GroEL·lysozyme1,
respectively, at 37 °C for 30 min. To examine the construction of
the complexes of GroEL·GroES·GAPDH2 and
GroEL·GAPDH2·GroES, denatured and reduced lysozyme was
diluted to 2 µM in the Tris buffer containing either
complex at 2 and 50 µM ADP and incubated for 30 min at
25 °C. Similarly, denatured GAPDH was diluted to 4 µM
in the Tris buffer containing 2 µM
GroEL·GroES·lysozyme1 or
GroEL·lysozyme1·GroES and 50 µM ADP, or 2 µM GroEL·lysozyme2, respectively, to
examine the construction of three complexes.
In all the reactions involving GroES-containing complexes, 50 µM ADP was added to stabilize the complexes (22). All the reactions involving GroES binding were carried out at 37 °C and contained 1 mM ADP.
Identification of the Complexes--
Each complex was loaded on
a Sephacryl S-200 (Amersham Pharmacia Biotech) gel filtration column
(1.2 × 50 cm) and eluted at 4 °C using the Tris buffer at a
flow rate of 0.5 ml/min. The elution buffer contained also 50 µM ADP if the complex reaction contained GroES. The
protein peak in void volume was collected, concentrated by Centricon-10
(Amicon), and analyzed by SDS-PAGE of 15% polyacrylamide gel.
Refolding of GAPDH and Lysozyme--
Refolding of denatured
GAPDH was initiated by 200-fold dilution to 0.69 µM at
4 °C in the Tris buffer containing 5 mM dithiothreitol, with or without different concentrations of GroEL,
GroEL·GroES, GroEL·lysozyme1,
GroEL·lysozyme2, GroEL·GroES·lysozyme1,
or GroEL·lysozyme1·GroES as specified. The activity
recovery was determined according to Liang et al. (2) after
incubation first at 4 °C for 30 min and then at 25 °C for 3 h after dilution (23), and the reactivation yield was defined as
percentage of the activity of native GAPDH. Oxidative refolding of
reduced and denatured lysozyme was carried out by 100-fold dilution to
2 µM in the Tris buffer containing 1 mM
oxidized glutathione and 2 mM reduced glutathione with
or without different concentrations of the complexes of GroEL,
GroEL·GroES, GroEL·GAPDH2,
GroEL·GroES·GAPDH2, or
GroEL·GAPDH2·GroES at 25 °C for 2 h. If the
complex contained GroES, 50 µM ADP was also present in
the buffer. Lysozyme activity was determined at 30 °C by following
absorbance decrease at 450 nm of a 0.25 mg/ml M. lysodeikticus suspension in 67 mM sodium phosphate
buffer (pH 6.2) containing 100 mM NaCl (19, 24).
Reactivation yield was defined as percentage of the activity of the
native lysozyme. Aggregation of GAPDH during refolding was
monitored continuously at 25 °C by 90o light scattering
at 488 nm in a Hitachi F4010 spectrofluorometer.
Identification of Complexes of GroEL with Different
Ligands--
By Sephacryl S-200 chromatography with a MM fractionation
range of 250-5 kDa, GroEL (14 × 60 kDa) was eluted in the void
volume, and GAPDH (4 × 36 kDa), GroES (7 × 10 kDa), and
lysozyme (1 × 14.3 kDa) were eluted successively in well
separated peaks. Fig. 1 shows the
SDS-PAGE patterns of the proteins collected in void volume peaks of a
Sephacryl S-200 column loaded with reaction products of GroEL with
GroES, folding intermediates of GAPDH, or lysozyme at various
proportions. The results identified the formation of all the stable
complexes of GroEL with the bound components, and the bands in each
lane appear at MM positions of the expected components. The
band of GroES subunit appeared above the lysozyme band at the position
corresponding to an apparent MM of 15 kDa but not 10 kDa as also noted
by Tilly et al. (25). The order of addition of the ligands
had no effect on the formation of all the ternary complexes.
Effects of GroEL and GroEL·GroES on the Reactivation and
Aggregation during Refolding of Denatured GAPDH--
As shown in Fig.
2A, the spontaneous
reactivation of denatured GAPDH at 0.69 µM decreased with
increasing concentration of GroEL in the dilution buffer and was
completely suppressed at a ratio of GroEL/GAPDH of 0.5. When the
GroEL·GroES complex was used instead of GroEL, the reactivation of
GAPDH was suppressed at the same ratio of 0.5. Denatured GAPDH
aggregated rapidly upon dilution as monitored by light scattering (Fig.
2B). Both the rate and the extent of aggregation decreased
with increasing concentration of GroEL or GroEL·GroES complex, with
full suppression of the aggregation at the same molar ratio of 0.5 in
both cases.
The suppression of both the spontaneous reactivation and the
aggregation of GAPDH during refolding in the presence of GroEL indicated the formation of a stable complex between GroEL and a folding
intermediate of GAPDH (13). The above results indicate that one
tetradecameric GroEL binds stoichiometrically with two monomeric or one
dimeric species of the GAPDH folding intermediate. The stable complexes
of GroEL·GroES, GroEL·GAPDH2, and
GroEL·GroES·GAPDH2 thus formed were eluted in the void
volume peak of Sephacryl S-200 chromatography and had the expected
components as shown in Fig. 1 (lanes 1, 2, and
3).
Effects of GroEL and GroEL·GroES on the Reactivation of Denatured
and Reduced Lysozyme--
In contrast to GAPDH, the lysozyme folding
intermediate binds to GroEL and the GroEL·GroES complex with
different stoichiometry. As shown in Fig.
3, the spontaneous reactivation of 18%
for lysozyme at 2 µM decreased with increasing
concentration of GroEL in the refolding solution and was fully
suppressed at a ratio of GroEL/lysozyme of 0.5. The reactivation of
lysozyme also declined in the presence of the GroEL·GroES complex but
became fully suppressed only when the molar ratio of
GroEL·GroES/lysozyme reached 1.0. The above indicates the formation
of stable complexes between one GroEL molecule and two lysozyme folding
intermediates, GroEL·lysozyme2, and between one
GroEL·GroES and one lysozyme folding intermediate, GroEL·GroES·lysozyme1. The components of the above
complexes have also been demonstrated directly (Fig. 1, lanes
4 and 6).
The formation of stable ternary complexes of GroEL·GroES with folding
intermediate of GAPDH or lysozyme indicates that the substrate binding
does not induce the release of GroES from the GroEL·GroES complex.
Moreover, the stability of the ternary complexes were not effected by
ADP from 50 µM to 1 mM present in the Tris buffer (data not shown).
Effects of GroEL·Lysozyme1,
GroEL·Lysozyme2,
GroEL·GroES·Lysozyme1, and
GroEL·Lysozyme1·GroES on GAPDH Reactivation--
As
shown in Fig. 4, the presence of
GroEL·lysozyme1 (Fig. 1, lane 5) in the
refolding buffer suppressed the refolding of denatured GAPDH at a molar
ratio of 0.5, indicating the formation of the complex of one
GroEL·lysozyme1 with one dimeric or two monomeric GAPDH
subunits, consistent with the result shown in Fig. 1, lane 7. However, the presence of GroEL·lysozyme2,
GroEL·GroES·lysozyme1, or
GroEL ·lysozyme1·GroES in the refolding buffer had no
effect at all on the reactivation of GAPDH up to a molar ratio of 0.5, indicating full occupancy on both ends of these GroEL complexes to
preclude binding with GAPDH. For the GroEL·lysozyme2
complex, hence, there must be one lysozyme intermediate on each end of the GroEL molecule, and for the ternary complexes of
GroEL· GroES·lysozyme1 and
GroEL·lysozyme1·GroES, a lysozyme at one end and a
GroES at the other were also consistent with the SDS-PAGE patterns of
the respective complexes (Fig. 1, lanes 4, 6, and
9). GAPDH band was not detected from the reaction products
of GroEL·lysozyme2,
GroEL·GroES·lysozyme1, or
GroEL·lysozyme1·GroES with GAPDH folding intermediates
(Fig. 1, lanes 13, 14 and 15).
Effects of GroEL·GAPDH2,
GroEL·GroES·GAPDH2, and
GroEL·GAPDH2·GroES on Lysozyme Reactivation--
In
contrast to GroEL·lysozyme2, the presence of
GroEL·GAPDH2 complex fully suppressed the refolding of
denatured and reduced lysozyme at the molar ratio of 1.0 (Fig.
5), indicating that the complex of
GroEL·GAPDH2 had one substrate binding site free for binding with one lysozyme, i.e. both subunits of the GAPDH
folding intermediates are bound at only one end of the GroEL molecule. The components of the complex of
GroEL·GAPDH2·lysozyme1 were confirmed by
SDS-PAGE in Fig. 1, lane 8. The complexes of
GroEL·GroES·GAPDH2 and GroEL·GAPDH2-GroES
(Fig. 1, lanes 3 and 10) even at a molar ratio of
1.0 had no effect at all on the refolding of lysozyme, and no lysozyme
band could be detected in the respective reaction products as shown in
lanes 11 and 12 in Fig. 1. Moreover, in the presence of
different concentrations of ADP from 50 µM to 1 mM, the complexes of GroEL·GroES·lysozyme1
and GroEL·GroES·GAPDH2 showed no effect on the
reactivation of GAPDH and lysozyme, respectively (data not shown).
GroES bound to complex of GroEL·GAPDH2 or
GroEL·lysozyme1 only at the trans end of the
complex but not at the cis end to cover the bound substrate,
therefore no free ends were available in GroEL·GAPDH2·GroES and
GroEL·lysozyme1·GroES complexes.
The three-dimensional structure of GroEL (26) reveals that
the GroEL molecule has two substrate-binding sites on each end of the
double ring. In the presence of ADP, one GroEL molecule binds with only
one GroES molecule to form the bullet-like complex of GroEL·GroES; no
football-like GroEL·GroES2 is detected even in the
presence of 3-fold excess of GroES (27). The complex of GroEL·GroES
has a free binding site and can bind with either lysozyme or GAPDH
folding intermediate to form a stable 1:1:1 complex of
GroEL·GroES·lysozyme1 or 1:1:2 complex of
GroEL·GroES·GAPDH2. The binding of GAPDH or lysozyme
intermediate in the presence of 50 µM ADP does not
trigger the release of GroES from GroEL. It is consistent with the
report by Sparrer and Buchner (28) for the complex of
GroEL·GroES·maltose binding protein (Y283D) but is different from
the report by Hartl and co-workers (29) that GroEL-bound substrate
polypeptide can induce GroES cycling on and off GroEL in the presence
of 0.2 mM ADP. In fact, ADP at concentrations from 50 µM to 1 mM has been found to show no effect on the stability of the ternary complexes of
GroEL·GroES·lysozyme1 and
GroEL·GroES·GAPDH2.
It has been reported that GroES binds with similar efficiency to either
ring of the GroEL·rhodanese complex in the presence of physiological
concentration of ADP, 0.2 mM, resulting in a mixture of
trans- and cis-complexes (29), however, the fact that the stable 1:1:1 complex of GroEL·lysozyme1·GroES
or the 1:2:1 complex of GroEL·GAPDH2·GroES can no
longer bind additional substrate strongly suggests that GroES binds
with the complex of GroEL·lysozyme1 or
GroEL·GAPDH2 in the presence of 1 mM ADP on
the free end only. Similarly, the complex of GroEL·malate
dehydrogenase·GroES also appears to be in a trans form
(27).
One GroEL molecule binds with one lysozyme folding intermediate to form
a stable 1:1 complex, GroEL·lysozyme1, which can further bind an additional lysozyme folding intermediate at the other side to
form the 1:2 complex. As this complex GroEL·lysozyme2 can
no longer bind with any additional substrate, the two lysozyme molecules bind, most likely, one on each end of the double ring. The
GroEL·lysozyme1 complex can also bind GroES or GAPDH
folding intermediate at the free side.
In contrast to the GroEL·lysozyme complex, although the
GroEL·GAPDH2 complex has the stoichiometry of 1:2 and no
longer binds with additional GAPDH folding intermediate, it can still
bind with either one lysozyme or one GroES molecule, indicating the GroEL·GAPDH2 complex has indeed a free binding site left
on the other end, and the two subunits of the GAPDH folding
intermediate are bound at the same end of the double ring. It seems
that as in the case of allosteric proteins, the binding with GAPDH
folding intermediate at one end of GroEL molecule prevents the binding of another GAPDH folding intermediate at the other end, or in other
words, the binding of GAPDH folding intermediate is a "half of the
sites" reaction. The volume of the cavity of the GroEL molecule is
estimated to be 85,000 Å3 and might accommodate a native
protein of 70 kDa or a much smaller nonnative polypeptide (22).
However, as Chen et al. (27) and Thiyagarajan et
al. (30) pointed out, portions of the bound polypeptide can
protrude from the cavity so as to allow the binding of a larger
nonnative molecule, such as two subunits of GAPDH folding intermediate
of 72 kDa or in a similar way alcohol oxidase of 75 kDa (31). It is
known that some other proteins, such as subtilisin with MM of 27.7 kDa
(32), rhodanese of 33 kDa (31, 33), malate dehydrogenase of 35 kDa
(34), rat liver ornithine transcarbamylase of 36 kDa (35), and
mitochondrial aspartate aminotransferase of 46 kDa (36) bind with GroEL
in a 1:1 stoichiometry, but it is not known whether the free end is
still available for binding of another substrate. On the other hand,
the relatively small molecules of lysozyme (14.3 kDa) and dihydrofolate
reductase (DHFR, 20 kDa) show multiple binding. However molecular mass
does not seem to be the only factor as the binding of maltose-binding protein (40 kDa) to GroEL shows a 1:2 stoichiometry. Presumably, molecular shape rather than molecular mass and the mode of interactions between GroEL and ligands are the crucial factors. "Half of the sites" binding probably results from the conformational change at the
other end of the GroEL molecule induced by the bound ligand at one end.
It is known that the closely packed crystalline homotetrameric GAPDH
exists as a dimer of dimers (37). During the unfolding at low
denaturant concentrations, the dimer of GAPDH subunit exists as a
partially unfolded and aggregation-prone form (2), which is most likely
the species formed in the burst phase during its refolding and prone to
GroEL binding (13). It is therefore likely that, in the
GroEL·GAPDH2 complex, the two GAPDH subunits exist as a
dimer but not two monomers.
It is interesting to note that one GroEL molecule can rapidly bind with
four barnase molecules composed of 110 amino acid residues with
barnase in great excess to GroEL, but the binding stoichiometry becomes
1:1 with GroEL in excess to barnase (38). Two or four molecules of
mutated E. coli DHFR can be bound to one GroEL depending on
different mutants (39). Also the binding stoichiometry of chymotrypsin
inhibitor 2, a very small protein of only 64 residues (12), with GroEL
varies from 1:1 for wild type to 1: 8 for some mutants with the
substrate in excess. However, in all the above cases, it has not been
stated whether the multiple molecules of substrate were bound at the
same end or both ends of the GroEL double ring. It has been pointed out
that the 1:4 complex of GroEL with mutant DHFR is not significantly
populated, only < 10%, even with the ligand in excess, and the
predominating complex formed has a ratio of 1:2 (39). The lysozyme
molecule is only slightly bigger than barnase and smaller than DHFR,
but only one lysozyme molecule can bind at each end of a GroEL molecule.
Scheme 1 is a model consistent with all
the observations for the unfolding and refolding of GAPDH and the
binding of the folding intermediate with GroEL, where M is
the denatured monomer; D, D*, and
D' are dimeric folding intermediates with different
conformations; T is tetrameric native GAPDH; and
A is aggregated enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Denaturation of GAPDH
was carried out by incubation of 140 or 200 µM enzyme in
the Tris buffer containing 3.0 M GdnHCl and 1 mM dithiothreitol for at least 4 h at 4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SDS-PAGE (15%) analysis of complexes.
Lane 1, GroEL·GroES; lane 2,
GroEL·GAPDH2; lane 3, the ternary complexes of
GroEL·GroES·GAPDH2; lane 4,
GroEL·lysozyme2; lane 5,
GroEL·lysozyme1; lane 6,
GroEL·GroES·lysozyme1; lane 7,
GroEL·lysozyme1·GAPDH2; lane 8,
GroEL·GAPDH2·lysozyme1; lane 9,
GroEL·lysozyme1·GroES; lane 10,
GroEL·GAPDH2·GroES; lane 11,
GroEL·GroES·GAPDH2 + denatured and reduced lysozyme;
lane 12, GroEL·GAPDH2·GroES + denatured and
reduced lysozyme; lane 13, GroEL·lysozyme2 + denatured GAPDH; lane 14,
GroEL·GroES·lysozyme1 + denatured GAPDH; lane
15, GroEL·lysozyme1·GroES + denatured GAPDH;
lane 16, lysozyme; lane 17, GroES; lane
18, GAPDH; lane 19, GroEL.
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Fig. 2.
Effects of GroEL and GroEL·GroES on the
reactivation and aggregation of denatured GAPDH. A,
reactivation of 0.69 µM denatured GAPDH in the presence
of different concentrations of GroEL ( ) or GroEL·GroES complex
(
). B, aggregation during the refolding of 0.69 µM denatured GAPDH monitored by light scattering at 488 nm. 1, no addition; 3 and 5, in the
presence of GroEL at the ratio of 0.25 and 0.5, respectively;
2, 4, and 6, with GroEL·GroES
complex at the ratio of 0.125, 0.25, and 0.5, respectively.
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Fig. 3.
Effects of GroEL and GroEL·GroES on the
reactivation of denatured and reduced lysozyme. Reactivation of
200 µM denatured and reduced lysozyme was triggered by
dilution to 2 µM in the presence of different
concentrations of GroEL ( ) or GroEL·GroES complex (
).
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Fig. 4.
Effects of
GroEL·lysozyme1,
GroEL·lysozyme2,
GroEL·GroES·lysozyme1, and
GroEL·lysozyme1·GroES on GAPDH
reactivation. Reactivation of 0.69 µM denatured
GAPDH in the presence of different concentrations of
GroEL·lysozyme1 ( ), GroEL·lysozyme2
(
), GroEL·GroES·lysozyme1 (
), and
GroEL·lysozyme1·GroES (
).
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Fig. 5.
Effects of
GroEL·GAPDH2,
GroEL·GroES·GAPDH2, and
GroEL·GAPDH2·GroES on lysozyme
reactivation. Reactivation of 2 µM denatured and
reduced lysozyme in the presence of different concentrations of
GroEL·GAPDH2 ( ), GroEL·GroES·GAPDH2
(
), and GroEL·GAPDH2·GroES (
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scheme 1.
A tetrameric GAPDH is fully denatured to be four unfolded
monomeric subunits by GdnHCl under the conditions employed. Upon dilution, a dimeric folding intermediate formed very fast in a burst
phase, which is partially folded with secondary structure between that
of the native and the denatured species and aggregation-prone (13).
GroEL recognizes and binds with this dimeric folding intermediate through hydrophobic interactions at one end of the double ring to form
a stable asymmetric complex and thus prevent the aggregation between
the folding intermediates. The intermediate is released only in the
presence of ATP or ATP/GroES for either further folding and association
to become an active tetrameric molecule or aggregation or rebinding
with GroEL.
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ACKNOWLEDGEMENT |
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We sincerely thank Prof. C. L. Tsou for continuous encouragement, helpful advice, and critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by the Pandeng Project of the Chinese Ministry of Science and Technology and Grant KJ982-J1-609 from Academia Sinica.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: National Laboratory of
Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun
Road, Beijing 100101, China. Tel.: +86-10-64888502; Fax: +86-10-64872026; E-mail: chihwang{at}sun5.ibp.ac.cn.
2 J. Chen, J.-L. Song, S. Zhang, Y. Wang, D.-F. Cui, and C.-C. Wang, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase; GroEL, chaperonin 60; GroES, chaperonin 10; DHFR, dihydrofolate reductase; GdnHCl, guanidine hydrochloride; MM, molecular mass; PAGE, polyacrylamide gel electrophoresis.
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