From the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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The decarboxylase component (E1) of the human
mitochondrial branched chain Molecular chaperones are generally protein agents that promote
proper folding of other polypeptides in an energy-dependent manner by preventing or reversing aggregation caused by off-pathway folding reactions (1-3). The family of molecular chaperones is large
and consists primarily of heat shock proteins
(Hsp).1 Among them, group I
chaperonins include GroEL/GroES in bacteria, Hsp60/Hsp10 in eukaryotic
mitochondria, and RUBISCO-binding protein/Cpn21 in the chloroplasts of
higher plants (4). This subset of molecular chaperones are highly
conserved and, for the most part, functionally interchangeable (5).
Crystal structures of bacterial chaperonins GroEL (6) and GroES (7, 8)
have been determined both at 2.8 Å. GroEL is a double-ring complex
with the two heptameric rings stacked upon one another back to back.
Each 57-kDa GroEL monomer contains an apical domain, an intermediate
domain, and an equatorial domain. The diameter of the opening in the
apical domain is 45 Å, which allows the passage of unfolded and
partially folded substrate polypeptides (6). The GroES structure is a single heptameric ring consisting of 10-kDa monomers, with a side view
showing a dome-like structure of 30 Å in height and 75 Å in width
(7). At the base of the GroES structure are mobile loops that provide
contact with the apical domains of GroEL. Recently, the crystal
structure of the
GroEL14-GroES7-ADP7 complex has
been determined (9). The complex structure shows that the binding of
GroES to one end of GroEL causes an en bloc domain movement within the complex, resulting in doubling of the volume in the cis ring cavity of GroEL. It is estimated that the enlarged
GroEL cavity when capped by GroES can accommodate substrate
polypeptides of up to 70 kDa in size (9).
The asymmetric GroEL14-GroES7 complex with only
one end of the GroEL double-ring complex capped by GroES has the
appearance of a "bullet" under the electron microscope (10). It has
been suggested that the asymmetric GroEL-GroES complex is the
functional hetero-oligomer that promotes protein folding reactions (11, 12). Unfolded polypeptides enter the uncapped ring through its open
apical domain, and folding is thought to occur, following GroES
binding, in this cis ring. However, under certain
conditions, for example, the presence of high Mg-ATP and high salt
concentrations, both ends of the GroEL double-ring complex can be
capped by GroES to produce a symmetric
GroEL14-(GroES7)2 complex, which
has the shape of a "football" when examined by electron microscopy
(10, 13). Although the functional significance of the symmetric complex is still a matter of debate, there is evidence to suggest that this
species represents an intermediate in the protein folding cycle (12,
14). During the cycle, it is believed that only one of the two GroEL
cavities or rings accommodates the unfolded polypeptide. However,
recent electron micrograph data indicate that both cavities of GroEL
harbor unfolded polypeptides (15, 16). Correlations of the symmetric
GroEL14-(GroES7)2 complex with the
protein folding activity of chaperonins have been shown (16, 17).
However, the mechanism of chaperoning action for this putative
symmetric hetero-oligomer is not clear.
Despite the advances described above, only limited information is
available concerning the role of chaperones during the assembly of
oligomeric proteins. Earlier studies with mitochondrial ornithine transcarboxylase (18), glutamate synthetase (19), and malate dehydrogenase (20) suggest that chaperonins GroEL and GroES mediate the
folding of assembly competent monomers, followed by spontaneous
assembly of monomers into active homo-oligomers. In the case of
heterodimeric proteins, for example, the native We are interested in understanding the chaperonin-mediated biogenesis
of mitochondrial macromolecular structures using the human branched
chain In this paper, we report in vitro reconstitution of
urea-denatured human E1, which shows an absolute requirement for
GroEL/GroES and Mg-ATP. Surprisingly, the kinetics of E1 reconstitution
is markedly slower than that of other proteins, for example,
mitochondrial malate dehydrogenase refolded under similar conditions.
Moreover, a novel ternary complex resulting from the interaction of
GroEL with a large (85.5 kDa) Bacterial Strains and Plasmids--
E. coli CG-712
cells (an ESts strain) and the pGroESL plasmid
overexpressing GroEL and GroES were kind gifts of Drs. George Lorimer
and Anthony Gatenby of the DuPont Experimental Station (Wilmington,
DE). The pTrcHisB expression vector was obtained from Invitrogen
(Carlsbad, CA). The pHisT-hE1 plasmid for co-expression of
His6- Expression and Purification of GroEL and GroES--
The pGroESL
plasmid was transformed into ESts CG-712 cells, which were
grown at 37 °C under chloramphenicol selection to
A600 of 0.6. The expression of GroEL and GroES
was induced by IPTG overnight at 37 °C. Cell lysates were prepared
by sonication in a lysis buffer containing 1 mM
phenylmethylsulfonyl fluoride and 1 mM benzamidine as
described previously (28). GroEL was purified from the lysates
according to the method described by Clark et al. (29) that
included a critical Reactive Red column step with the following
modifications. Prior to DEAE-Sepharose column fractionation, the
protein sample was treated with 10 mM Mg-ATP at 37 °C
for 2 h. The collected GroEL fractions from the ion exchange
column were concentrated and treated with 10 mM Mg-ATP
again at 37 °C for 2 h, followed by purification on Sephacryl
S-400HP column with Mg-ATP omitted from the column buffer. Eluted GroEL
fractions were pooled, concentrated, and purified on a Reactive Red
column. In a concurrent purification scheme, fractions from the above DEAE-Sepharose column containing GroES were pooled, and GroES was
further purified on a phenyl-Sepharose column as described previously
(30). Concentrations of GroEL and GroES were determined spectrophotometrically using published extinction coefficients of
1.22 × 104 M Expression and Purification of Recombinant Human E1
Proteins--
CG-712 cells co-transformed with pGroESL and pHisT-hE1
plasmids were grown in YTGK (32) medium. Expression of
His6- Denaturation and Reconstitution of Human E1
Assays for E1 Activity in a Reconstituted BCKD System--
The
assay mixture contained 100 mM potassium Pi, pH
7.5, 2.5 mM NAD+, 2 mM
MgCl2, 100 mM NaCl, 0.6 mM CoA, 0.2 mM TPP, 0.1% Triton X-100, 7 nM in
vitro lipoylated recombinant bovine E2, and 0.4 µM
recombinant human E3. To 0.3 ml of the assay mixture, 20 µl of the
aliquot from the refolding reaction was added. The enzyme reaction of
the BCKD complex was initiated by addition of 10 µl of 6.4 mM (final concentration, 0.2 mM)
Preparation of Individual GroEL-His6- Expression and Purification of the Determination of Second Order Folding Rate Constants--
An
assumption was that the recovery of E1 activity represented the
formation of the tetramer. During folding, activity of nondenatured E1
incubated under the same conditions served as 100% control. At a given
time point, the percentage of E1 activity recovered relative to the
control was converted to active Reactivation of Urea-denatured His6-tagged Human
E1--
Recombinant His6-tagged E1 was denatured in a
buffer containing 8 M urea. The denatured E1 tetramer was
rapidly diluted 100-fold into a refolding buffer containing a 4-fold
and 8-fold molar excess of GroEL and GroES, respectively, and 1 mM DTT. Fig. 1 shows that the
time course for the recovery of E1 activity follows strikingly slow
kinetics during the 30-h incubation at 23 °C with a second order
rate constant of 290 M Differential Dependence of E1 Subunits on GroEL for E1
Reconstitution--
The requirement for chaperonins for reconstitution
of E1 activity was demonstrated by experiments in which GroEL addition to the folding reaction mixture was delayed. The His6- Effects of Polypeptide/GroEL Stoichiometry on Reconstitution of E1
Activity--
To further investigate differential dependence on
GroEL/GroES between the Differential Release of Assembly State of E1 Folding Intermediates--
The complete
refolding mixture with urea-denatured His6-tagged E1 as a
substrate was sampled at different times during a 24-h incubation at
23 °C. Aliquots were treated with Ni-NTA resin, and bound proteins
were eluted with a buffer containing 250 mM imidazole.
Sucrose density gradient centrifugation was routinely used to separate
protein species in the refolding mixture because: 1) It was capable of
handling multiple samples with relatively large volumes (0.5-1 ml),
which normally resulted after diluting out the denaturant for
refolding. Sample concentration required for gel filtration often led
to the precipitation of assembly intermediates (see Fig. 5). 2) The
method caused a minimal dilution of samples, which was usually
associated with gel filtration as a result of diffusion. 3) Folded or
assembled E1 species were stable on the sucrose density gradient, and
their identities were confirmed by calibrated FPLC gel filtration (see
Fig. 7).
As shown in Fig. 5, at zero time, a
detectable amount of aggregated His6- Expression and Purification of the Dimeric Assembly
Intermediate--
To produce a large amount of
The combined Binding of Slow Reconstitution of Active
The assembly state of E1 subunits released from the GroEL- Most investigations into chaperonin-mediated protein folding to
date have focused on small monomeric proteins or synthetic polypeptides
(1, 2). Although these studies have provided significant insights
regarding the mechanisms of GroEL/GroES action, relatively little is
known in the area of chaperonin-dependent folding and
assembly of large hetero-subunit proteins. To address this problem, we
have chosen the E1 component of the human mitochondrial BCKD complex as
a model system. Here, we show that the reconstitution of the
A striking feature of chaperonin-dependent E1
reconstitution is the slow kinetics. The rate constant of 290 M The reconstitution of E1 activity from individual GroEL- The GroEL- Fig. 11 depicts our current working
model for the chaperonin-dependent E1 assembly pathway. The
folding of individual -ketoacid dehydrogenase multienzyme
complex (~4-5 × 103 kDa) is a thiamine
pyrophosphate-dependent enzyme, comprising two 45.5-kDa
subunits and two 37.8-kDa
subunits. In the present study,
His6-tagged E1
2
2 tetramers
(171 kDa) denatured in 8 M urea were competently
reconstituted in vitro at 23 °C with an absolute
requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the
kinetics for the recovery of E1 activity was very slow with a rate
constant of 290 M
1 s
1.
Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-
and GroEL-
complexes as combined
substrates. However, the
subunit was markedly more prone to
misfolding than the
in the absence of GroEL. The
subunit was
released as soluble monomers from the GroEL-
complex alone in the
presence of GroES and Mg-ATP. In contrast, the
subunit discharged
from the GroEL-
complex readily rebound to GroEL when the
subunit was absent. Analysis of the assembly state showed that the
His6-
and
subunits released from corresponding
GroEL-polypeptide complexes assembled into a highly structured but
inactive 85.5-kDa
dimeric intermediate, which subsequently
dimerized to produce the active
2
2
tetrameter. The purified
dimer isolated from Escherichia
coli lysates was capable of binding to GroEL to produce a stable
GroEL-
ternary complex. Incubation of this novel ternary complex
with GroES and Mg-ATP resulted in recovery of E1 activity, which also
followed slow kinetics with a rate constant of 138 M
1 s
1. Dimers were regenerated
from the GroEL-
complex, but they needed to interact with
GroEL/GroES again, thereby perpetuating the cycle until the conversion
from dimers to tetramers was complete. Our study describes an
obligatory role of chaperonins in priming the dimeric intermediate for
subsequent tetrameric assembly, which is a slow step in the
reconstitution of E1
2
2 tetramers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dimer of
bacterial luciferase, a prevailing model is that GroEL binds separately
to the folding intermediate of either subunit and the folded
and
subunits released in the presence of GroES and Mg-ATP assemble to
form the active enzyme (21). In a recent study, it was shown that
GroEL/GroES modulate the kinetic partitioning of the
subunit
intermediate of bacterial luciferase between two alternate pathways to
increase the yield of native
dimers (22). As for
Bacillus pyruvate dehydrogenase, the
and
subunits individually expressed in Escherichia coli, when incubated
in vitro, spontaneously assemble into the native
2
2 structure (23).
-ketoacid dehydrogenase (BCKD) complex as a model system. The
mitochondrial BCKD complex (~5 × 103 kDa) is
organized around a cubic core comprising 24 dihydrolipoyl acyltransferase (E2), to which branched chain
-ketoacid
decarboxylase (E1), dihydrolipoyl dehydrogenase (E3), a specific
kinase, and a specific phosphatase are attached through ionic
interactions (24). We reported earlier that GroEL/GroES are essential
for in vitro reconstitution of the 24-meric inner core of
the E2 component (25). The E1 component is a thiamine pyrophosphate
(TPP)-dependent enzyme, which consists of two 45.5-kDa
subunits and two 37.8-kDa
subunits. We have shown previously that
co-transformation of GroEL/GroES into E. coli expressing
mammalian E1 subunits resulted in an over 500-fold increase in the
yield of the active E1
2
2 tetramer,
compared with the single transformant without overexpression of
chaperonins (26, 27). However, the precise steps in chaperonin-mediated assembly pathway of E1 remain to be elucidated.
dimeric intermediate during
assembly is described. We show that the
chaperonin-dependent conversion of inactive dimers to
active tetramers is a slow step in E1 assembly. These findings provide
evidence for an obligatory role of chaperonins in priming the assembly
intermediate for subsequent higher order oligomerization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and untagged
subunits of human E1 was
described previously (28). The His6-
subunit contained a
His6 tag and a tobacco etch virus protease cleavable linker
fused to the N terminus of the
subunit.
1
cm
1 (30) and 1.2 × 103
M
1 cm
1 (31), respectively.
and untagged
subunits and GroEL/GroES was
induced by IPTG. Assembled human His6-E1 was isolated from
cell lysates and purified by Ni-NTA (Qiagen, Chatsworth, CA) column
chromatography as described previously (28). To remove the
His6 tag, the purified fusion human E1 was digested with
the tobacco etch virus protease at 4 °C overnight. The released
His6 tag was removed by Ni-NTA extraction.
2
2 Tetramers--
Purified
His6-tagged or untagged human E1 was denatured for
1 h at 23 °C in a denaturing buffer containing 8 M
urea, 50 mM potassium Pi, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 0.1% Tween 20, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride
at a final E1 tetramer concentration of 12.5 µM.
Denatured E1 was quickly diluted 100-fold on a Vortex mixer into a
refolding buffer consisting of 50 mM potassium
Pi, pH 7.5, 100 mM KCl, 0.1 mM
EDTA, 0.1% Tween 20, 2 mM TPP, 2 mM
MgCl2, 1 mM DTT, and 1 mM
phenylmethylsulfonyl fluoride with or without 2 µM GroEL
(14-mers) and 4 µM GroES (7-mers). The refolding reaction
was initiated by addition of Mg-ATP to a final concentration of 10 mM. The reaction mixture was incubated at 23 °C for up
to 30 h. At indicated times, aliquots were taken to which CDTA was
added to a final concentration of 25 mM to discontinue the
folding reaction. Samples were kept at
20 °C until analysis. As a
control, purified human E1 without urea treatment was incubated in the
refolding buffer under the same conditions. For samples to be extracted
with Ni-NTA resin, 1 mM DTT was replaced with 20 mM
-mercaptoethanol in the refolding buffer.
-keto[1-14C]isovalerate (specific radioactivity,
1500-1800 cpm/nmol). After incubation at 37 °C for 20 min, the
reaction was quenched by addition to a final concentration of 6%
trichloroacetic acid. Released radiolabeled CO2 was trapped
in a paper wick soaked with 1 N NaOH and measured in a
Beckman scintillation counter. The assay was linear for up to 1 µg of
the purified human E1 protein. The rate of decarboxylation with
-keto[1-14C] isovalerate as substrate by E1 in the
reconstituted BCKD system was 15-fold higher than that by E1 alone
(33). Assay components in the BCKD system had no measurable effects on
E1 renaturation (data not shown).
and
GroEL-
Complexes--
His6-tagged human E1 was
denatured in the above denaturing buffer containing 10 mM
-mercaptoethanol. The His6-
subunit was extracted
with Ni-NTA resin pre-equilibrated in the same denaturing buffer. The
untagged
subunit remained in the supernatant. The His6-
subunit was eluted from Ni-NTA resin with 250 mM imidazole in the denaturing buffer. Separated individual
subunits were concentrated in Centricon-30 concentrators and rapidly
diluted into the refolding buffer containing various stoichiometric
amounts of GroEL. The GroEL-polypeptide complexes were purified on a
10-30% sucrose density gradient in Beckman SW41 rotor at 210,000 × g for 18 h at 4 °C. Fractions containing the
GroEL-His6-
or the GroEL-
complex were pooled,
concentrated, and stored at
80 °C. The stoichiometries of
GroEL-polypeptide complexes were determined by densitometry scanning of
Coomassie Blue-stained SDS-PAGE gels. GroEL, His6-
, and
untagged
monomers that were used as standards and run on the same
gel were linear up to 30, 5.5, and 4.5 µg, respectively. The amount
of sample to be determined was adjusted to be within the linear range.
Dimeric
Intermediate--
CG-712 cells co-transformed with pHisT-hE1 and
pGroESL plasmids were grown at 37 °C in the C2 broth minimal medium
and induced for expression of E1 subunits with IPTG for 3 h. The
C2 broth minimal medium was modified from the C broth medium with low
sulfate and amino acid contents as described previously (34). The
growth medium in the present study contained per liter: 2 g of
NH4Cl, 6 g of
Na2HPO4·7H2O, 3 g of
KH2PO4, 3 g of NaCl, 6 g of yeast extract, 40 µM TPP, 50 mg of carbenicillin, and 50 mg of
chloramphenicol. Lysates prepared from harvested cells were extracted
with Ni-NTA resin and washed with a lysis buffer containing 15 mM imidazole (28). Bound proteins were eluted with the same
buffer containing 250 mM imidazole. Eluted fractions
containing the
dimer and the
2
2
tetramer were concentrated by centrifugation in Millipore Ultra free-15
filter concentrators. Proteins were separated on a 10-25% sucrose
density gradient spun at 210,000 × g for 18 h. Fractions were analyzed by SDS-PAGE and assayed for E1 activity. Fractions that contained the
dimers were combined and further purified by FPLC on a HiLoad Superdex 200 column (2.6 × 60 cm).
2
2 tetramers formed, based on the initial concentration of tetramers in
the control. The concentration of tetramers formed was used to
calculate the concentrations of remaining monomers (
or
) or
dimers (
). Data were fit to the second order rate equation: kt = 1/[A]
1/[A]0, where
k = the second order rate constant, t = reaction time (s), [A] = the concentration (M) of
remaining monomers or dimers at a given time and [A]0 = the initial concentration (M) of monomers or dimers.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 s
1.
With the same GroEL/GroES preparations, refolding of urea-denatured mitochondrial malate dehydrogenase reached a plateau of 80% recovered activity in 40 min with a second order rate constant of 1.2 × 104 M
1 s
1, similar
to that reported previously (20). The reconstitution of
His6-tagged E1 was completely dependent on GroEL/GroES and Mg-ATP. The 100% control E1 activity (2.08 µmol/min/mg E1 protein) assayed in the presence of E2 and E3 was comparable with that reported
previously (35). No E1 activity was reconstituted when Mg-ATP or
GroEL/GroES and Mg-ATP were omitted from the refolding mixture (Fig.
1). When urea-denatured untagged E1 was used as a substrate, its
reactivation kinetics was very similar to that obtained with
urea-denatured His6-tagged E1 (data not shown).
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Fig. 1.
GroEL/GroES-dependent
reconstitution of urea-denaturated human E1. Recombinant
His6-tagged E1 (12.5 µM, tetramer), with a
His6 tag and a tobacco etch virus protease-cleavable linker
fused to the N terminus of the subunit, was denatured in a
denaturing buffer containing 8 M urea. Denatured E1 was
rapidly diluted 100-fold into a refolding buffer at pH 7.5 in the
presence or absence of 2 µM GroEL and 4 µM
GroES. The refolding reaction was initiated by addition of Mg-ATP to a
final concentration of 10 mM and incubated at 23 °C. At
indicated time points, aliquots were taken, and the reaction was
terminated by addition of CDTA to 25 mM. Samples were
assayed for the reconstituted BCKD activity in the presence of excess
in vitro lipoylated recombinant E2 and recombinant E3 with
-keto[1-14C] isovalerate (1, 800 cpm/nmol) as a
substrate (see "Experimental Procedures"). The 100% control
activity represents the activity of the same amount of E1 without
denaturation at 2.08 µmol CO2 released/min/mg E1.
,
GroEL/GroES and Mg-ATP;
, Gro/EL/GroES;
, no addition.
subunit denatured in 8 M urea was rapidly diluted into the
refolding buffer at 23 °C without chaperonins. At different times,
aliquots were taken, and a 4-fold molar excess of the GroEL/GroES
mixture (molar ratio 1:2) relative to His6-
monomers was
added. The mixture was then combined with an 1:1 stoichiometric amount
of the GroEL-
complex with respect to the His6-
subunit to reconstitute E1 activity. E1 activity regained with
GroEL-His6-
(no delay in GroEL addition) and GroEL-
as substrates was 100% (Fig. 2, 0 min).
In a converse experiment, GroEL was added to the denatured untagged
subunit in a time-dependent fashion, and stoichiometric amounts of the GroEL-His6-
complex were added to
reconstitute E1 activity. Delayed additions of GroEL to denatured
His6-
caused a sharp concentration-dependent
decline in the ability of the latter to regain E1 activity with the
GroEL-
complex (Fig. 2). The second order rate constant for the
reduction in regained E1 activity resulting from delayed addition of
GroEL to the denatured His6-
subunit was 2.75 × 104 M
1 s
1 at
23 °C. The delayed addition of GroEL to denatured
resulted in a
more precipitous fall in renatured E1 activity with a second order rate
constant of 9.79 × 104 M
1
s
1, which was 3.5-fold higher than that obtained with the
denatured His6-
. Without added GroEL, the denatured
His6-
and
subunits rapidly aggregated upon dilution
of the denaturant, as measured by light scattering at 488 nm (data not
shown).
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Fig. 2.
Loss of reconstituted E1 activity caused by
delayed addition of GroEL to denatured E1 subunits. The
His6- subunit in 8 M urea was diluted
100-fold into the refolding buffer without chaperonins. At the
indicated elapsed time, GroEL/GroES (molar ratio, 1:2) at a 4-fold
molar excess were added to different concentrations of the denatured
His6-
monomer. The preformed GroEL-
complex was added
at a stoichiometry of His6-
:
= 1:1. The refolding
reaction was initiated by the addition of 5 mM Mg-ATP.
After incubation at 23 °C for 18 h, the refolding mixture was
assayed for reconstituted E1 activity. In a reverse experiment,
GroEL/GroES were added to different concentrations of denatured
subunit preincubated in the refolding buffer at different elapsed
times. The preformed GroEL-His6-
complex was added, and
refolding was initiated by the addition of 5 mM Mg-ATP. The
concentrations of the denatured His6-
subunit were 0.08 µM (
) and 0.16 µM (
). The
concentrations of the denatured
subunit were 0.125 µM
(
) and 0.25 µM (
). Reconstituted E1 activity
obtained with both preformed GroEL-subunit complexes, without elapsed
times, is 100%.
and
subunits for proper folding,
different molar ratios of the polypeptide-GroEL (abbreviated as EL)
complex were used in the refolding studies. To produce complexes with
different (subunit)1:(EL) 14 stoichiometry,
increasing amounts of the denatured His6-
or
subunit
in 8 M urea were diluted at 4 °C into the refolding
buffer containing GroEL. After purification on a sucrose density
gradient, the (
)1:(EL)14 or
(
)1:(EL)14 ratio was determined by
densitometry scanning of Coomassie Blue-stained SDS-PAGE gels. As shown
in Fig. 3, when
(
)1:(EL)14 = 1:2 and
(
)1:(EL)14 = 2:1 complexes were incubated
with GroES and Mg-ATP, efficient E1 activity was reconstituted
(Experiment 1, Fig. 3, left panel). In this experiment, open
GroEL cavities were present in the 1:1
-EL complex but absent in the
2:1
-EL complex. In Experiment 2, the unfolded
subunit in 8 M urea was diluted 100-fold by rapid mixing into the
refolding buffer containing the (
)1:(EL)14 = 2:1 complex. The recovery of E1 activity was comparable with that
detected in Experiment 1, indicating that the
subunit did not
rapidly aggregate even when all GroEL cavities were initially occupied by the
subunit in the 2:1
-EL complex. Efficient reconstitution of E1 activity was also achieved, when the
subunit denatured in 8 M urea was rapidly diluted into a refolding mixture
containing the (
)1:(EL)14 = 1:2 complex
(Experiment 3). The presence of excess unoccupied GroEL rings was
likely responsible for the formation of the GroEL-
complex and
efficient recovery of E1 activity. In Experiment 4, which was converse
to Experiment 2, very little, if any, E1 activity was recovered, when
the unfolded
subunit was diluted into a refolding buffer containing
the (
)1:(EL)14 = 2:1 complex. The failure to
reconstitute E1 activity apparently resulted from the absence of GroEL
with open cavities for the
subunit. Taken together, this series of
experiments demonstrate that the
subunit is more dependent than the
on the immediate availability of unoccupied GroEL ends for
preventing misfolding.
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Fig. 3.
Effects of GroEL:polypeptide stoichiometry on
reconstitution of E1 activity. The or
monomer complexed
with the GroEL 14-mer at different stoichiometries (right
panel) was prepared as described under "Experimental
Procedures." The molar ratio of the monomer (
or
subunit) to
the 14-mer (GroEL) was determined by SDS-PAGE and densitometry scanning
of the Coomassie Blue-stained protein bands. Actual molar
concentrations (in µM) of each stoichiometric complex
(right panel) were as follows:
(
)1:(EL)14 = 1:2 (0.58:1.2);
(
)1:(EL)14 = 2:1 (0.74:0.35);
(
)1:(EL)14 = 2:1 (1.48:0.63). The
concentrations of urea-denaturated
or
subunit were both at 1 µM. To carry out the refolding reaction in Experiments
1-4, 10 µM GroES and 1 mM Mg-ATP were added
to each refolding mixture. Refolding was allowed to proceed at 23 °C
for 16 h. E1 activity (left panel) was assayed and
expressed as radioactivity (in cpm) of 14CO2
released from substrate
-keto[1-14C] isovalerate in 20 min at 37 °C.
and
Subunits from Corresponding
GroEL-Peptide Complexes--
The release of E1 subunits from
GroEL-polypeptide complexes was studied by incubation of the
GroEL-His6-
or the GroEL-
complex alone with GroES
and Mg-ATP at 23 °C for 4 h. The incubation mixtures were
separated on a sucrose density gradient, and fractions were analyzed by
SDS-PAGE. As shown in Fig. 4A,
a significant portion of the His6-
subunit was released
from the GroEL-His6-
complex. The discharged
His6-
subunit was present as soluble monomers as
determined with a calibrated FPLC gel filtration column (see Fig. 7). A
small fraction of GroEL monomers resulting from centrifugation in the
presence of Mg-ATP (36) co-sedimented, but were not complexed, with
His6-
monomers. In contrast, when the GroEL-
complex
was incubated with GroES and Mg-ATP, no free soluble
protein was detected on the sucrose density gradient (Fig. 4B). To ask
whether the
subunit was capable of being released from the
GroEL-
complex alone, the C-terminally tagged His6-
subunit was used to form the GroEL-His6-
complex. When
Ni-NTA was present in the refolding mixture containing
GroEL-His6-
, GroES, and Mg-ATP, the His6-
subunit was extracted (data not shown). This indicates that the
subunit is indeed released from the GroEL-
complex transiently but
cannot exist as a soluble species without assembly with the
and
rebinds to GroEL.
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Fig. 4.
Differential release of and
subunits from corresponding
GroEL-polypeptide complexes. The GroEL-
(0.68 µM)
or GroEL-
(0.68 µM) at 1:1 stoichiometry in 200 µl
of a 50 mM potassium Pi buffer, pH 7.5 containing 100 mM KCl, 5 mM DTT, 1 mM EDTA, 2 mM MgCl2, and 2 mM TPP was incubated with GroES (3.6 µM) and
10 mM Mg-ATP for 4 h at 23 °C. The reaction
mixtures were separated in a 10-30% sucrose density gradient, and the
fractions were analyzed by SDS-PAGE and Coomassie Blue staining.
subunits and a
small fraction of the GroEL-His6-
complex extractable
with Ni-NTA resin sedimented to near the bottom of the sucrose density
gradient. At 45 min into the refolding reaction, small amounts of
putative species of His6-
monomers (fractions 4 and 5),
His6-tagged
dimers (fractions 4-6), and
2
2 tetramers (fractions 7-10) were
present in the gradient. Because His6-
monomers and
His6-tagged
dimers partially overlapped on the
sucrose density gradient, the appearance of the untagged
in the
Ni-NTA extract served as a true indicator for the assembly state of
folding intermediates. At the 2-h time point, an appreciable amount of
the
subunit was present in fractions 4-8. However, unassembled
His6-
monomers still persisted, resulting in
significantly higher abundance of the
subunit than
in dimer
fractions 4-6. The data suggest that the assembly of the
subunit
with the
is rate-limiting in the formation of
dimers. During
the prolonged incubation (6-24 h), there was not only more Ni-NTA
extractable
present but also a significant shift in the position of
the
subunit from that of dimers to tetramers (fractions 7-9). The data depict a slow but ostensible conversion from putative dimers to
tetramers. Enzyme assays indicated a close correlation between the
recovery of E1 activity and the amount of tetramers formed during the
refolding (data not shown). The apparent substoichiometric amounts of
the
subunit relative to the
in assembled dimers and tetramers
resulted from the poorer binding of the
subunit to Coomassie Blue
than the
(molar dye intensity
:
= 1:1.4).
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Fig. 5.
Formation of the
dimeric intermediate and
2
2
tetramers as analyzed by sucrose density gradient centrifugation.
His6-
human E1 (12.5 µM of tetramers)
denatured in 8 M urea was diluted 100-fold into the
refolding buffer containing 1 µM GroEL and 4 µM GroES. The refolding reaction at 23 °C was started
by additions of Mg-ATP (final concentration, 5 mM). At each
time point, an aliquot was taken and kept on ice. A portion was used
for enzyme assays, and the remainder was extracted with Ni-NTA resin.
After washing for three times with the refolding buffer (without
GroEL/GroES and Mg-ATP), bound protein species were eluted with the
same buffer containing 250 mM imidazole. The eluates were
separated on a 10-30% sucrose density gradient spun at 210,000 × g for 18 h. Fractionated samples were precipitated
with 6% trichloroacetic acid and separated on 12% SDS gels, followed
by staining with Coomassie Blue. The molecular mass markers used for
calibrations (in kDa) were: bacterial lipoylated enzyme LplA (35),
bovine serum albumin (60), human E3 (110), maltose-binding
protein-human BCKD kinase (340), and GroEL (840).
dimers for
characterization, ESts CG-712 host co-transformed with the
pHisT-hE1 and pGroESL plasmids were grown in the C2 minimal medium with
the expression of E1 subunits induced by IPTG for 3 h, and the
lysates were purified by Ni-NTA extraction. Fig.
6 (top panel) shows that the
assembled
dimer (fractions 4 and 5) is the major species in the
sucrose density gradient and is partially separated from the
2
2 tetramer (fractions 6-11), with
aggregated E1 subunits sedimented at the bottom of the gradient.
Reconstituted BCKD enzyme assays indicated that fractions containing
the tetramer but not the dimer possessed E1 activity (Fig. 6,
bottom panel). Residual E1 activity in fraction 5 was the
result of a spill-over from the tetramer fractions.
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Fig. 6.
Accumulation of inactive
dimers in E. coli
grown in C2 broth minimal medium. EStsCG-712
cells were co-transformed with the pGroESL plasmid and the pHisT-hE1
plasmid carrying the His6-
and the untagged
cDNAs. Cells grown in the C2 broth minimal medium were induced for
expression of E1 subunits with IPTG for 3 h. Cell lysates were
extracted with Ni-NTA resin. After washing the resin with a phosphate
buffer at pH 7.5 containing 500 mM KCl and 15 mM imidazole, His6-
and associated untagged
subunits were eluted with 250 mM imidazole in the same
buffer. The concentrated eluate was subjected to sucrose density
gradient centrifugation, and the fractions were analyzed by SDS-PAGE
(upper panel). E1 activity in eluted fractions was assayed
(lower panel). One m-unit is equivalent to 1 nmol
CO2 released/min. The molecular mass markers used for
calibration (in kDa) were: ovalbumin (44), bovine serum albumin (67),
human E3 (110), aldolase (158), and catalase (232).
dimer fractions from the sucrose density gradient
was further purified by FPLC gel filtration. Purified
His6-tagged tetramers, His6-tagged
dimers, and His6-
monomer were eluted from a calibrated
HiLoad Superdex 200 column at peak fractions 39, 42, and 46, respectively (Fig. 7). The elution
profiles confirmed the identities of folded or assembled E1 species
separated on sucrose density gradients (Figs. 4-6). The FPLC-purified
dimer was free of contamination by His6-
monomers
and assumed a native-like structure as indicated by its tryptophane
fluorescence at 345 nm, similar to the
2
2
tetramer (data not shown).
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Fig. 7.
FPLC gel filtration profiles of
2
2 tetramers
(A),
dimers
(B), and
monomers
(C). His6-tagged
2
2 tetramers,
dimers were
expressed in E. coli and partially purified by Ni-NTA
extraction and sucrose density gradient centrifugation.
His6-
monomers expressed alone in the same host were
isolated in a similar manner. The purified
2
2 tetramers (10 mg),
dimers (0.5 mg), or
monomers (2.1 mg) in 50 mM potassium
Pi buffer, pH 7.5 containing 350 mM NaCl, 2 mM EDTA, 0.5 mM DTT, and 0.2 mg/ml Pluronic
F-68 (BASF Corporation, Parsippany, NJ) was applied to a HiLoad
Superdex 200 column (2.6 × 60 cm) on an Amersham Pharmacia
Biotech FPLC system. Proteins were eluted with the same buffer at a
flow rate of 1.5 ml/min and collected in 5-ml fractions. The peak
fractions of His6-tagged
2
2
tetramers (171 kDa), His6-tagged dimers (85.5 kDa), and
His6-
monomers (47.7 kDa) were 39, 42 and 46, respectively. The molecular mass markers used for calibration (in kDa)
were: chymotrypsinogen A (23), ovalbumin (44), bovine serum albumin
(67), human E3 (110), aldolase (158), and GroEL (840).
Dimers to GroEL--
In our earlier studies,
when His6-tagged E1 carrying a T265R human mutation was
expressed in the ESts CG-712 host co-transformed with the
pGroESL plasmid, a ternary complex comprising GroEL, the mutant
His6-
, and untagged
subunits was isolated from
the bacterial lysate by Ni-NTA extraction (data not shown). The
accumulation of the
dimer in E1 assembly strongly suggested that
the GroEL-
ternary complex may have resulted from binding of the
assembly intermediate to GroEL. To address this question, the FPLC
purified His6-
dimer (5 µM) from
E. coli lysates (Fig. 7B) was incubated with
GroEL (5 µM) for 4 h at 23 °C. The incubation
mixture was then separated by HPLC gel filtration. The
dimer and
GroEL were co-eluted as a large single peak at 6.5 min (Fig.
8, peak 1). SDS-PAGE analysis
showed the GroEL-
ternary complex with a 1:1
to
subunit
stoichiometry (Fig. 8, inset). The excess unbound
dimer was eluted as a minor peak at 8.9 min (Fig. 8, peak
2). Incubation of the His6-tagged
2
2 tetramer with GroEL did not form a
complex as determined by sucrose density gradient centrifugation (data
not shown).
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Fig. 8.
Purification of the
GroEL- complex by HPLC. The
FPLC-purified E1
dimer (4 µM) (see Fig. 7) was
incubated with GroEL (2 µM) for 4 h at 23 °C.
Following the incubation, the reaction mixture (200 µl) was applied
to a TSK-G3000SWxl gel filtration column in a Waters HPLC
system. Proteins were eluted isocratically with a potassium
Pi buffer, pH 7.5, containing 250 mM KCl, 1 mM EDTA, and 0.2% NaN3 at a flow rate of 1 ml/min and monitored by absorbance at 280 nm. Peak 1 (retention time, 6.5 min), GroEL-
complex; peak 2 (retention time, 8.9 min), the excess unbound
dimer.
Inset, SDS-PAGE and Coomassie Blue staining of Peak 1 fractions.
2
2
Tetramers with the GroEL-
Complex, GroES, and Mg-ATP--
The
isolation of the stable GroEL-
complex prompted us to ask whether
this ternary complex was an obligatory intermediate in the E1 assembly
pathway and whether GroES was essential for the conversion of dimers
into tetramers. The purified GroEL-
complex (Fig. 8) was
incubated with GroES and 5 mM Mg-ATP at 23 °C. Aliquots
collected at different times were assayed for E1 activity. Fig.
9 shows that E1 activity was
reconstituted from the GroEL-
complex, but again with very slow
kinetics with a second order rate constant of 138 M
1 s
1. The reconstitution of E1
activity from the GroEL-
complex was also absolutely dependent on
a complete chaperonin system. No E1 activity was reconstituted when
Mg-ATP or GroES was omitted or when Mg-ADP alone was added to the
refolding mixture (Fig. 9).
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Fig. 9.
GroEL/GroES-dependent
reconstitution of E1 activity from the
GroEL- ternary complex. The
refolding reaction was carried out at 23 °C in the refolding buffer
containing 1 µM HPLC purified GroEL-
complex (Fig.
8), 2 µM GroES, and 5 mM Mg-ATP. At indicated
time points, aliquots were taken and E1 activity was assayed. For the
definition of the activity unit, see the legend to Fig. 6.
, with
GroES and Mg-ATP;
, with Mg-ATP only;
, with GroES only;
,
with Mg-ADP only.
complex was also studied. The sucrose density gradient profile showed
that the dimer released from GroEL slowly converted to the tetramer
during an 18-h incubation at 23 °C, similar to that observed with
the urea-denatured E1. The Ni-NTA-extracted refolding mixture taken at
the 2-h time point consisted primarily of the
dimer and a trace
amount of the
2
2 tetramer as separated on
the sucrose density gradient. Incubation of this refolding mixture
without chaperonins for 16 h at 23 °C did not cause the conversion of the dimer to tetramer (Fig.
10A). However, a similar incubation with GroEL/ES and Mg-ATP resulted in a complete conversion of
dimers to
2
2 tetramers (Fig.
10B).
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Fig. 10.
GroEL/GroES-dependent conversion
of dimers regenerated from the
GroEL-
complex to
2
2
tetramers. The GroEL-
complex (2 µM) was
incubated at 23 °C with GroES (4 µM) and 5 mM ATP. At the 2 h-time point, aliquots were taken,
extracted with Ni-NTA, and re-incubated at 23 °C either with no
addition (A) or with GroEL (1 µM), GroES (4 µM), and 5 mM Mg-ATP (B) for
16 h. Following the second incubation, the refolding mixtures were
extracted with Ni-NTA and separated in a 10-25% sucrose density
gradient. Fractions were analyzed by SDS-PAGE and Coomassie Blue
staining. The molecular mass markers (in kDa) used were: ovalbumin
(44), bovine serum albumin (66), E3 (110), aldolase (158), and catalase
(232).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2 tetramer of E1 is absolutely dependent
on GroEL/GroES. In contrast, other proteins such as rhodanese (36, 37),
bacterial luciferase (22), and mitochondrial malate dehydrogenase (30) can either spontaneously refold or be assisted by the chaperonins during folding. Both the denatured
and
subunits depend on GroEL/GroES for their ability to reconstitute E1 activity. The system
thus allows for an elucidation of how these chaperonins mediate the
cross-talk between the
and the
subunits during E1
2
2 assembly. Moreover, the BCKD complex
is deficient in patients with heritable maple syrup urine disease
(MSUD), resulting in severe ketoacidosis, neurological derangements,
and mental retardation (38). We have shown previously that a subset of
MSUD mutations in the
subunit impair E1 assembly in vivo
(39, 40) and in E. coli (28). The availability of the
reconstituted E1 system will facilitate studies on the mechanism by
which MSUD mutations perturb the normal interaction between chaperonins
and E1 subunits.
1 s
1 is 2 orders of magnitude
slower than that for the refolding of mitochondrial malate
dehydrogenase (Ref. 20 and this study). The slow rate of folding and
assembly for the E1 tetramer in vitro is consistent with the
sluggish expression of recombinant E1 in E. coli. In the
latter experiment, the levels of the assembled recombinant
His6-
and untagged
subunits reached a plateau after induction with IPTG for 7 h at 37 °C; however, the maximal E1 activity was not observed until 12 h after the induction (data not
shown). The slow pace of E1 biosynthesis also coincides with the
relatively low turnover rate of the enzyme components of mammalian mitochondrial
-ketoacid dehydrogenase complexes. In a previous study, half-lives for the
and
subunits of mitochondrial
pyruvate dehydrogenase were found to be identical at 41 h in
murine 3T3-L1 preadipocytes and 49 h in differentiated adipocytes
(41). At present, however, we cannot rule out the involvement of
additional chaperones or assembly factors that might enhance the rate
of E1 reconstitution in vitro. For example, COX14 (42) and
COX15 (43) are mitochondrial membrane proteins essential for assembly of yeast cytochrome oxidase. Peptidyl prolyl isomerase was shown to
accelerate the reactivation of the antibody Fab fragment (44), but our
preliminary data to date indicate that this enzyme/chaperone has no
effect on E1 assembly either in the presence or absence of GroEL/GroES.
It is also plausible that the slow kinetics of E1 refolding may be due
to the absence of an additional mitochondrial chaperone Hsp70 in the
refolding mixture. The mitochondrial Hsp70, upon anchoring to matrix
TIM44, is proposed to function as an ATP-driven import motor that binds
to the incoming precursor and causes unfolding and inward translocation
of the protein (45, 46). Successive action of DnaK (a bacterial Hsp70
homologue), DnaJ, and GroEL has been shown to augment the efficiency of
in vitro refolding through stabilization of folding
intermediates (47).
and
GroEL-
complexes permitted studies on the differential dependence of
the two distinct E1 subunits on GroEL for folding. The more stringent
requirement of the
subunit than the
for the chaperonin was
indicated by: 1) the more rapid loss of regained E1 activity when the
addition of GroEL to the denatured
subunit was delayed and 2) the
inability of the
subunit to reconstitute E1 activity with the
1:EL14 = 2:1 complex, in which most, if not
all, GroEL cavities were initially occupied by the
subunit (Fig.
3). In this context, it is significant that in the absence of the
folded
subunit, the
released from the GroEL-
complex rebound
to GroEL, resulting in no net discharge of the
subunit from the chaperonin (Fig. 4B). This result explains our earlier
observation that the bovine
subunit overexpressed alone in E. coli was largely insoluble and a fraction of the recombinant
subunit formed a stable complex with GroEL (26, 48). Moreover, we
recently found that the rate of import of the precursor
subunit
into mitochondria is markedly faster than its
counterpart.2 This raises
the possibility that part of the
subunit released from the
mammalian chaperonin Hsp60 may accumulate in the mitochondrial matrix
and exists as assembly competent monomers as suggested by the in
vitro data (Fig. 4A). The presence of a free
subunit pool in mitochondria would be advantageous because it ensures a
timely discharge of the
subunit from the Hsp60-
complex for assembly with the
, thereby preventing the prolonged "tie-up" of
the mitochondrial chaperonin by the
subunit. Further studies are
needed to establish this model for chaperonin-mediated biogenesis of
hetero-oligomeric protein inside mitochondria.
ternary
complex reported here is a
novel folding intermediate and differs from the previously described
interaction between GroEL and a folding intermediate (Dc) of the Fab
fragment (49). The transformation of the disulfide bonded Dc
intermediate to the native Fab fragment can occur spontaneously
in vitro, but GroEL is capable of binding Dc to form the
GroEL-Dc complex (49). Addition of ATP alone leads to discharge of Dc
from the GroEL-Dc complex, followed by the conversion of Dc to the
functional Fab fragment. In contrast, the GroEL-
complex is
formed through interaction of the chaperonin with an assembly
intermediate comprising two distinct polypeptides. The reconstitution
of
2
2 tetramers from the GroEL-
complex absolutely requires both GroES and Mg-ATP. Moreover, the
binding of the relatively large
dimer to GroEL poses a topology
problem, because the 85.5-kDa dimer exceeds the 70-kDa size constraint
for a protein to reside in the cis cavity of an asymmetric
GroEL14-(GroES7)1 complex (9). It
is likely that the highly structured
dimer and GroES bind to
opposite ends of the GroEL double-ring complex to circumvent the size
problem associated with the dimer. In line with this trans
model, we report in the accompanying paper that a large 86-kDa unfolded
maltose-binding protein-
fusion polypeptide on GroEL cannot be
sequestered by GroES in a cis configuration (50). Work is in
progress to delineate the topology of the GroEL-
complex by
cryoelectron microscopy.
and
monomers occurs on two separate GroEL
scaffolds. The folded assembly competent polypeptides that are released
from GroEL readily associate in solution to form the
dimeric
intermediate, which subsequently dimerizes through obligatory
interactions with GroEL complex to generate the native
2
2 tetramer. Our earlier pulse-chase labeling studies indicated that the folded and assembled
and
subunits can be observed within 10-20 min after a chase in E. coli (28). This time scale is similar to that observed in
vitro, where the assembled
and
subunits first appeared at
45 min of the refolding reaction (Fig. 5). Both dimerization of the
intermediate and reconstitution of E1 activity follow the second order reaction with similarly slow rate constants. It is therefore tempting to suggest that the conversion from the dimer to the tetramer
is the rate-limiting step in E1 assembly. The
dimer dissociated
from the GroEL-
complex needs to interact again with GroEL/GroES
(Fig. 10), similar to that established with refolding of small
polypeptides such as rhodanese and citrate synthetase (51, 52), thus
perpetuating the cycle until the conversion from dimer to tetramers is
complete. Our recent data indicate that the interaction of the
dimer with GroEL is complex in that GroEL/GroES and Mg-ATP promote
dissociation of the
dimer. This results in reassembly of E1
subunits into new
dimers with a fraction capable of
spontaneously converting to the native
2
2 tetramer.3 As described
above, the T265R mutation in the
subunit of a classic MSUD patient
results in an accumulation of the GroEL-
complex by presumably
impeding the release of the mutant dimer from GroEL (Fig. 11). Another
human mutation, i.e. Y393N in the
subunit that occurs in
homozygous affected Mennonite MSUD patients, prevents dimerization of
the
dimeric intermediate, leading to the production of exclusive
mutant E1 dimers both in vitro (data not shown) and in
E. coli (28). Characterization of these E1 assembly mutants
may shed light on the mechanism for the slow conversion from dimers to
tetramers during human E1 assembly.
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Fig. 11.
A working model for the pathway of
chaperonin-promoted E1 assembly. Our study has established that:
1) the folding and assembly of E1 tetramers has an absolute requirement
for chaperonins GroEL and GroES, 2) the folding of the and
subunits occurs on two different GroEL scaffolds, 3) the assembly of
the active E1 tetramers (
2
2) proceeds
through an inactive dimeric intermediate (
), and the dimerization
of
dimers mediated by GroEL/GroES is an obligatory step, and 4)
human mutations on the
subunit that block specific steps in the
chaperonin-mediated E1 assembly pathway have been identified (indicated
by X).
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ACKNOWLEDGEMENTS |
---|
We thank Clay Clark and Carl Frieden for generous gifts of highly purified GroEL preparations, which were used in the initial phase of refolding studies and also served as activity standards for our GroEL preparations. Helpful discussions with Clay Clark concerning GroEL purification are gratefully acknowledged. We also thank Cindy Cote for technical assistance.
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FOOTNOTES |
---|
* This work was supported by Grant DK-26758 from the National Institutes of Health, Grant I-1286 from the Welch Foundation, and Grant 95G-074R from the American Heart Association, Texas Affiliate.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: Dept. of Biochemistry,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75235-9038. Tel,: 214-648-2457; Fax: 214-648-8856; E-mail: chuang01{at}utsw.swmed.edu.
3 R. M. Wynn, J.-L. Song, and D. T. Chuang, manuscript in preparation.
2 J. L. Chuang, and D. T. Chuang, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
Hsp, heat shock
protein;
BCKD, branched chain -ketoacid dehydrogenase;
CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid;
DTT, dithiothreitol;
E1, branched chain
-ketoacid
decarboxylase;
E2, dihydrolipoyl transacylase;
E3, dihydrolipoyl
dehydrogenase;
FPLC, fast protein liquid chromatography;
HPLC, high
performance liquid chromatography;
IPTG, isopropyl
-D-thiogalactoside;
MSUD, maple syrup urine disease;
Ni-NTA, nitrilotriacetic acid;
TPP, thiamine pyrophosphate;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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