 |
INTRODUCTION |
Molecular chaperones are a class of proteins which assist folding
of other proteins in the cell by preventing or reversing aggregation
caused by off-pathway folding reactions (1-3). Many of the chaperones
are heat shock proteins
(Hsp),1 named for their
induced synthesis in cells during heat-shock stress. Group I
chaperonins (4, 5) include GroEL/GroES in bacteria, Hsp60/Hsp10 in
eukaryotic mitochondria, and ribulose-P2 carboxylase-binding protein/cpn21 in plant chloroplasts (6, 7). These
proteins exhibit remarkable structural and functional conservation from
bacteria to plants to humans (8-10). The conserved function in the
chaperonin family is the basis that bacterial GroEL and GroES are
widely used in promoting refolding of various proteins, including those
from mitochondria and chloroplasts both in vitro and in
Escherichia coli (1-3). The biogenesis of eukaryotic mitochondrial matrix proteins is proposed to follow a complex chaperone-mediated pathway (11-13). Unfolded subunit polypeptides are
imported into mitochondria as aided by Hsp70 family chaperones, and the
final stage of folding and assembly of mitochondrial oligomeric proteins is promoted by chaperonins Hsp60/Hsp10 (11-13).
Our laboratory has previously shown the obligatory role of GroEL and
GroES in facilitating folding and assembly of the decarboxylase (E1)
and the transacylase (E2) components of human branched-chain
-ketoacid dehydrogenase complex in vitro (14, 15) and in E. coli (16). E1 is a thiamine
pyrophosphate-dependent enzyme, consisting of two 45-kDa
and two 38-kDa
subunits (17). In an earlier study (18), we
overexpressed MBP-E1 in which the mature sequence of maltose-binding
protein (MBP) was fused to the N terminus of the mature
subunit
(abbreviated MBP-
). Although MBP was shown to rapidly and
efficiently refold in vitro without chaperonins (18), the
overexpression of MBP-E1 in E. coli required co-expression
of GroEL/GroES (16, 19). The results have established that fusion of a
spontaneously refolded protein to the
sequence from E1 does not
change chaperonin-dependent folding characteristics of the
latter sequence.
The native conformation of a large polypeptide is often folded into
several compact regions or domains (20). Previous studies with
spontaneously refolded proteins, e.g. tryptophan synthetase (21, 22),
dihydrofolate reductase (23), and
aspartokinase-homoserine-dehydrogenase (24, 25), show that individual
domains in the entire polypeptide chain refold differentially and can
be expressed as soluble functional units, suggesting that the domain
alone is a folding unit. Co-translational independent domain folding in
eukaryotes has been recently demonstrated using a Ras-dihydrofolate
reductase fusion polypeptide (26). Molecular chaperones do not contain
information for specifying correct folding. The information for folding
into the functional three-dimensional structure of a protein is solely
present in its amino acid sequence (27). Thus, one can reasonably
expect that independent domain folding also occurs on the GroEL scaffold.
The crystal structure of unliganded GroEL double-ring complex has
indicated that, in the absence of GroES, a polypeptide of up to ~35
kDa in size can be accommodated within a single ring of GroEL (28, 29).
Binding of GroES, however, induces a large conformational change in
GroEL, leading to an approximate doubling of the volume in the central
cavity of that ring. This allows the accommodation of polypeptides of
~70 kDa in size (30). Because of the size constraint of the GroEL
cavity, it has been proposed that GroES promotes the productive release
of polypeptides larger than 70 kDa from a trans
configuration, in which GroES and the unfolded polypeptide bind to the
opposite rings of GroEL (31). However, evidence for the productive
trans release of large polypeptides from GroEL is lacking.
In the present study, we investigate GroEL/GroES-assisted refolding of
the large 86-kDa MBP-
fusion polypeptide in vitro. The
kinetic data show that the MBP moiety folds appreciably more rapidly
than the
subunit. This explains the isolation of an MBP-
·GroEL
binary complex from the MBP-E1 refolding mixture and E. coli
lysates with amylose resin. Moreover, we provide evidence that GroES
can only cap the MBP-
·GroEL complex in trans in
relation to the fusion polypeptide. This is in contrast to that
observed in the
·GroEL complex where GroES binds to GroEL in both
cis and trans configurations. These findings
provide a paradigm for independent domain folding during
chaperonin-assisted folding of large multidomain polypeptides.
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EXPERIMENTAL PROCEDURES |
Materials--
CG712 (an E. coli ESts
strain) and the expression plasmid pGroESL overexpressing GroEL and
GroES (32) were generous gifts from Drs. George Lorimer and Anthony
Gatenby of DuPont Experimental Station (Wilmington, DE). The pH1
plasmid carrying MBP-
and -
sequences, the pHisT-hE1 plasmid
harboring (His)6-
and -
sequences, and the
pMAL-c-hE1
plasmid carrying MBP-
sequence were previously described (19, 33). The MBP or (His)6 tag was fused to the N terminus of the mature
sequence through a linker containing the
factor Xa or tobacco etch virus protease-specific site, respectively. GroEL and GroES were overexpressed in E. coli and purified
as described previously (14). Amylose resin was prepared according to a
previously reported method (34).
Expression and Purification of MBP-E1, MBP-
, and
E1--
CG-712 cells co-transformed with pGroESL and the pH1,
pMAL-c-hE1
, or pHisT-hE1 plasmids were grown overnight at 37 °C
in YTGK media containing 2 × YT medium (35), 10 mM
KCl, 1% glycerol, and antibiotics (100 µg/ml ampicillin and 50 µg/ml chloramphenicol). The overnight culture was diluted 6:1000 into
1 liter of YTGK medium with added antibiotics. Cultures were grown on a
shaker at 220 rpm and 37 °C to a measured
A600 of 0.6, and expression was induced with
0.75 mM isopropyl-1-thio-
-D-galactosidase.
After induction, cultures were grown at 37 °C for 16 h. The
extraction of MBP-E1 and MBP-
from the E. coli lysate
with amylose resin was described previously (19). The purified proteins
were separated on a 10-30% sucrose density gradient. Purification of
(His)6-E1 on the Ni-NTA column was also previously
described (33). To prepare untagged E1, dialyzed (His)6-E1
was digested with the tobacco etch virus protease. Undigested
(His)6-E1 was removed by Ni-NTA extraction.
GroEL/GroES-assisted and Spontaneous Refolding of Substrate
Proteins--
MBP-E1, E1, or MBP was incubated for 1 h at
22 °C in a denaturing buffer (50 mM potassium
Pi, pH 7.5, 100 mM KCl, 8 M urea, 0.1% Tween 20, and 1 mM dithiothreitol). Refolding was
initiated at 22 °C by a 100-fold dilution of the denatured protein
into a refolding buffer (50 mM potassium Pi, pH
7.5, 100 mM KCl, 1 mM thiamine pyrophosphate, 5 mM dithiothreitol, and 5 mM MgCl2) containing GroEL/GroES (molar ratio of GroEL:GroES:monomeric
substrate = 2:4:1). Unless otherwise specified, the final
concentration of the denatured monomer was 0.5 µM. Rapid
mixing was accomplished by vortexing of a denatured protein drop into
the refolding buffer, which was immediately followed by an addition of
10 mM ATP. At indicated times, an aliquot of 50 µl was
withdrawn and frozen at
20 °C until assays for E1 activity. E1
activity was assayed radiochemically in a reconstituted branched-chain
-ketoacid dehydrogenase system with the addition of excess
recombinant E2 and E3 components as described elsewhere (14). An
identical concentration of nondenatured E1 or MBP-E1 was also added
into the separate refolding buffer and incubated at 22 °C to serve
as the 100% activity control. The folding efficiency was defined as
the percent recovery of activity compared with the 100% activity
control at the final time point.
Amylose Resin Extraction and Analysis by Sucrose Density Gradient
Centrifugation--
Unless otherwise stated, at indicated times,
aliquots (1 ml) of the refolding mixture was withdrawn, and the
refolding reaction was terminated by the addition of 50 mM
CDTA to chelate Mg2+ ions. The refolding mixture was
incubated with 0.5 ml of amylose resin for 10 min. After a brief
spinning, the supernatant was removed and the resin was washed three
times with 1 ml of the refolding buffer. Proteins bound to the resin
were eluted twice with 0.5 ml of the refolding buffer containing 20 mM maltose. The eluted protein sample was loaded on a
10-30% sucrose density gradient, and separated by centrifugation at
210,000 × g for 18 h.
Determination of Rate Constants for the E1 Refolding Reaction
(36, 37)--
To determine rate constants for the folding of MBP and
moieties, the data were fit to the first-order rate equation:
ln[A] = ln[A]0-kt,
where [A] = molar concentration of the reactant, i.e. the MBP or the
moiety at a given time;
[A]0 = initial concentration of the reactant;
k = rate constant for the folding reaction, and t = the reaction time. To determine rate constants for
the reconstitution of E1 activity from denatured E1 or MBP-E1, the data
were fit to second-order rate equation kt = 1/[A]
1/[A]0, where
[A] =
, MBP-
or -
monomers at a given time. The
percent of E1 activity recovered was used to calculate the amount of E1
or MBP-E1 tetramers formed. The same amount of E1 or MBP-E1 not treated
with the denaturant was incubated in the refolding buffer for 24 h, and the enzyme activity served as a 100% refolding control. The
percent of folded E1 or MBP-E1 tetramers at different time points was
calculated. The concentration of remaining E1 monomers was derived by
subtracting a 2-fold concentration of the E1 tetramer formed from the
initial concentration of the monomer (
, MBP-
or -
). The
assumption was that the
and
subunits participating in the E1
refolding reaction either stayed as monomers or complexed with each
other to form heterodimers or heterotetramers according to the assembly pathway: 2
+ 2
2 
2
2 (14). The concentrations of
and
monomers were identical at any time during the refolding. Therefore, V = k [
][
] was treated
as V = k[
]2 = k[
]2, where V = velocity.
The second-order equation, kt = 1/[
]
1/[
]0 = 1/[
]
1/[
]0 was
derived after integration.
Proteinase K Digestion of Folding Intermediates--
At
indicated times of the refolding reaction, a 100-µl aliquot of the
refolding mixture containing 0.5 µM MBP was removed. The
sample was digested with 4 µg/ml PK for 10 min at 22 °C, and the
digestion then quenched with 5 mM PMSF. The same amounts of native MBP-E1 and MBP were also treated with PK and served as a 100%
refolding control. The protease-digested samples were precipitated with
7.5% trichloroacetic acid containing 125 µg/ml sodium deoxycholate, and analyzed by SDS-PAGE and Western blotting.
Preparation of MBP-
·GroEL, (His)6-
·GroEL,
and
·GroEL Complexes--
MBP-E1 (3 mg) was denatured in 400 µl
of the denaturation buffer (50 mM sodium acetate, pH 4.5, and 8 M urea) for 15 min. The denatured protein was
incubated with 200 µl of the carboxymethyl-Sepharose CL-6B resin
(Pharmacia) equilibrated with the denaturation buffer for 15 min. The
resin was washed with the denaturation buffer containing 60 mM KCl. Denatured MBP-
bound to the resin was eluted with the denaturation buffer containing 300 mM KCl. The
eluted MBP-
was repeatedly diluted in 50 mM potassium
Pi, pH 7.5, containing 100 mM KCl and 8 M urea and concentrated in a Microcon-30 concentrator (Amicon). The denatured protein was diluted into 50 mM
potassium Pi, pH 7.5, containing 100 mM KCl and
1:1 molar ratio of GroEL to make 1:1 stoichiometric substrate-GroEL
complexes. The mixture was separated on a 10-25% sucrose density
gradient and the MBP-
·GroEL complex collected. The preparation of
(His)6-
·GroEL and
·GroEL complexes was described
(14).
 |
RESULTS |
Kinetic Measurements for Renaturation of E1 and MBP-E1--
The
refolding of untagged E1 or MBP-E1 denatured in 8 M urea
was initiated by dilution of the denatured protein into a refolding buffer at 22 °C containing GroEL/GroES and Mg2+-ATP.
Fig. 1 shows the time course for
reactivation of untagged E1. No E1 activity was recovered when GroEL,
GroES, or Mg2+-ATP was omitted from the refolding mixture.
The folding kinetics of E1 at concentrations of 0.125 µM
and 0.0625 µM were fit to the second-order equation, with
rate constants of 512 and 417 M
1
s
1, respectively. The folding of MBP-E1 also showed an
absolute requirement for GroEL/GroES and Mg2+-ATP (Fig.
2). The kinetic data at 0.2, 0.1, and
0.05 µM denatured MBP-E1 were fit to the second-order
equation. This resulted in rate constants of 324, 397, and 407 M
1 s
1, respectively, for the
reactivation of E1 activity. The average rate constant for untagged E1
was 465 M
1 s
1 and for MBP-E1
was 376 M
1 s
1.

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Fig. 1.
Kinetics for
GroEL/GroES-dependent reconstitution of untagged human
E1. Recombinant human E1 was denatured in 8 M urea and
reconstituted at 22 °C in the presence of GroEL/GroES and
Mg2+-ATP as described under "Experimental Procedures."
The final concentrations of denatured E1 tetramers for refolding,
following a 100-fold dilution, were 0.25 µM ( ), 0.125 µM ( ), and 0.0625 µM ( ). The data at
0.125 and 0.0625 µM of E1 were fit to the second-order
function to yield rate constants. Folding efficiencies at 0.25, 0.125, and 0.0625 µM denatured E1 were 68, 60, and 83%,
respectively. , GroEL, GroES, or Mg2+-ATP omitted. One
milliunit (mU) was defined as 1 nmol of
14CO2 evolved per min.
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Fig. 2.
Time course for chaperonin-mediated
reactivation of urea-denatured MBP-E1. The denaturation and
reconstitution at 22 °C of the MBP-E1 fusion protein were as
described in the legend to Fig. 1. The concentrations of the denatured
MBP-E1 tetramer for refolding were 0.2 µM ( ), 0.1 µM ( ), and 0.05 µM ( ). The data at
all three protein concentrations were fit to the second-order function
to produce folding rate constants. Folding efficiencies at 0.2, 0.1, and 0.05 µM denatured MBP-E1 were 67, 99, and 84%,
respectively. , GroEL, GroES, or Mg2+-ATP absent.
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|
Folded State of Fused and Unfused Maltose-binding Protein--
The
urea-denatured MBP was refolded for 1 h at 22 °C in the absence
or presence of GroEL·GroES·Mg2+-ATP. The refolded MBP
was digested with PK (4 µg/ml) and separated on a sucrose density
gradient, and the fractions were analyzed by SDS-PAGE. The appearance
of MBP on the top of the sucrose density gradient either without
(panel A) or with (panel B) chaperonins indicated
that the refolded MBP was soluble and resistant to PK digestion (Fig.
3). The results showed that limited
proteolysis with PK was a feasible approach to monitor MBP refolding.
The GroEL 14-mer sedimented to the bottom of the gradient
(panel B). The presence of GroEL monomers at the top of the
gradient resulted from a partial dissociation of the GroEL 14-mer
during centrifugation in the presence of Mg2+-ATP (38).

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Fig. 3.
Resistance of refolded MBP to proteinase K
digestion. MBP denatured in 8 M urea was diluted into
the refolding buffer (final concentration 0.5 µM) and
refolded for 1 h at 22 °C in the absence (panel A)
or presence (panel B) of GroEL/GroES/Mg2+-ATP.
The refolding mixture was subsequently treated with 4 µg/ml PK also
at 22 °C for 10 min and the digestion quenched with PMSF. The
digested MBP refolding mixture (100 µl in panel A and 240 µl in panel B) was separated on a 10-25% sucrose density
gradient and fractions analyzed on 12% SDS gels, followed by Coomassie
Blue staining.
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|
The refolding kinetics of unfused and fused forms of MBP denatured in 8 M urea was studied. Aliquots taken at different times during the refolding at 22 °C were treated with PK, followed by analysis by SDS-PAGE. To increase sensitivity of the folding assay, the
PK-resistant MBP moiety was probed with the antibody to MBP by Western
blotting. Radioactivity associated with the PK-resistant folded MBP
moiety was compared with the intensity of the PK-treated native MBP or
MBP-E1 control present at the same level as that used in the refolding
reaction. Either control was expressed as 100% folded MBP. As shown in
Fig. 4, refolding of unconjugated MBP
reached 99.8 and 99.5% of the native MBP control in the absence or
presence of GroEL·GroES·Mg2+-ATP, respectively.
The plateau levels for refolding of the MBP moiety on the
MBP-
fusion polypeptide from the denatured MBP-E1 were 68.3 and
27.2% in the presence or absence of
GroEL·GroES·Mg2+-ATP, respectively (Fig. 4). The
rate constants for the first-order reaction for MBP folding in the
absence or presence of chaperonins were 0.020 and 0.036 s
1, respectively. They were in the same order of
magnitude as previously reported value of 0.025 s
1 for
spontaneous MBP refolding (39), as determined by tryptophan fluorescence measurements. The rate constants for refolding of the MBP
moiety on MBP-
in the absence or presence of chaperonins are
1.1 × 10
3 and 1.9 × 10
3
s
1, respectively (Fig. 4). These values are an order of
magnitude lower than those measured with unlinked MBP.

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Fig. 4.
Folding kinetics of unfused and fused forms
of MBP in the presence or absence of GroEL/GroES. The
urea-denatured MBP or MBP-E1 was refolded at 0.25 and 0.125 µM, respectively, at 22 °C with or without
GroEL/GroES/Mg2+-ATP. At different times, aliquots were
withdrawn and digested with 4 µg/ml PK at 22 °C for 10 min. The
same amount of native MBP or MBP-E1 treated with PK was used as a
control (100% standard). After quenching with PMSF, the digested
mixture was separated on SDS-PAGE, transferred to Immobilon-P membrane
(Millipore), and probed with the MBP antibody and
125I-protein A. The radioactivity associated with protein
bands was quantified by PhosphorImaging. , unfused MBP without
chaperonins; , unfused MBP with GroEL/GroES/Mg2+-ATP;
, MBP moiety in MBP- fusion without chaperonins; , MBP moiety
in MBP- fusion with GroEL/GroES/Mg2+-ATP.
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Folded State of the
Moiety during Renaturation of
MBP-E1--
Since the folded
subunit does not possess enzyme
activity, the refolding of the
moiety on MBP-
was monitored by
the release of MBP-
monomers from GroEL and subsequent assembly of
the folded
moiety with
to form the (MBP-
)
dimer or the
(MBP-
)2
2 tetramer (14). Aliquots
collected at different times during refolding were separated on a
sucrose density gradient, and fractions analyzed by SDS-PAGE and
Coomassie Blue staining. The reaction mixture was not extracted with
amylose resin so that measurement of the released MBP-
fusion was
not dependent on the prefolded MBP moiety. Fractions 3 to 8 corresponding to MBP-
monomers, (MBP-
)
dimers, and
(MBP-
)2
2 tetramers were quantified by
densitometry scanning (Fig.
5A). As shown in Fig.
5A, at the 0-min time point, all of MBP-
or -
was
complexed with GroEL and sedimented close to the bottom of the gradient
(fractions 13-15). The bands in fractions 3 and 4 were dissociated
GroEL monomers. At the earlier time points (5 min to 1 h), the
majority of MBP-
was present as unassembled MBP-
monomers
(fractions 3 and 4) and assembled (MBP-
)
dimers (fractions 4-6).
At later time points (2 to 24 h), the dimers were gradually
converted to (MBP-
)2
2 tetramers, which
peaked in fractions 6-8. The identities of these E1 folding
intermediates as separated by the sucrose density gradient were
confirmed by fast protein liquid chromatography gel filtration
(14).

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Fig. 5.
Kinetics of the moiety refolding on the MBP- fusion
polypeptide in the presence of GroEL/GroES. A,
sedimentation profiles on a sucrose density gradient. The
reconstitution of MBP-E1 denatured in 8 M urea was carried
out as described under "Experimen-tal Procedures." Aliquots
collected at different times without amylose resin extraction were
separated on a 10-25% sucrose gradient, and fractions analyzed by
SDS-PAGE, followed by Coomassie Blue staining. Due to space limitations, except for the 0-min point, only the
MBP- portion of the gel is shown. To normalize variations in
Coomassie Blue staining, 1.5 µg of MBP-E1 was included in each gel
(the Std. lane). The molecular mass markers (in kDa) used
for calibration were: 40, MBP; 67, bovine serum albumin; 110, human E3;
158, aldolase; 232, catalase; 800, GroEL. B, the time course
of folded MBP- . The dye intensities of MBP- bands in fractions
3-8 that contained MBP- monomers, (MBP- ) dimers, and
(MBP- )2 2 tetramers were measured by
densitometry and summed to represent folding of the moiety. The
signal was linear with up to 10 µg of MBP-E1. The highest amount of
MBP- in combined fractions at the 4-h time point was expressed as
100%. C, time course for transition from dimers (fractions
4 and 5) ( ) to tetramers (fractions 7 and 8) ( ) in
panel A and the recovery of E1 activity ( ) from a
separate experiment (Fig. 2, 0.1 µM) during the
refolding.
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|
The densities of all folded MBP-
species (fractions 3-8) were
summed and the maximum at the 4-h time point was expressed as 100%.
The percent of folded MBP-
species was plotted against the folding
time to depict the refolding kinetics of the
moiety. The folding of
the
moiety reached a plateau in about 4 h (Fig. 5B), which was much slower than the MBP (15 min) on the same
fusion polypeptide (Fig. 4). The rate constant for folding of the
moiety fit to the first-order reaction was 2.95 × 10
4 s
1. This rate constant is independent
of assembly with the
subunit, since both unassembled and assembled
MBP-
species were counted. To measure the percent formation of
dimers and tetramers during the refolding, subtotals of the normalized
MBP-
signal in fractions 4-5 (in the dimeric state) and fractions
7-8 (in the tetrameric state) were expressed as percents of the
maximal intensity and plotted against the folding time (Fig.
5C). No significant E1 activity was recovered (about 10%)
until 2 h when an appreciable amount of tetramers accumulated. The
time course for recovery of E1 activity was in approximate parallel
with that for the formation of MBP-E1 tetramers (Fig. 5C).
The slight disconcordance between the two rates was the result of two
separate experiments. The data show that the tetramer, but not the
dimer, is the enzymatically active form, and confirm that the slow
process in E1 refolding is the formation of tetramers from dimers
(14).
An MBP-
·GroEL Complex Isolated from the Transformed Bacterial
Lysate--
During the overexpression of the MBP-
fusion
polypeptide with GroEL·GroES in E. coli, a significant
amount (10 mg/liter) of the MBP-
·GroEL complex (Fig.
6, lanes 11-14) and
aggregated MBP-
(Fig. 6, lane 15) were isolated with the
amylose resin when analyzed by sucrose density gradient centrifugation.
The data were consistent with the notion that the MBP moiety was folded and capable of binding to the resin. The
moiety on the MBP-
fusion was apparently unfolded and associated with GroEL, resulting in
the formation of the MBP-
·GroEL complex. The MBP-
* polypeptide was a preterminated translation product. The MBP-
·GroEL complex isolated from the E. coli lysate produced enzymatically
active MBP-E1 when incubated with the
·GroEL complex and
GroES·Mg2+-ATP (data not shown). The result indicated
that the MBP-
·GroEL complex was a productive folding intermediate
when MBP-
was expressed in E. coli.

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Fig. 6.
Isolation of the
MBP- ·GroEL complex with amylose resin from
E. coli lysate in the absence of expression. CG712 cells were co-transformed with the
pMAL-c-hE1 and the pGroESL plasmids and the expression of MBP- ,
GroEL, and GroES was induced by
isopropyl-1-thio- -D-galactosidase. Cell lysates were
extracted with amylose resin, and proteins eluted with 20 mM maltose were subsequently separated on a 10-30%
sucrose density gradient. The bottom of the gradient (fraction 15)
contained 2 M sucrose. MBP- * was a
preterminated translation product.
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|
To ask whether the MBP-
/GroEL intermediate also occurred during
MBP-E1 folding, aliquots collected from the refolding mixture were
extracted with amylose resin and separated on a sucrose density gradient. As shown in Fig. 7, at the
10-min time point, when the majority of the MBP moiety was folded (Fig.
4) with a less than 50% maximum of the
moiety folded or complexed
with
(Fig. 5 B), a significant amount of the MBP-
·GroEL
complex was isolated with the resin (fractions 9-12) (Fig. 7,
upper panel). At the 16-h time point, only (MBP-
)
dimers (fractions 3 and 4) and (MBP-
)2
2
tetramers (fractions 5 and 6) were extracted with the resin (Fig. 7,
lower panel). The data establish that the MBP-
·GroEL complex is indeed a productive folding intermediate during the refolding of urea-denatured MBP-E1.

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Fig. 7.
Isolation of the
MBP- /GroEL intermediate from the refolding
mixture with amylose resin. Reconstitution of urea-denatured
MBP-E1 was carried out at 22 °C in the presence of
GroEL/GroES/Mg2+-ATP as described under "Experimental
Procedures." At 10-min and 16-h points, aliquots (1 ml) of the MBP-E1
refolding reaction were incubated with 500 µl of amylose resin for 10 min at 22 °C. Proteins eluted with 20 mM maltose were
separated on a 10-30% sucrose density gradient. Fractions were
analyzed by SDS-PAGE and Coomassie Blue staining.
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The trans-MBP-
·GroEL Complex as a Folding Competent
Species--
Due to the size constraint associated with the GroEL
cavity, we suspected that during the folding reaction GroES can cap the MBP-
·GroEL complex only at the open end unoccupied by the large 86-kDa fusion polypeptide, resulting in the exclusive trans
configuration. To address this question, we first studied the binding
of a smaller polypeptide 48-kDa (His)6-
to GroEL, and
compared the results with those obtained with the larger MBP-
fusion. The 1:1 (His)6-
·GroEL complex with only one
ring of GroEL occupied by the small polypeptide was incubated with
GroES without added nucleotides or in the presence of 10 mM
ADP or ATP, followed by digestion with PK. The digestion was quenched
with 5 mM PMSF, and to the mixture containing GroES and the
digested (His)6-
·GroEL complex,
·GroEL and 5 mM Mg2+-ATP were added to reconstitute E1
activity. As shown in Fig. 8A,
in the absence of nucleotides and PK, the
(His)6-
·GroEL was intact and maintained the monomeric
molar ratio of GroEL:(His)6-
= 14:1 (lane 1).
The undigested (His)6-
·GroEL complex and GroES in the
mixture, when incubated with
·GroEL and 5 mM
Mg2+-ATP, resulted in a full recovery of E1 activity, which
served as a 100% control (Fig. 8B, lane 1). In the presence
of PK but absence of nucleotides, GroES was incapable of binding to
GroEL. (His)6-
bound to GroEL was not protected and
mostly digested (Fig. 8A, lane 2), which was consistent with
the complete failure of the digested (His)6-
to
reconstitute E1 activity with
·GroEL (Fig. 8B, lane 2).
In the presence of GroES, ADP, and PK, GroES was able to cap the
(His)6-
·GroEL complex in either trans or cis configuration (31). About half of (His)6-
sequestered under GroES in cis was protected from PK
digestion (Fig. 8A, lane 3). The proteolytically protected
(His)6-
in the presence of GroES and ADP was able to
refold with the added
·GroEL complex and Mg2+-ATP,
resulting in 42% of the control E1 activity (Fig. 8B). The data depict equal probabilities for GroEL to form cis and
trans complexes with GroES and a small polypeptide. When
GroES, 10 mM ATP, and PK were present, the
(His)6-
·GroEL complex underwent multiple rounds of
polypeptide release and rebinding (40, 41). The dynamic alternations
between cis and trans configuration eventually led to digestion of most of the (His)6-
polypeptide
through binding to GroEL in trans (Fig. 8A, lane
4). This result was supported by the absence of reconstituted E1
activity when the digested mixture was incubated with
·GroEL and
Mg2+-ATP (Fig. 8B, lane 4).

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Fig. 8.
Capping of
(His)6- ·GroEL complex by GroES
in cis. The (His)6- ·GroEL complex (molar
ratio 1:1) (2.3 µM) was incubated with GroES (4.6 µM) in the absence of nucleotides (lane 2) or
in the presence of 10 mM ADP (lane 3) or 10 mM ATP (lane 4). Except lane 1, the
complex mixtures were treated with 4 µg/ml PK for 15 min at 22 °C
and the digestion quenched by the addition of 5 mM PMSF.
A, one-half of the digests treated with PMSF were analyzed
by SDS-PAGE, followed by Coomassie Blue staining. B, the
other half of PMSF-quenched reaction mixtures was used to reconstitute
E1 activity with a stoichiometric amount of the ·GroEL complex and
5 mM Mg2+-ATP. The recovery of E1 activity from
the complex without PK treatment (lane 1) was expressed as
100%.
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In a parallel experiment, the MBP-
·GroEL complex was incubated
with GroES in the presence or absence of nucleotides and subjected to
PK digestion. E1 activity reconstituted with undigested MBP-
·GroEL complex (lane 1) in the absence of nucleotides and PK (Fig.
9A, upper panel)
and the
·GroEL complex was expressed as 100% (Fig. 9B, lane
1). In the absence of nucleotides but with (Fig. 9A, upper
panel, lane 3) or without GroES (lane 2), MBP-
in
the MBP-
·GroEL complex was completely digested by PK, with the MBP
moiety remaining intact. Similar results was obtained when the
MBP-
·GroEL complex was incubated with GroES and PK in the presence
of ADP (lane 4) or ATP (lane 5). The complete
digestion of MBP-
by PK was consistent with the absence of
reconstituted E1 activity when the digested mixture was incubated with
·GroEL and Mg2+-ATP (Fig. 9B, lanes 3-5).
The residual E1 activity (Fig. 9B, lanes 2-5) represented
background of the radiochemical assay. The capping of GroEL by GroES
was examined by loading a fraction of the digestion mixture on the gel
as shown in the Fig. 9A (bottom panel). The
C-terminal 16 amino acid residues of the GroEL ring not capped by GroES
were digested by PK, resulting in smaller cut monomers without
affecting the GroEL function (31). The 1:1 ratio of uncut and cut GroEL
(lane 4) confirmed that GroEL·GroES formed the 1:1
asymmetric complex in the presence of ADP as established by x-ray
crystallographic studies (30). These data, taken together, establish
that the formation of cis MBP-
·GroEL complex is
sterically impossible, supporting the thesis that refolding of MBP-
is mediated exclusively through the trans configuration of
the chaperonin complex.

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Fig. 9.
Inability of GroES to cap
MBP- ·GroEL complex in
cis. As a parallel experiment to Fig. 8, the
MBP- ·GroEL complex (molar ratio 1:1) (1.7 µM) was
either untreated (lane 1) or digested with PK (4 µg/ml) in
the presence of 3.4 µM GroES without added nucleotides
(lane 3), with 10 mM ADP (lane 4) or
with 10 mM ATP (lane 5). The MBP- ·GroEL
complex was also digested with PK in the absence of GroES (lane
2). A, untreated and PK-treated mixtures were separated
by SDS-PAGE, followed by Coomassie Blue staining. The gel in the
top panel was loaded with 45% of the incubation mixtures,
and the gel in the bottom panel with 5% of the mixtures.
B, the remainder of the incubation mixtures was incubated
with a stoichiometric amount of the ·GroEL complex and 5 mM Mg2+-ATP to reconstitute E1 activity. The
recovery of E1 activity from the MBP- ·GroEL complex without the PK
digestion (lane 1) was expressed as 100%.
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GroES/Mg2+-ATP-dependent Release of MBP-
Monomers from the MBP-
·GroEL Complex--
To determine whether
GroES was involved in the folding of MBP-
, the release and folding
of the fusion polypeptide from the MBP-
·GroEL complex was studied.
The MBP-
·GroEL complex was incubated with Mg2+-ATP
alone or Mg2+-ATP and GroES for different durations. After
incubation, MBP-
monomers separated on a sucrose density gradient
were quantified by SDS-PAGE and densitometric scanning. Fig.
10 shows that, in the presence of GroES
and Mg2+-ATP, the MBP-
fusion is readily released from
the MBP-
·GroEL complex, and exists as soluble monomers. The level
of the released monomers remains constant from 10 min to 2 h. In
contrast, when the complex is incubated with Mg2+-ATP
alone, the level of the discharged MBP-
monomers is less than 1/25
of that obtained when the complex is incubated with GroES and
Mg2+-ATP for the same durations. The results indicate that
GroES is required for the efficient release and folding of MBP-
monomers from the MBP-
·GroEL complex.

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Fig. 10.
Dependence on GroES for efficient release of
MBP- monomers from the
MBP- ·GroEL complex. The MBP- ·GroEL
complex (molar ratio 1:1) (60 µg) was incubated with 10 mM ATP in the presence (shaded bar) or absence
(solid bar) of GroES for different lengths of time. After
incubation, the mixtures were separated on a 10-25% sucrose density
gradient, and fractions analyzed by SDS-PAGE and Coomassie Blue
staining. The amount of MBP- monomers (fractions 3-5) released from
GroEL was quantified by densitometric scanning. The minimal density
obtained from the 10-min incubation in the presence of
Mg2+-ATP alone (solid bar) was treated as unity.
The amounts of MBP- monomers released in other samples were
expressed as relative to the minimum.
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DISCUSSION |
Reconstitution of MBP-E1 with GroEL/GroES/Mg2+-ATP
in vitro provides a novel system to study the question of
differential domain folding in the presence of chaperonins. The kinetic
data from this study indicate that the refolding of MBP-E1 begins with
the proper local folding of MBP and
moieties on the MBP-
chimeric polypeptide. This is ensued by the association of MBP-
(with the
moiety folded) and
into a dimer, and subsequent
dimerization of two (MBP-
)
dimers into an
(MBP-
)2
2 tetramer. The time course for
reconstitution of E1 activity essentially superimposes with that for
the formation of MBP-E1 tetramers, suggesting that the dimerization
step is rate-limiting in the renaturation of E1. This result is similar
to the refolding of (His)6-tagged E1 (14). The transition
of dimers to tetramers as the rate-limiting step is also observed in
homotetrameric proteins, e.g. bacterial phosphofructokinase (42) and lactate dehydrogenase, although their rates of folding are
approximately 100-fold faster than that of E1 dimers. In the case of
mitochondrial malic dehydrogenase, the rate constant for the
association of two monomers into a native dimer is 3 × 104 M
1 s
1 (43),
which is 2 orders of magnitude higher than that of E1 refolding with
rate constants of 376 M
1 s
1 for
MBP-E1 and 465 M
1 s
1 for
untagged E1. Moreover, malate dehydrogenase can refold at the same rate
either in the presence or absence of GroEL/GroES, although the yield of
spontaneous refolding is only 15%, which is considerably lower than
that of GroEL/GroES-dependent folding (80-90%) of the
same enzyme (43). In contrast, the folding of E1 has an absolute
requirement for the presence of GroEL/GroES/Mg2+-ATP, and
an excess molar ratio (GroEL:E1 monomer) of GroEL is necessary to
achieve the maximal degree of refolding (14).
MBP has been shown to function as a chaperone, similar to Hsp70
proteins, in assisting the refolding of citrate synthase. In the
presence of DnaK (the bacterial homologue of Hsp70) or MBP, 22 and
13%, respectively, of refolding for citrate synthase can be achieved
(44). Although less efficient than GroEL and GroES as chaperones (45),
MBP or Hsp70 may help the refolding of citrate synthase by
sequestering hydrophobic surfaces on the unfolded polypeptide. These
results imply that the periplasmic MBP may interact with unfolded
proteins in that compartment, resulting in an increase in productive
folding. In the present study, fusion of MBP moiety to the
sequence
does not promote folding of the MBP-E1 proteins. In the MBP-
fusion,
the rate constant for refolding of the MBP moiety (1.1 × 10
3 s
1) is 20-fold slower than that
measured with unfused MBP (0.020 s
1), suggesting that
there is intramolecular interference between MBP and
sequences
during refolding of the fusion polypeptide. This observation is similar
to the folding kinetics of individual domains during the spontaneous
folding of multidomain proteins. The refolding of an individual domain
is usually more rapid than when it occurs in a multidomain protein
(21-25). The intermolecular interference in folding is of functional
significance during protein transport. For example, the precursors of
MBP (18) and ribulose-binding protein (46), the presence of leader
sequences at N termini of these proteins reduces the rate of folding by
30-40-fold in comparison with their mature forms. It is proposed that
the targeting sequence of some exported proteins could behave as an
inhibitor of folding. The retarded folding of an exporting protein
allows a sufficient time for the partially folded precursor to be
transported to a correct target site in the cell with the help of Hsp70
(46). On the other hand, negative interactions between concurrently folding domains may result in intramolecular misfolding. As a case in
point, only 10% of total Ras-dihydrofolate reductase was in the
soluble native form when expressed in E. coli (26). It has
been suggested that co-translational domain folding has evolved in
eukaryotic cytosol to mitigate the adverse intramolecular interference during the folding of large multidomain polypeptides (26). However, this mechanism is not relevant to bacterial or mitochondrial proteins which fold post-translationally or after import and processing (11-13).
Molecular chaperones may play the role of an unfoldase by reversing the
off-pathway or that of a foldase by decreasing the activation energy of
a folding intermediate along the folding pathway, and this results in
an increase in the yield and/or rate of a folding reaction (6, 47-49).
The similar rates for refolding of the MBP moiety on MBP-
in the
absence and presence of GroEL/GroES indicate that GroEL and GroES do
not act as a catalyst for MBP folding. It is noteworthy, however, the
yield of MBP refolding in the present study increases from 25 to 30%
in spontaneous refolding to 70-90% when assisted by GroEL/GroES. In
contrast, folding of the
moiety (rate constant, 2.95 × 10
4 s
1) absolutely requires the presence of
GroEL/GroES, and is about 1 order of magnitude slower than folding of
the MBP moiety (rate constant, 1.9 × 10
3
s
1) on MBP-
. The differential requirement for
chaperonin-assisted folding remains unchanged for MBP and
moieties
either as individual proteins or in the form of a fusion polypeptide.
Moreover, refolding of MBP-
as assisted by GroEL and GroES produces
an intermediate, i.e. the MBP-
·GroEL complex, in which
part of the polypeptide chain, i.e. the MBP moiety, has
already reached a native conformation which is resistant to PK and
capable of binding to amylose resin, while the other segment of the
chain, i.e. the
moiety, is still unfolded and associated
with GroEL. This binding topology is expected to also occur in
mitochondria between Hsp60 and large mature polypeptides with different
folding characteristics between domains, as a means to circumvent aggregation.
The interaction between GroEL and GroES in mediating the folding of
large polypeptides such as MBP-
is unknown. Studies on chaperonin-assisted refolding of small polypeptides, e.g.
ornithine transcarbamylase monomers (36 kDa), have shown that
productive folding occurs exclusively through the cis
complex, in which GroES and the unfolded polypeptide bind to the same
ring of GroEL (31). However, this mechanism raises the question as to
the size constraint for a polypeptide whose folding can be assisted by
GroEL in a GroES-dependent manner. It has been speculated
that GroES may play a role in promoting the productive release of large
polypeptides from a trans configuration, that is, GroES and
unfolded polypeptide bind to the opposite rings of GroEL (31). Here, PK
digestion data provide evidence that the large MBP-
fusion
polypeptide indeed binds to the asymmetric GroEL·GroES complex
exclusively in trans (Fig. 9). One can argue that the
MBP-
fusion forms a stable complex with GroEL but its folding is not
assisted by GroES, similar to that observed with the large 124-kDa
phytochrome photoreceptor (50) and the 72-kDa tail-spike protein of
phage P22 (51). However, the level of MBP-
monomers released from
the MBP-
·GroEL complex with GroES and Mg2+ATP is
sharply higher than that obtained with Mg2+-ATP alone
(Fig. 10). Thus, our data strongly suggest that interactions between GroEL and GroES are needed for efficient folding of the MBP-
fusion. The availability of the stable MBP-
·GroEL binary complex in vitro provides a system to investigate how GroES
promotes the productive release of large polypeptides from GroEL.