From the Biochemisches Institut, Universität
Zürich, CH-8057 Zürich, Switzerland, and § F.
Hoffmann-La Roche Ltd., Pharmaceutical Research-Metabolic Diseases,
CH-4070 Basel, Switzerland
Received for publication, October 11, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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DnaK, a Hsp70 acting in concert with its
co-chaperones DnaJ and GrpE, is essential for Escherichia
coli to survive environmental stress, including exposure to
elevated temperatures. Here we explored the influence of temperature on
the structure of the individual components and the functional
properties of the chaperone system. GrpE undergoes extensive but fully
reversible conformational changes in the physiologically relevant
temperature range (transition midpoint at ~48 °C), as
observed with both circular dichroism measurements and differential
scanning calorimetry, whereas no thermal transitions occur in DnaK and
DnaJ between 15 °C and 48 °C. The conformational changes in GrpE
appear to be important in controlling the interconversion of T-state
DnaK (ATP-liganded, low affinity for polypeptide substrates) and
R-state DnaK (ADP-liganded, high affinity for polypeptide
substrates). The rate of the T Chaperone systems of the Hsp70 family facilitate the folding of
nascent polypeptide chains and denatured proteins, preventing the
formation of protein aggregates (for a comprehensive review, see Ref.
1). DnaK, a Hsp70 homolog of Escherichia coli, binds peptides and segments of denatured proteins in extended conformation (2, 3). In its chaperone action, DnaK cooperates with two cohort
heat-shock proteins, DnaJ and GrpE (4). The DnaK chaperone system has
been studied extensively in vitro at ambient temperatures. The model cycle of the system may be summarized as follows (Fig. 1A; Refs. 5-11): DnaK
alternates between two states, the ATP-liganded T state and the
ADP-liganded R state. The conversion of DnaK from its T state to the R
state is mediated by DnaJ, which accelerates the hydrolysis of
DnaK-bound ATP. The conversion from the R state back to the T state is
triggered by GrpE, which facilitates the exchange of DnaK-bound ADP for
ATP. The affinity of T-state DnaK for peptide and protein substrates is
low, and both binding and release of substrates are fast. In contrast,
the substrate affinity of R-state DnaK is high, and the rates of
binding and release of substrates are too slow to be of physiological
significance. Thus, a substrate is first bound by T-state DnaK, which
is then converted to the high-affinity R state in a DnaJ-triggered
reaction. With the assistance of GrpE, DnaK is re-converted into the
low-affinity T state, releasing the substrate.
R conversion of DnaK due to
DnaJ-triggered ATP hydrolysis follows an Arrhenius temperature
dependence. In contrast, the rate of the R
T conversion due to
GrpE-catalyzed ADP/ATP exchange increases progressively less with
increasing temperature and even decreases at temperatures above
~40 °C, indicating a temperature-dependent reversible
inactivation of GrpE. At heat-shock temperatures, the reversible
structural changes of GrpE thus shift DnaK toward its high-affinity R state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Control of the DnaK chaperone cycle by the
co-chaperones DnaJ and GrpE. A, low-affinity T-state
DnaK (ATP-liganded) rapidly binds and releases the target polypeptide
( ). DnaJ triggers the hydrolysis of DnaK-bound ATP and thus the T
R conversion of DnaK. High-affinity R-state DnaK (ADP-liganded)
slowly binds and releases the polypeptide. GrpE catalyzes the exchange
of ADP for ATP, and in this way triggers the completion of the
functional cycle (5-11). B, crystal structure of the
nucleotide exchange factor GrpE. Dimeric GrpE has been crystallized in
complex with the ATPase domain of DnaK (22).
-Helices and
-sheets
in GrpE are represented by ribbons; residues not resolved in
the crystal structure are denoted with dotted lines. The
contour of the ATPase domain of DnaK is indicated with a solid
line. The atomic coordinates are from Protein Data Bank entry
1DKG.
The expression of DnaK and the co-chaperones DnaJ and GrpE is
controlled by the transcription factor 32 (for a review,
see Ref. 12). The expression levels of chaperones and co-chaperones are
enhanced by heat shock, resulting in an approximately 2-fold increase
in the cellular concentrations of DnaK (13) and the co-chaperones.1 Here we
report a study of the effect of heat-shock conditions on the DnaK
chaperone system itself. We found the co-chaperone GrpE to undergo a
reversible conformational transition within the physiologically
relevant temperature range that appears to be of functional significance.
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EXPERIMENTAL PROCEDURES |
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Materials--
DnaK was purified as described previously (6) and
stored at 80 °C. To prepare stock solutions for experimentation,
samples containing ~12 mg of DnaK were thawed, concentrated by
ultrafiltration (Centricon-30; Amicon) to 500 µl, and transferred to
assay buffer (25 mM Hepes/NaOH, 100 mM KCl, 10 mM MgCl2, pH 7.0, or 25 mM
potassium phosphate, pH 7.0, for circular dichroism experiments) by
size exclusion chromatography (NAP-10; Amersham Pharmacia Biotech). The
protein concentration was determined photometrically with a molar
absorption coefficient of
280 = 14,500 M
1 cm
1
(14). The concentrations of DnaK stock solutions were ~100 µM. The nucleotide content was <0.1 mol nucleotide/mol
DnaK (15). DnaJ (110 µM; in 50 mM
Tris-HCl, pH 7.7) and GrpE (200 µM; in 50 mM
Tris-HCl, pH 7.7) were prepared as described elsewhere (16, 17). For
circular dichroism measurements and differential scanning calorimetry,
GrpE was dialyzed against 25 mM potassium phosphate, pH
7.0.
The amino acid sequence of peptide ala-p5 (ALLLSAPRR) is derived from
the 23-residue prepiece of mitochondrial aspartate aminotransferase of
chicken, which has previously proven to be a high-affinity ligand for
DnaK (6, 18). Peptide a-ala-p5 is labeled with acrylodan
(6-acryloyl-2-dimethylaminonaphtalene) at the -amino group.
Synthesis and labeling of the peptide (purity > 95%) are described elsewhere (10). The concentrations of the stock solutions of
ala-p5 and a-ala-p5 were 1.9 mM in water and 240 µM in 20% (v/v) acetonitrile, respectively.
N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP)2
was a gift from Dr. J. Reinstein (Max Planck Institut für
molekulare Physiologie, Dortmund, Germany) and had been
synthesized as described elsewhere (9). A fresh ATP stock solution (50 mM disodium salt, pH 7.0; Fluka) in assay buffer was
prepared before every experimental series. The ADP/Pi stock
solution was 38 mM ADP (disodium salt; Fluka), 50 mM potassium phosphate, pH 7.0.
Circular Dichroism Measurements--
Circular dichroism was
measured with a Jasco J-715 spectropolarimeter (Jasco, Tokyo, Japan)
using a thermostated cuvette of 1 or 0.2 mm path length. The
temperature was controlled with a programmable water bath. At fixed
temperatures, four spectra (2 nm bandwidth) between 250 and 185 nm were
recorded at a scan rate of 5 nm min1 and
averaged. The time course of temperature-induced conformational changes
was followed by continuously monitoring the ellipticity at 222 nm at a
scan rate of 1 degree min
1.
Differential Scanning Calorimetry--
A VP-DSC
microcalorimeter (MicroCal, Northampton, MA) equipped with twin
coin-shaped cells of 0.52 ml volume was used. Technical details and
performance of the instrument have been described elsewhere (19). The
protein was dialyzed for 18 h against the same batch of buffer
that was used to establish the baseline (25 mM potassium
phosphate, pH 7.0). Instead of degassing the sample, two successive
pre-scan cycles of heating and rapid cooling were performed between
5 °C and 35 °C. The scanning rate was 1 degree min1. The data were corrected for the
buffer-buffer baseline and normalized for the concentration.
Fast Kinetic Measurements--
An Applied Photophysics SX18 MV
stopped-flow apparatus served to record the changes in intrinsic
fluorescence of DnaK (excitation at 290 nm; emission high-pass filter
305 nm), acrylodan fluorescence of peptide a-ala-p5 (excitation at 370 nm; emission high-pass filter 455 nm), or MABA fluorescence of MABA-ADP
(excitation at 360 nm; emission high-pass filter 455 nm). The
temperature was controlled with a water bath. The instrument was
equilibrated at the respective temperature (± 0.5 °C) for at least
3 min before performing the experiments. ADP/Pi, MABA-ADP,
and peptide were preincubated with DnaK for ~1 h at ambient
temperature. All experiments were performed in assay buffer (25 mM Hepes-NaOH, 100 mM KCl, 10 mM
MgCl2, pH 7.0) and were started by mixing equal volumes of
the two reaction solutions (~70 µl each). The data were evaluated with the software provided by the manufacturer of the instrument. The
standard error of the rate determinations was between 0.5% and 6%,
except when the reaction (T R conversion) was not monophasic due to
accelerated spontaneous ATP hydrolysis at high temperature (Fig. 8,
B and C; standard error 13% and 10%,
respectively, at 48 °C). Arrhenius curves were fitted to the rate
constants with the Origin program from MicroCal Software (Northampton, MA).
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RESULTS |
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Temperature-induced Structural Transitions in DnaK, DnaJ, and
GrpE--
The thermal transitions in the secondary structure of DnaK
and its co-chaperones were monitored with circular dichroism
spectroscopy. GrpE was further examined by differential scanning
calorimetry. In ADP-liganded DnaK, no structural changes were observed
between 15 °C and 48 °C. At temperatures above the
physiologically relevant range, two thermal transitions take place
(Fig. 2); the midpoint of the
low-temperature transition is at 58 °C, and the midpoint of the
high-temperature transition is at 75 °C. In the first thermal transition, ~40% of the total ellipticity at 222 nm is lost; the residual secondary structure is abolished in the high-temperature transition. The structural transitions become partially irreversible when DnaK is heated to 95 °C. The stability of nucleotide-free DnaK
is, as observed previously (20, 21), significantly lower than that of
the nucleotide-liganded form; in the absence of nucleotide the
first transition midpoint is shifted toward lower temperature by
9 °C (data not shown). Similar to nucleotide-liganded DnaK, DnaJ
undergoes no structural changes between 15 °C and 48 °C (Fig. 3). A single thermal transition with a
midpoint at ~58 °C is observed. The denaturation is irreversible
when DnaJ is heated to 95 °C.
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GrpE undergoes two well-separated temperature-induced conformational
transitions, with midpoints at ~48 °C and 75-80 °C, as evident
from both circular dichroism measurements
and differential scanning calorimetry (Figs.
4 and 5).
Experiments at varying concentrations (5-75 µM) of GrpE,
which exists as a dimer (Fig. 1B; Refs. 16 and 22),
indicated the low-temperature transition midpoint to be
concentration-independent, consistent with a monomolecular unfolding
process (data not shown). The unfolding, as monitored by both loss of
ellipticity and change in molar heat capacity, was completely
reversible after heating up to 60 °C (Fig. 5). The low-temperature
heat capacity peak was broad and asymmetric, indicating that either
part of the protein unfolds in a noncooperative manner or several
tightly coupled cooperative transitions occur. In the low-temperature
transition, ~25% of the total heat is absorbed. Unlike DnaK and
DnaJ, GrpE loses a significant fraction of ellipticity below 48 °C
(~40% of total). A rigorous thermodynamic analysis of the complete
unfolding mechanism of GrpE is beyond the scope of the present study
and will be the subject of further investigation.
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Temperature Dependence of the Rates of T/R Interconversion--
We
examined the effects of temperature within the physiologically relevant
range on the co-chaperone-induced interconversion of DnaK between its
low-affinity T state and its high-affinity R state. The rates of these
conversions were determined at fixed temperatures during a stepwise
increase in temperature from 15 °C to 48 °C and then at fixed
temperatures during stepwise cooling of the same solution. Three
different types of measurements were performed: (i) DnaK possesses a
single tryptophan residue at position 102 that allows spectroscopic
monitoring of conformational changes (5, 10, 21); (ii) the increase in
fluorescence intensity of acrylodan-labeled peptide a-ala-p5 upon
binding of the peptide to DnaK (6) affords another possibility for
monitoring the T/R interconversion of DnaK, which is accompanied by
changes in the affinity for target peptides (Fig. 1A; Ref.
23); and (iii) the release of fluorescence-labeled ADP allows the
monitoring of the rate-determining step of the nucleotide exchange,
which takes place during the R T conversion (9).
When the DnaJ-triggered T R conversion was followed by the increase
in the intrinsic fluorescence of DnaK, the rates of the conversion as a
function of temperature complied with the Arrhenius equation (Fig.
6A). The conversion reactions
were performed at 0.5 and 1 µM DnaJ; at all temperatures,
their rates proved to be a linear function of the concentration of
DnaJ. If we followed the increase in acrylodan fluorescence of peptide
a-ala-p5 during the T
R conversion (Fig. 6B), we
observed the same pattern of temperature dependence seen when the
intrinsic fluorescence was monitored, although the observed rate
constants were somewhat slower.
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In contrast, if the temperature dependence of the rate of the
GrpE-catalyzed R T conversion was examined, a reversible
temperature-dependent inactivation of GrpE became apparent
that above ~40 °C resulted in decreasing reaction rates (Fig.
7A). The reactions, followed by the decrease in the intrinsic fluorescence of DnaK, were performed at different concentrations of GrpE; at all temperatures, their rates
were a linear function of the concentration of GrpE. At all
concentrations of GrpE, the fastest rates were measured at ~40 °C.
Similar reaction rates and the same non-Arrhenius temperature dependence were observed when the decrease in acrylodan fluorescence of
peptide a-ala-p5 was followed during the GrpE-catalyzed R
T
conversion (Fig. 7B). The non-Arrhenius temperature
dependence was also observed when the release of fluorescent MABA-ADP
was followed (Fig. 7C).
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The conformation and peptide binding properties of nucleotide-free
DnaK are comparable with those of ADP-liganded, i.e.
R-state, DnaK (9, 10). The R T conversion of DnaK can thus be
triggered by the addition of ATP to nucleotide-free DnaK rather than by the addition of GrpE. This experimental setup allows the examination of
whether the non-Arrhenius temperature dependence observed in the R
T conversion (Fig. 7) is inherent to the R
T conversion or has to
be attributed to the participation of GrpE in this process. The rates
of the R
T transition triggered by the addition of ATP to
nucleotide-free DnaK in the absence of GrpE and monitored by the
decrease in the intrinsic fluorescence of DnaK exhibited an Arrhenius
temperature dependence (Fig.
8A). The rates were more than
1 order of magnitude faster than those of the R
T conversion
induced by the GrpE-catalyzed exchange of nucleotide (Fig.
7A), in which, under the chosen conditions, the
GrpE-catalyzed release of nucleotide is rate-limiting. When the R
T
conversion occurred without the need for release of bound ADP (Fig.
8A), the concentration of added ATP (5 and 2.5 mM) did not influence the observed rates. Apparently, the
change in conformation of DnaK rather than the binding of ATP is
rate-limiting for the observed change in the fluorescence signal. When
ADP-liganded DnaK was used (Fig. 8B), and the R
T
conversion occurred through spontaneous exchange of DnaK-bound ADP with
ATP in the absence of GrpE, we still observed the Arrhenius temperature
dependence of the rates of the R
T conversion, although in this
case the rates were several orders of magnitude slower than that seen
in the case of nucleotide-free DnaK (Fig. 8A). Again, the
reaction rates did not depend on the ATP concentration, which in this
case indicates that the spontaneous release of ADP from DnaK is
rate-limiting. When the R
T conversion induced by spontaneous
ADP/ATP exchange was followed through the decrease in acrylodan
fluorescence of the substrate peptide (Fig. 8C), an
Arrhenius temperature dependence was again found, with the observed
rates being about the same as those observed when the intrinsic
fluorescence of DnaK was followed (Fig. 8B). When the
spontaneous release of MABA-ADP was followed (Fig. 8D), the
rates also exhibited an Arrhenius temperature dependence. However, the
reaction rates were clearly slower than those observed when either the
intrinsic fluorescence or the acrylodan fluorescence was followed
(Figs. 8, B and C). Because all experiments in
Fig. 8 were performed in the absence of GrpE, and in all experiments the R
T conversion showed an Arrhenius temperature dependence, we
conclude that the non-Arrhenius temperature dependence of the GrpE-catalyzed R
T conversion (Fig. 7) is due to the participation of GrpE.
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DISCUSSION |
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Our experiments addressed the question of how temperature influences the structure of the components of the DnaK chaperone system (DnaK, DnaJ, and GrpE) and the kinetics of the co-chaperone-controlled T/R interconversion of DnaK. The experiments were performed over a temperature range from 15 °C up to the extreme heat-shock temperature of 48 °C, which approximately corresponds to the upper temperature boundary for growth of E. coli (24).
Nucleotide-liganded DnaK proved to be stable against thermal denaturation within this temperature range. In accordance with the stability of nucleotide-liganded DnaK, the steady-state ATPase activity of DnaK in the absence of co-chaperones shows Arrhenius behavior within the physiologically relevant temperature range (25). At higher temperatures, ADP-liganded DnaK shows two structural transitions (Fig. 2). The first transition has been assigned to the NH2-terminal ATPase domain because the isolated ATPase domain undergoes at a similar temperature a thermal transition that is absent in the isolated peptide-binding domain (20), a notion that has not remained undisputed (26). The co-chaperone DnaJ was also found to be stable up to 48 °C (Fig. 3), a finding that is consistent with previous studies (27). The very minor loss in ellipticity that was observed during heating and cooling of DnaK and DnaJ between 15 °C and 48 °C might be due to the fact that the midpoint of the first thermal transition, as determined by differential scanning calorimetry, is at 54.4 °C and 53 °C for DnaK (20) and DnaJ (27), respectively, i.e. close to 48 °C. These midpoint temperatures are lower than those indicated by the circular dichroism measurements (Figs. 2A and 3A) and infrared spectroscopy (27). We also cannot exclude that the observed decrease in circular dichroism is due in part to interactions of the proteins with the surface of the cuvette.
GrpE is the only constituent of the DnaK chaperone system that
undergoes extensive structural alterations within the physiologically relevant temperature range. In both circular dichroism measurements and
differential scanning calorimetry, the first of the two thermal transitions becomes evident at ~35 °C and reaches its midpoint at
48 °C (Figs. 4 and 5). This low-temperature transition is fully reversible. Because we did not observe a
concentration-dependent shift of the first transition in
GrpE, we assume that this transition is not accompanied by a change in
the state of oligomerization. This notion is confirmed by the
observation that the maximum rates of the GrpE-catalyzed R T
conversion are always reached at ~40 °C, irrespective of the
concentration of GrpE (see below). A single unfolding transition in
GrpE has been reported previously (28). The circular dichroism
measurements in that study were performed up to a temperature of
~80 °C, whereby the second thermal transition may not have become apparent.
In accord with the thermal stability of DnaK (Fig. 2) and DnaJ (Fig.
3), the DnaJ-catalyzed T R conversion of DnaK shows an Arrhenius
temperature dependence within the physiologically relevant temperature
range (Fig. 6A). The rates of increase in fluorescence of
peptide a-ala-p5, which accompanies the conversion of DnaK from its
low-affinity state to its high-affinity state, showed a similar
temperature dependence (Fig. 6B). In contrast, the
GrpE-induced R
T conversion substantially deviated from the
Arrhenius temperature dependence (Fig. 7A). The same
deviation from normal temperature dependence applied for the
GrpE-triggered release of peptide (Fig. 7B) and the release
of fluorescence-labeled nucleotide (Fig. 7C). However, the R
T conversion, unless triggered by GrpE, complied with the Arrhenius
equation. The non-Arrhenius rate-temperature curve was only and always
observed if the R
T conversion was catalyzed by GrpE; the deviant
temperature dependence must thus be due to GrpE. The obvious
sensitivity of the functionality of GrpE toward temperature correlates
with the reversible structural transition of GrpE in the same
temperature range. Experiments are underway to assign the thermal
transitions to specific structural features of GrpE.
The decrease in efficacy of GrpE in catalyzing the ADP/ATP exchange at
higher temperatures may be due to either a decreased affinity for DnaK
or a decreased specific activity. Dimeric GrpE forms a tight complex
with DnaK; at ambient temperature, the dissociation equilibrium
constant of the GrpE/DnaK complex is estimated to be 1 nM
in the absence of nucleotide and 0.22 µM in the presence of MABA-ADP (29). Varying the concentration of GrpE (0.1-1
µM) does not shift the temperature at which the rate of
nucleotide exchange is at its maximum, indicating that the stability of
GrpE/DnaK complexes is not significantly impaired at higher
temperatures. The crystal structure of GrpE complexed with the ATPase
domain of DnaK (22) shows several noncontiguous contact areas between DnaK and GrpE, with the two largest being in the -sheet domain of
GrpE. Contact areas are also located at the COOH-terminal end of the
long helix of GrpE (Fig. 1B). Temperature-dependent
changes in these contact areas of GrpE might underlie its
temperature-sensitive functional behavior. However, limited
temperature-dependent structural changes in DnaK itself
that might modulate its interaction with GrpE cannot be excluded. An
exposed, conserved loop in the ATPase domain of DnaK is needed for
stable interaction with GrpE, as has been shown by deletion of this
loop (30). Besides, there might be an additional interaction of the
extended helix of GrpE with the peptide-binding domain (22). However,
there is no evidence for a significant thermal transition in DnaK in
the physiologically important temperature range. In view of the thermal
transition of GrpE, which is extensive, occurs in the relevant
temperature range, and is reversible, GrpE seems to be the prime
candidate to control the kinetics of the R
T conversion of DnaK
in a temperature-dependent manner.
The differential temperature dependence of the
DnaJ-dependent T R conversion and the
GrpE-dependent R
T conversion leads, with increasing
temperature, to a progressive shift of DnaK toward its high-affinity R
state. This shift becomes particularly prominent at heat-shock
temperatures due to the decrease in the rate of the R
T conversion.
In our experimental setup, i.e. equimolar concentrations of
DnaK, DnaJ, and GrpE, the changes in interconversion rates between
15 °C and 48 °C indicate a 10-fold shift in favor of the R state
of DnaK at heat-shock temperatures (from 0.7% to 7%; Table
I). The fraction of R-state DnaK (7% of
the total) seems modest. However, together with the increase by
more than 2 orders of magnitude in the rate of ATP hydrolysis,
i.e. of the T
R conversion, observed in the presence of
a protein substrate (11), the shift might result in the sequestering of
protein substrates at heat-shock temperatures. During heat shock, DnaK and DnaJ have indeed been reported to cooperatively retain thermally unfolded substrate protein in a folding competent state both in vivo (31, 32) and in vitro (31, 33), whereas GrpE is
required for the reactivation of the substrate protein after the heat
shock (31, 33). The occurrence of a sequestering mechanism at
heat-shock temperatures does not preclude the possibility of an
additional mechanism of action in which DnaK uses the energy of ATP
hydrolysis to exert conformational work upon polypeptide substrates
that have undergone off-pathway folding (10, 34).
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GrpE homologs exist in bacteria, eukaryotic mitochondria, and
chloroplasts, but not in the eukaryotic cytosol and the endoplasmic reticulum. Whereas apparently not all Hsp70 systems depend on a
separate nucleotide exchange factor, GrpE is essential for bacterial viability at all temperatures (35). A mutant of DnaK, which exhibits
impaired interaction with GrpE (30), has only marginal chaperone
activity (36). In vitro, efficient refolding of firefly luciferase requires GrpE in addition to DnaK and DnaJ (7, 31). The
yield of chaperone-assisted refolding of firefly luciferase attains a
maximum at a specific molar ratio of GrpE to DnaJ and DnaK (29).
Obviously, the balance of ATP hydrolysis and nucleotide exchange,
accelerated by DnaJ and GrpE, respectively, and thus the ratio of
T-state to R-state DnaK, are important for effective refolding of
denatured proteins. The amount of the components of the DnaK heat-shock
system is known to be controlled through the regulation of
transcription, stability, and activity of 32 (for a
review, see Ref. 12). The differential temperature dependence of the T
R and R
T conversions described in this report might be the
basis of a mechanism for adapting the DnaK/DnaJ/GrpE chaperone system
to heat-shock conditions by a modulation of functionality rather than by a regulation of quantity.
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ACKNOWLEDGEMENTS |
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We thank Ezra Pierpaoli for helpful discussions and critical reading of the manuscript, Jochen Reinstein for MABA-labeled ADP, and Hans Schmid and Bas van Wieringen for technical assistance.
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FOOTNOTES |
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* 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: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-55-60; Fax: 41-1-635-59-07; E-mail: christen@biocfebs.unizh.ch.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009290200
1 X. Liu, P. Christen, H.-J. Schönfeld, and E. Sandmeier, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviation used is: MABA-ADP, N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate.
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