From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390
Received for publication, September 21, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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We described previously that during the
assembly of the Chaperonins GroEL/GroES, which are homologues of mitochondrial
Hsp60/10, respectively, have been shown to promote proper folding and
assembly of a variety of proteins (for review see Refs. 1 and 2). GroEL
is a double-ring complex with two heptameric rings, consisting of
identical 57-kDa subunits, which are stacked back to back. The
efficient folding for most proteins has been shown to occur inside the
cavity of the cis ring that houses the unfolded or partially
folded protein, following encapsulation or capping of the
cis ring by dome-shaped heptameric GroES in the presence of
Mg-ATP (3). The subsequent binding of Mg-ATP to the unoccupied or
trans ring of GroEL results in the collapse of the
cis ring assembly with the concomitant release of GroES and
partially or completely folded protein from the cis cavity. It has been generally accepted that only proteins of 55-57 kDa or
smaller in size can fit inside the cavity encapsulated by GroES, as
shown by in vivo (4, 5) and in vitro
(6) studies. For certain proteins larger than this size, for example,
75-kDa methylmalonyl-CoA mutase (3) and 72-kDa phage P22 tailspike protein (7) that are capable of binding to GroEL, chaperonin-assisted folding is independent of GroES. It was shown recently, however, that
both chaperonins GroEL/GroES are required for the productive folding of
an 86-kDa maltose-binding protein fusion (8) and 82-kDa mitochondrial
aconitase (9). Interestingly, for this group of large proteins,
productive folding is achieved through binding of GroES to the
trans ring of GroEL. GroEL was also shown to trap
heat-induced inactive 98-kDa citrate synthase homodimers (10) and
thermally induced partially active 66-kDa rhodanese dimers (11). In the
presence of GroES and Mg-ATP, the trapped dimers of citrate synthase or
rhodanese are released from GroEL in the monomeric form. It is not
clear whether GroES binds in cis or in trans to
the complex formed between GroEL and these relatively large folded intermediates.
Our laboratory is interested in the potential role of chaperonins in
promoting the assembly of mitochondrial macromolecular multi-enzyme
complexes. We have shown that chaperonins GroEL/GroES are indispensable
for both folding and assembly of the Because of the large size associated with the Materials--
Bacterial chaperonins GroEL/GroES and recombinant
human BCKD were prepared as described previously (13).
1-Anilino-8-naphthalenesulfonate (ANS), trypsin (type III, from bovine
pancreas), trypsin-chymotrypsin inhibitor (from soybean), Reactive Red
120-agarose (type 3000-CL), and Expression and Purification of SR1--
The expression plasmid
for SR1 was transformed into BL21 (DE3) cells. Cells were grown in the
LB medium containing 100 µg/ml ampicillin at 37 °C overnight, and
expression for SR1 was induced with 1 mM
isopropyl- Engineering and Expression of BCKD Preparation of GroEL-Protein and SR1-Protein
Complexes--
KSCN-induced
GroEL- Limited Digestion of Chaperonin-Protein Complex with
Trypsin--
GroEL-protein and SR1-protein complexes at 0.8 µg/µl
in Buffer C with or without 0.67 µg/µl GroES, 13 mM
Mg-ADP, or Mg-ATP were subjected to limited digestion with 20 µg/ml
trypsin at 23 °C for 10 min. The reaction was terminated by adding
trypsin-chymotrypsin inhibitor to a final concentration of 27 µg/ml.
Samples were analyzed by SDS-PAGE and Western blotting using a
combination of anti- Fluorescence Assays of BCKD and BCKD Fusion Proteins with
ANS--
The urea-denatured Co-purification of GroES with SR1- GroEL/GroES-mediated Recovery of BCKD Activity--
The
renaturation of urea-denatured BCKD and Expression and Characterization of Large Target Proteins--
The
His6-tagged
Conformations of the target proteins were characterized by fluorometry
using the ANS as a hydrophobic probe. Native and urea-denatured proteins were incubated with 50 µM ANS. The reaction
mixtures were excited at 365 nm, and fluorescence emission measured as a function of wavelength (Fig. 1). ANS
bound to the Capping of the 86-kDa Native-like Heterodimeric Intermediate inside
GroEL and SR1 Cavities by GroES--
Double-ring GroEL or its
single-ring variant SR1 was incubated with a molar excess of
KSCN-induced
Under identical conditions, the SR1- Unfolded 86-kDa Both Subunits of the
The enclosure of the SR1- GroEL/GroES-dependent Refolding of Wild-type and
Chaperonin-mediated recovery of BCKD activity was also studied using 8 M urea-denatured wild-type BCKD or The The crystal structure of GroEL-GroES-ADP7 complex shows a
2-fold enlargement of the cis cavity over the
trans to a volume of 175,000 Å3 (20). This
volume theoretically is capable of accommodating a globular protein of
~142 kDa beneath GroES, assuming a perfect fit to the actual folded
protein volume (20). Nonetheless, the upper limit for an unfolded
protein to be encapsulated inside the GroEL cavity by GroES has been
shown to be 57 kDa in both in vitro (6) and in
vivo (4, 5) studies. This size constraint of 57 kDa for an
unfolded protein may reflect that an unfolded polypeptide is more
extended than the fully folded protein of similar size. As a result,
the space larger than the actual protein volume is needed for an
unfolded protein inside the cis cavity to be encapsulated by
GroES. As a case in point, we show that the urea-denatured 86-kDa
2
2 heterotetramer
of human mitochondrial branched-chain
-ketoacid dehydrogenase
(BCKD), chaperonins GroEL/GroES interact with the kinetically trapped
heterodimeric (
) intermediate to facilitate conversion of the
latter to the native BCKD heterotetramer. Here, we show that the 86-kDa
heterodimeric intermediate possesses a native-like conformation as
judged by its binding to a fluorescent probe
1-anilino-8-naphthalenesulfonate. This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and
its single-ring variant SR1 and is encapsulated by GroES as indicated
by the resistance of the heterodimer to tryptic digestion. The
SR1-
-GroES complex is isolated as a stable single species by gel
filtration in the presence of Mg-ATP. In contrast, an unfolded BCKD
fusion protein of similar size, which also resides in the GroEL or SR1
cavity, is too large to be capped by GroES. The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-
complex, GroES, and Mg-ATP. The 86-kDa
native-like heterodimeric intermediate in the BCKD assembly pathway
represents the largest protein substrate known to fit inside the GroEL
cis cavity underneath GroES, which significantly exceeds
the current size limit of 57 kDa established for unfolded proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2
heterotetramer of branched-chain
-ketoacid dehydrogenase
(BCKD),1 a component of the
4 × 106-Da human mitochondrial BCKD complex, both in
Escherichia coli (12) and in vitro (13).
GroEL binds to an ensemble of 86-kDa
heterodimeric
intermediates, which are kinetically trapped during assembly, to
produce a stable GroEL-
complex. In the presence of GroES and
Mg-ATP, the heterodimeric intermediate undergoes multiple rounds of
dissociation and reassociation to facilitate the conversion of the
heterodimeric intermediate to the native BCKD heterotetramer (14, 15).
Our data have established the central role of chaperonin GroEL/GroES in
promoting oligomeric protein assembly through iterative annealing of
non-productive assembly intermediate.
assembly
intermediate of BCKD, a question arises as to whether the productive folding of the GroEL-
complex proceeds through the cis
or trans capping of the GroEL cavity by GroES. We have shown
previously that the GroEL-
complex is resistant to protease
digestion in the presence of GroES and Mg-ADP, suggesting the enclosure
of the 86-kDa heterodimer inside the GroEL cis cavity by
GroES (15). However, the interpretation of these data was complicated
by the presence of the trans ring in GroEL. In the present
study, we revisited this issue by investigating the encapsulation of
the heterodimer inside GroEL along with its single-ring variant SR1. We
showed that the 86-kDa heterodimeric intermediate inside the cavities
of both GroEL and SR1 was encapsulated by GroES. In contrast, an
unfolded fusion protein of similar size, which also resides in the
GroEL or SR1 cavity, is too large to be capped by GroES. The 86-kDa
heterodimeric intermediate represents the largest protein substrate
ever known to fit inside the GroEL cis cavity underneath GroES. This finding indicates that for assembly intermediates with
compact native-like conformations, the size limit for cis folding is significantly larger than 57 kDa established for unfolded proteins (4-6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactalbumin (type III,
calcium-depleted, from bovine milk) were obtained from Sigma.
Bovine lactoferrin was purchased from ICN Biochemicals, Inc. (Aurora,
OH). A pET vector for the expression of single-ring GroEL variant SR1
was a generous gift from Dr. Arthur Horwich.
-D-thiogalactopyranoside for 3 h at
37 °C. The SR1 protein was purified at 4 °C using a previously
described protocol with modifications (16). Briefly, cells from 4-liter culture were collected and re-suspended in 150 ml of buffer containing 20 mM Tris, pH 7.4, 50 mM KCl, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.4 mg/ml
lysozyme. After cells were broken by sonication, the lysate was
fractionated by centrifugation in a Ti60 rotor at 120,000 × g for 35 min at 4 °C. The supernatant was applied to a
Q-Sepharose Fast-Flow column (Amersham Biosciences), and proteins were
eluted with a gradient of 0 to 1 M NaCl in Buffer A
(50 mM Tris, pH 7.4, 1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride). SR1-containing fractions were collected, and protein was precipitated with 65% ammonium sulfate. Following centrifugation, the protein pellet was dissolved in Buffer A,
and solubilized proteins were fractionated on an FPLC Superdex 200 gel
filtration column equilibrated in the same buffer. At this stage, the
SR1 protein was >98% pure as judged by SDS-PAGE. To remove minor
contaminants, it was dialyzed against 20 mM Tris, pH 7.4, and 5 mM MgCl2 and purified further on a
Reactive Red 120-Agarose (type 3000-CL) dye column equilibrated in the
same buffer (17).
-11-
Fusion
Protein--
For the construction of an expression plasmid for the
-11-
fusion, three fragments from the pHisT-E1 vector (18) were produced: a 45-bp NcoI/XhoI fragment containing
the His6 tag and the tobacco etch-virus protease cleavage
site, a 751-bp XhoI/SmaI fragment encoding a 5'
portion of the
subunit, and a 1202-bp PstI/EcoRI fragment encoding a C-terminal segment
of the
subunit. The linker region of 11 amino acids (GSEALEAAERS)
between the
and
subunit was derived from the sequence
connecting the
and
domains of tryptophan synthase (19). The
nucleotide sequence encoding this linker was
5'-GGATCCGAAGCATTAGAAGCCGCTGAGAGATCT-3', which contained a
BamHI site at the 5' end and a BglII site at the
3' terminus. The BCKD-
cDNA with the linker immediately
downstream of the 3' cDNA end was amplified, followed by digestion
of the PCR product to produce a SmaI/BamHI
fragment. The BCKD-
cDNA with the linker attached to the 5'
cDNA end was also amplified, and digestion of this PCR product
resulted in a PstI/BamHI fragment. The
SmaI/BamHI and PstI/BamHI
fragments, along with the above three fragments derived from the
pHisT-E1 plasmid, were ligated into the pTrcHisB expression vector
(Invitrogen) pre-digested with NcoI and EcoRI.
The resultant plasmid pHisT-
-11-
and the pGroESL plasmid
overexpressing GroEL and GroES (12) were co-transformed into BL21
cells. Transformed cells were grown in the LB medium at 37 °C until
A600 nm at 0.75 was reached. Expression of the
BCKD fusion protein was induced with 1 mM
isopropyl-
-D-thiogalactopyranoside, followed by an
overnight growth at 37 °C. The His6-tagged BCKD
-11-
fusion protein was isolated from the cell lysate with
nickel-nitrilotriacetic acid-agarose as described previously (18).
heterodimers were produced by
incubating His6-tagged BCKD (11.5 µM,
2
2 heterotetramer) with 400 mM KSCN in Buffer B (50 mM potassium phosphate,
pH 7.5, 250 mM KCl) containing 2 mM
dithiothreitol for 45 min at 23 °C (14). The incubation mixture was
diluted 2-fold into Buffer B containing 8 µM GroEL or
SR1. Following incubation for 4 h at 23 °C, the complex formed was purified on an FPLC Superdex 200 gel filtration column. Fractions containing the GroEL-
or the SR1-
complex were collected
and concentrated in a Millipore (Bedford, MA) Ultrafree-15 filter device with a 30-kDa cut-off membrane.
-11-
and SR1-
-11-
complexes were prepared by diluting
the 8 M urea-denatured
-11-
fusion protein into
Buffer C (50 mM potassium phosphate, pH 7.5, 100 mM KCl) containing GroEL or SR1 on ice. The mixture was
kept at 4 °C overnight, concentrated, and purified on an FPLC
Superdex 200 column as described above.
subunit and anti-
subunit antibodies as a probe.
-11-
fusion protein was generated by
incubation with 8 M urea at 23 °C for 1 h. Native
BCKD, the KSCN-induced heterodimer, or the urea-denatured
-11-
fusion protein at 0.1 mg/ml was mixed with 50 µM ANS in
Buffer C. Emission spectra were recorded from 400 to 550 nm with the
excitation wavelength at 365 nm on a PerkinElmer Life Sciences
luminescence spectrometer LS 50B. Each spectrum was an average of three
consecutive scans and was corrected for contributions from the buffer solution.
or SR1-
Complexes by
HPLC and Quantification of Protein Components by Densitometry--
The
SR1-
complex was prepared as described previously except that SR1
was used instead of GroEL (13). The SR1-
or SR1-
complex (60 µg) was incubated with 100 µg of GroES in Buffer C containing 5 mM Mg-ATP for 5 min at 23 °C. The mixture was
fractionated on an HPLC G3000SWXL gel filtration column in
the same buffer containing 0.1 mM Mg-ATP. As a control, the
SR1-
complex was incubated with GroES in the absence of Mg-ATP and
separated on HPLC with Mg-ATP omitted from the column buffer. 5 min
after the sample injection, fractions were collected every 20 s
and analyzed by SDS-PAGE. To determine the stoichiometry of protein
components in the SR1-
-GroES or the SR1-
-GroES complex,
different amounts of SR1, BCKD, or GroES were run on the same gel.
After staining with Coomassie Blue, gels were scanned on a Molecular
Dynamics densitometer (model 300A) and analyzed by ImageQuant. Standard curves for SR1, BCKD, and GroES were generated by plotting the known
amount of the protein against the intensity of the band on the same
gel. The amount of each protein component in the SR1-
(or
-
)-GroES complex was calculated using the standard curve for each protein.
-11-
fusion protein was
carried out as described previously (13). BCKD activity was also
recovered by incubating 0.8 µM of the GroEL-
or the GroEL-
-11-
complex with 2 µM GroES and 10 mM Mg-ATP as described previously (14).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimeric intermediate in the BCKD
assembly pathway was produced by incubating the N-terminally
His6-tagged BCKD
2
2
heterotetramer in 400 mM KSCN for 45 min at 23 °C,
followed by a 2-fold dilution. The size of the resultant
heterodimer was 86 kDa, as determined by FPLC gel filtration and
dynamic light scattering in the presence of 200 mM KSCN
(14). A second target protein, the His6-tagged
-11-
fusion, was generated by fusing the N-terminally
His6-tagged
subunit to the
subunit through an
11-residue linker derived from E. coli tryptophan synthase (19). The
-11-
fusion migrated as a 170-kDa species in FPLC gel
filtration, similar to the size of wild-type BCKD heterotetramer. The
-11-
fusion shows Km values of 0.5 µM for thiamin pyrophosphate and 62 µM for
substrate
-ketoisovalerate, essentially indistinguishable
from those obtained with the wild-type BCKD (Table
I). The kcat value
for the
-11-
fusion is 4.0 s
1, which is
approximately one-half that obtained with the wild-type BCKD. The size
of the 8 M urea-denatured
-11-
fusion was determined by sedimentation equilibrium centrifugation (data not shown). The data
were fitted to a single species model with a size of 86,671 Da for the
unfolded
-11-
fusion polypeptide. The above data, taken together,
indicate that the native
-11-
exists as a homodimeric
protein.
Kinetic constants for wild-type 2
2 and
-11-
fusion BCKD
assembly intermediate or the
2
2 native BCKD exhibits similar spectra with a maximum at 473 nm. The data indicate that the heterodimeric assembly intermediate possesses a folded structure similar to native
BCKD. In contrast, the urea-denatured
-11-
fusion with the
unfolded conformation fail to bind to ANS, as indicated by the absence
of fluorescence emission after subtracting emission from the unbound
ANS.
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Fig. 1.
Fluorescence emission spectra of ANS-bound
wild-type BCKD ( 2
2), its
heterodimeric intermediate (
), and
urea-denatured
-11-
fusion. ANS (50 µM) were mixed with wild-type
His6-tagged BCKD
(
2
2) in Buffer C,
KSCN-induced heterodimeric intermediate (
) in 200 mM
KSCN, or urea-denatured
-11-
fusion in 8 M urea (see
"Experimental Procedures"). Following a 5-min incubation at
23 °C, the emission spectrum was recorded at the excitation
wavelength of 365 nm. Each spectrum was an average of three consecutive
scans and was corrected for contributions from the buffer
solution.
heterodimer; the resultant GroEL-
and
SR1-
complexes were purified by FPLC gel filtration. Identical
amounts of the GroEL-
or the SR1-
complex were incubated with GroES and trypsin in the presence or absence of nucleotides for 10 min at 23 °C. Digestion mixtures were analyzed by SDS-PAGE (upper panel) and Western blotting (lower panel)
(Fig. 2). In the absence of GroES and
nucleotide, the
heterodimer bound to GroEL was digested
completely after incubation of the complex with trypsin (Fig. 2,
both panels, lane 2). The heterodimer
was also digested when GroES alone (lane 3), or GroES and
Mg-ATP (lane 5) were added to the GroEL-
complex (Fig.
2, both panels). Under these conditions, GroES was unable to
form a stable complex with GroEL, resulting in the degradation of the
heterodimer by the protease. The smaller bands under GroEL,
which are stained with Coomassie Blue (upper panel), do not
represent the undigested
heterodimer, as indicated by the
absence of cross-reacting materials in Western blotting with the
combined anti-
and anti-
antibodies as probes (Fig. 2,
lower panel, lanes 3 and
5). These smaller bands, which migrate slower than GroES,
are proteolytic products of GroEL. In the presence of Mg-ADP and GroES
(lane 4), a significant portion of the heterodimer bound to
GroEL was protected from tryptic digestion (Fig. 2, both
panels). The amounts of
and
subunits resistant to trypsin
digestion were 56.3 and 39.7%, respectively, of the starting material
(lane 1) as determined by densitometry of the Western blot
(Fig. 2, lower panel). The data are explained by the fact
that GroES binds equally to either the cis or the
trans ring of GroEL, resulting in approximately half of the
cis cavity being capped by GroES. Only the
heterodimer inside the GroES-enclosed cis cavity is
resistant to tryptic digestion.
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Fig. 2.
Limited tryptic digestion of
GroEL- and
SR1-
complexes in the presence
and absence of GroES and nucleotides. GroEL-
and SR1-
complexes at 0.8 µg/µl were digested with 20 µg/ml trypsin under
specified conditions at 23 °C for 10 min. The reaction was
terminated by adding 27 µg/ml trypsin-chymotrypsin inhibitor. Samples
were analyzed by SDS-PAGE (upper panel) and Western blotting
(lower panel) using a combination of anti-
subunit and
anti-
subunit antibodies as a probe.
complex was digested with
trypsin in the presence of GroES with or without nucleotides. Only in
the presence of GroES and Mg-ATP, where a stable SR1-GroES complex was
found (3), was the SR1-bound
heterodimer resistant to tryptic
digestion (Fig. 2, both panels, lane
10). The
and
subunits protected from the protease
digestion (lane 10) were 89.4 and 101.4% of the starting
materials (lane 6), respectively, as measured by
densitometry of the Western blot (Fig. 2, lower panel). The
results corroborate that 86-kDa
native-like heterodimer, despite
its large size, fits inside the single SR1 cavity encapsulated by
GroES, similar to that observed with GroEL.
-11-
Fusion in GroEL and SR1 Cavities Cannot
Be Capped by GroES--
Similar protease protection assays were
carried out with the complex formed between urea-unfolded
-11-
fusion and GroEL or SR1. Incubation of the urea-denatured
-11-
fusion with GroEL or SR1 produced stable GroEL-
-11-
and
SR1-
-11-
complexes, which were isolated as single species by FPLC
gel filtration (data not shown). As shown in Fig.
3, the
-11-
fusion bound to GroEL was digested completely as indicated by Coomassie Blue staining, even
when both GroES and Mg-ADP were present (lane 4). These
conditions facilitate the formation of a stable
GroEL-GroES-ADP7 complex. The data indicate that unfolded
-11-
fusion, despite its similar size to the
heterodimer,
cannot be enclosed by GroES inside GroEL cavities. As positive
controls, the unfolded
-11-
fusion bound to GroEL was also
digested completely when GroES (lane 2) or the nucleotide
(lane 3) was absent or when GroES and Mg-ATP were present
(lane 5) (Fig. 3). In the same experiment, the unfolded
-11-
bound to the single-ring SR1 was also digested completely by
trypsin in the presence of GroES and Mg-ATP (Fig. 3, lane
10). In the presence of Mg-ATP, GroES binds to unoccupied SR1 to
form a stable complex. The data indicate that unfolded
-11-
in
the SR1 cavity prevents the capping of SR1 by GroES, resulting in degradation of the unfolded
-11-
fusion. The bands under GroEL (lanes 3-5) and SR1 (lanes 8-10) are
degradation products of the respective chaperonin (Fig. 3), similar to
those shown in Fig. 2. These bands only appear when GroES is present,
which is consistent with the finding that interactions between GroEL
and GroES result in conformational changes in the former (20).
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Fig. 3.
Limited tryptic digestion of
GroEL- -11-
and
SR1-
-11-
fusion
complexes. GroEL-
-11-
and SR1-
-11-
fusion complexes at
0.8 µg/µl were digested under specified conditions as described for
Fig. 2. Samples were analyzed by SDS-PAGE followed by Coomassie Blue
staining.
Heterodimer Remain inside the SR1
Cavity Enclosed by GroES--
We have shown previously that in the
presence of Mg-ATP, a portion of the
heterodimer in the
GroEL-
complex undergoes dissociation, resulting in the release
of the
subunit into the bulk solvent (15). The SR1-
preparation showed that only 55% of SR1 cavities were filled with the
heterodimer (see Fig. 5B). It is possible that in the
presence of GroES and Mg-ATP, the
heterodimer in the SR1-
complex also dissociates into individual subunits with the released
subunit binding to a new unoccupied SR1 cavity prior to the capping by
GroES. In this scenario, the individual SR1-
and SR1-
complexes
would present no size constraints for GroES capping and would be
protected from tryptic digestion. To rule out this possibility,
remaining empty cavities in the SR1-
preparation were filled by
incubation with excess calcium-depleted reduced 14-kDa
-lactalbumin
that binds to chaperonins with high affinity (21). Fully occupied SR1
cavities were then capped by GroES in the presence of Mg-ATP. After
tryptic digestion, the levels of protected
and
subunits were
determined by SDS-PAGE and Western blotting and quantified by
densitometry. Fig. 4 shows that most, if
not all, of the
and
subunits in the fully occupied SR1-
-GroES preparation are recovered at the levels of 81 and 83%, respectively, of the amount in the undigested complex. Because all SR1 cavities are occupied, if
and
subunits were released into the bulk solution, they would not be able to bind to new SR1
cavities and would therefore be degraded by the protease. The above
results exclude the possibility that the heterodimer dissociates into
individual
and
subunits with the
subunit escaping from the
original SR1 cavity prior to capping of the same cavity by GroES.
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Fig. 4.
Recovery of BCKD subunits from the
trypsin-digested SR1- complex
saturated with calcium-free reduced
-lactalbumin. The SR1-
complex was
incubated with a large excess amount of calcium-depleted reduced
-lactalbumin in the presence of 2 mM dithiothreitol and
0.5 mM EGTA for 10 min at 23 °C. The
-lactalbumin-saturated SR1-
complex preparation was purified
on an FPLC Superdex 200 column, followed by tryptic digestion in the
presence of GroES and Mg-ATP as described for Fig. 2. The same amount
of the SR1-
complex with trypsin omitted served as a control.
Samples were analyzed by Western blotting using anti-
antibody or
anti-
antibody, respectively. Densities of the
subunit and the
subunit on the same blot were scanned and quantified. The densities
of
and
subunits without trypsin digestion were set as
100%.
complex by GroES was deciphered
further. As a control, in the absence of Mg-ATP, SR1-
and excess GroES do not form a complex and migrate separately in HPLC-gel filtration (Fig. 5A, top
panel). In the presence of Mg-ATP, a fraction of GroES co-migrates
with SR1-
to produce a stable SR1-
-GroES complex (Fig.
5A, middle panel). Molar stoichiometry of
SR1:
:GroES in the ternary complex is 1:0.86:0.90 as determined by
scanning densitometry (Fig. 5B). The data confirm that
Mg-ATP promotes the stable binding of GroES to SR1, without causing the
release of the protein substrate. In a parallel experiment, the
SR1-
complex was incubated with excess GroES and Mg-ATP, followed
by separation by HPLC-gel filtration. As also shown in Fig.
5A (bottom panel), a stoichiometric amount of
GroES binds to SR1-
complex. The molar ratio of SR1:
:GroES
was estimated to be 1:0.55:1.08 (Fig. 5B). The result
indicates that 45% of the SR1 cavity is not occupied by the protein
substrate. This was corroborated by the additional binding of
calcium-depleted reduced
-lactalbumin to the same SR1-
preparation at a subunit ratio of SR1:
-lactalbumin = 1:0.40
(data not shown). The combined data indicate that the
heterodimer remains inside the same SR1 cavity during the capping of
SR1 by GroES and that all of the SR1 cavities including those occupied
by the
heterodimer are encapsulated by GroES.
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Fig. 5.
HPLC purification and subunit stoichiometry
of SR1- -GroES and
SR1-
-GroES complexes.
A, purification of SR1-
-GroES and SR1-
-GroES
complexes. SR1-
(middle panel) and SR1-
(bottom panel) complexes were incubated with excess amount
of GroES and 5 mM Mg-ATP for 5 min at 23 °C and
fractionated on an HPLC G3000SWXL gel filtration column in
the presence of 0.1 mM Mg-ATP. SR1-
-GroES- or
SR1-
-GroES-containing fractions were collected and analyzed by
SDS-PAGE. As a control, the SR1-
complex (top panel) was
incubated with GroES in the absence of Mg-ATP and separated on HPLC
with Mg-ATP omitted from the column buffer. B, the
stoichiometry of protein components in the SR1-
-GroES or the
SR1-
-GroES complex. Subunits of the purified SR1-
(or
-
)-GroES complex were separated by SDS-PAGE, along with subunit
standards on the same gel. After staining with Coomassie Blue, gels
were scanned and analyzed by ImageQuant. The amount of each subunit in
the SR1-
(or -
)-GroES complex was calculated using the
standard curve for each polypeptide.
-11-
Fusion BCKD--
Because the single-ring SR1 was unable to
refold BCKD proteins, a complete system comprising the double-ring
GroEL complex, GroES, and Mg-ATP was utilized in refolding studies.
Incubation of the GroEL-
complex with GroES and Mg-ATP resulted
in a recovery of 72% of BCKD activity (Fig.
6A). The activity of BCKD
heterotetramer based on the amount of heterodimer present in the
GroEL-
complex was set at 100%. The heterodimeric intermediate
does not possess enzyme activity. The renaturation of BCKD activity at
30% with the GroEL-
-11-
fusion complex in the presence of GroES
and Mg-ATP was markedly less than that obtained with the GroEL-
complex. The activity of the active
-11-
homodimer equivalent to
the amount of the unfolded monomer in the GroEL-
-11-
fusion
complex was set at 100%. Essentially no BCKD activity was recovered
with either the GroEL-
complex or the GroEL-
-11-
fusion
complex when Mg-ATP or GroES alone was added to the refolding
mixture.
View larger version (15K):
[in a new window]
Fig. 6.
Chaperonin-mediated recovery of BCKD activity
with GroEL-protein intermediate complexes (A) or denatured
proteins (B). A, the GroEL- or the
GroEL-
-11-
complex at 0.8 µM was incubated with 2 µM GroES and 10 mM Mg-ATP at 23 °C for
16 h. BCKD activity was assayed as described previously (14). The
activity of BCKD or the
-11-
fusion equivalent to the amount of
the
heterodimer or the unfolded
-11-
fusion in the
GroEL-protein complex was set at 100%. B, the refolding of
the urea-denatured BCKD and
-11-
fusion was carried out as
described previously (13). Wild-type BCKD or the
-11-
fusion
protein at 2 mg/ml was denatured in 8 M urea at 23 °C
for 1 h, followed by a rapid 100-fold dilution into the refolding
mixture containing 1 µM GroEL. Refolding was initiated by
adding 2 µM GroES and 10 mM Mg-ATP to the
folding mixture. BCKD activity was assayed following incubation at
23 °C for 16 h. Enzyme activity of the same amount of BCKD or
the
-11-
fusion protein without denaturation was set at
100%.
-11-
fusion
protein, instead of the GroEL-protein complex, as substrate (Fig.
6B). The recovery of BCKD activity with urea-denatured
wild-type BCKD was also high at 90% in the presence of GroEL/GroES and
Mg-ATP. By comparison, the recovery of BCKD activity at 25% with the
urea-denatured
-11-
fusion protein in the presence of GroEL/GroES
and Mg-ATP was significantly lower than that with urea-denatured
wild-type BCKD. No BCKD activity was recovered from the denatured
-11-
fusion protein when GroES was omitted from the refolding mixture.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimeric intermediate can be isolated from
E. coli during expression and assembly of the native BCKD
heterotetramer (18). The heterodimer represents an ensemble of trapped
energy minima, which in the absence of chaperonins does not dimerize to
form the native heterotetramer in vitro (14). We have
described previously that the heterodimer does not dissociate into
monomers in a measurable equilibrium and binds to GroEL as an intact
species with an apparent dissociation constant (KD)
of 1.1 × 10
7 M (15). The 86-kDa
heterodimeric intermediate bound to GroEL was shown to be partially
protected from protease digestion in the presence of Mg-ADP and GroES
(15). The data strongly suggest that GroES is capable of capping this
large assembly intermediate inside the GroEL cis cavity. In
the present study, we extended the investigation by determining the
ability of GroES to encapsulate the heterodimeric intermediate inside
the GroEL single-ring variant SR1, taking advantage of the absence of
the trans ring. Mutations of R452E, E461A, S463A, and V464A
in the GroEL equatorial domain prevent the back to back stacking of the
two GroEL rings, resulting in the formation of the single-ring complex
SR1 (16). The heterodimeric intermediate binds to SR1 to produce a
stable SR1-
complex. In the presence of Mg-ATP, but not Mg-ADP,
the SR1-
complex interacts with GroES, resulting in the existence
of a stable SR1-
-GroES ternary complex. The absence of the
trans ring in SR1 confers complete protection of the
heterodimer inside the SR1 cavity (Fig. 2). However, it is reasonable
to ask whether the heterodimer inside the single SR1 cavity dissociates
into individual
and
subunits prior to the capping by GroES in
the presence of Mg-ATP, followed by the release and rebinding of these
individual subunits to different unoccupied chaperonins. In this
scenario, new SR1 cavities would harbor the smaller 48.5-kDa
(with
the His6 tag) or 37.5-kDa
subunit, rather than the
larger 86-kDa
heterodimer, to be capped by GroES. We approached
this question by studying the binding of GroES to fully occupied SR1
(Fig. 4). Empty cavities in the preparation of the SR1-
complex
were filled by incubation with an excess amount of reduced and
calcium-depleted
-lactalbumin (19). The fully occupied SR1
preparation was encapsulated by GroES in the presence of Mg-ATP. The
close to complete recovery of both
and
subunits at 1:1
stoichiometry following the tryptic digestion strongly supports the
notion that both subunits of the
heterodimer remain inside the
original SR1 cavity when the latter is capped by GroES. The equal molar
ratio of SR1:GroES in the SR1-
-GroES ternary complex indicates
further that the heterodimer in the SR1-
complex is enclosed
uniformly by GroES (Fig. 5B). Thus, our data validate the
encapsulation of the large heterodimeric intermediate inside GroEL and
SR1 cavities by GroES. The 86-kDa heterodimer represents the largest
partially folded intermediate that is known to be capped by GroES
inside the chaperonin cavity.
-11-
fusion cannot be capped by GroES inside GroEL and SR1
cavity, which is consistent with the above size limit for unfolded
proteins. Capping of the native-like 86-kDa
-11-
monomeric
intermediate has not been studied, because this species cannot be
isolated. On the other hand, the
heterodimeric assembly
intermediate with a size similar to the
-11-
fusion possesses a
native-like conformation as indicated by ANS fluorescence analysis. The
three-dimensional structure of the heterodimeric intermediate has not
been determined. Based on the recently solved crystal structure of the
native human BCKD heterotetramer, the heterodimeric intermediate
contains folded
/
domains in both
and
subunits (22). In
each subunit, the central
sheet is buried by outer
helices in
various orientations. Proteins with this kind of folded conformation
have been shown to be dependent on chaperonin for proper folding (5).
The estimated volume of 132,800 Å3 for the heterodimeric
intermediate enables it to fit completely inside the GroEL
cis cavity, so as to be encapsulated by GroES as illustrated
by simulated packing (Fig. 7).
View larger version (94K):
[in a new window]
Fig. 7.
Simulated encapsulation of the 86-kDa
heterodimer inside the
cis GroEL cavity by GroES. The
ribbon representation of the GroEL-GroES-ADP7
complex was derived from PDB coordinates (accession number 1AON)
with GroEL in green and GroES in yellow. The
86-kDa
heterodimer representation, with the
subunit in
red and the
subunit in blue, was derived from
PDB coordinates (accession number 1DTW). The graphics were created
using Swiss-PDB Viewer and POV-ray.
Volume changes upon protein unfolding may also explain the striking
difference between the SR1- and the SR1-
-11-
complexes with
respect to their ability to be capped by GroES. The partial specific
volume change
° accompanying protein denaturation can be
expressed as a sum of three terms,
° =
V +
T +
I, where
V
represents changes due to the loss of intramolecular voids;
T represents changes in thermal volume, which result
from thermally induced mutual molecular vibrations between the unfolded
protein and the solvent; and
I is the interaction
volume that represents changes in the solvent volume resulting from
interactions of water molecules with charged and polar groups of the
unfolded protein (23). The terms
V and
I are inherently negative upon protein unfolding,
whereas
T is a positive term associated with the increase in the accessible surface area of the unfolded protein. For a
protein of 70 kDa in size, the complete unfolding results in a 30%
increase in the partial specific volume mainly because of the positive
change in the thermal volume,
T (23). It is predicted
that the larger the protein, the greater increase in the partial
specific volume of the unfolded state. Chemical denaturant-induced unfolding such as urea denaturation may still result in measurable residual structures (24). However, it is reasonable to expect a
significant increase in the specific volume of the urea-denatured 86-kDa
-11-
fusion protein compared with its folded counterpart, i.e. the native-like
heterodimer of similar size and
essentially identical sequences. The enlarged volume, along with the
extended structure, may account for the inability of the unfolded
-11-
fusion to be enclosed by GroES inside the SR1 or GroEL cavity.
We have shown previously that chaperonins GroEL/GroES and Mg-ATP
promote dissociation/reassociation cycles of the trapped heterodimeric
intermediate to facilitate its conversion to the functional
heterotetramer (14, 15). The encapsulation of the heterodimeric
intermediate suggests strongly that the unfolding and dissociation of
the heterodimer into individual and
subunits occurs inside the
encapsulated GroEL cis cavity. The subsequent binding of
Mg-ATP to the open trans ring triggers the collapse of the
cis assembly. The individual
and
subunits released into the bulk solvent reassemble to produce new heterodimeric intermediates with a fraction capable of dimerizing into the native heterotetramer. The dissociation/reassociation cycle perpetuates until
all the trapped heterodimeric intermediates are converted to
heterotetrameric BCKD. The apparent cis folding throughout the BCKD assembly pathway explains the high levels of BCKD activity recovered with either urea-denatured BCKD or the GroEL-
complex as substrate. By contrast, the failure of GroES to encapsulate the
unfolded
-11-
fusion dictates the chaperonin-mediated
trans folding of this protein, similar to that described for
86-kDa maltose-binding protein fusion (8) and 82-kDa aconitase (9). The
trans folding mechanism may confer, in part, the markedly lower recovery of BCKD activity with the GroEL-
-11-
complex than
that using the GroEL-
complex as the starting material.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Arthur Horwich for kindly supplying
the SR1 expression plasmid and Max Wynn and Diana Tomchick for help in
simulated molecular graphics of the GroEL--GroES complex.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant DK26758 from the National Institutes of Health and Grant I-1286 from the Welch Foundation.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.
Present address: Program in Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA 01655.
§ To whom correspondence should be addressed. Tel.: 214-648-2457; Fax: 214-648-8856; E-mail: david.chuang@utsouthwestern.edu.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M209705200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BCKD, branched-chain -ketoacid dehydrogenase;
ANS, 1-anilino-8-naphthalenesulfonate;
FPLC, fast protein liquid
chromatography;
HPLC, high performance liquid chromatography.
![]() |
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