From the Department of Physiology and Biophysics, University of California, Irvine, California 92697
Received for publication, October 18, 2000
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ABSTRACT |
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IscU, a NifU-like Fe/S-escort protein, binds to
and stimulates the ATPase activity of Hsc66, a hsp70-type molecular
chaperone. We present evidence that stimulation arises from
interactions of IscU with the substrate-binding site of Hsc66. IscU
inhibited the ability of Hsc66 to suppress the aggregation of the
denatured model substrate proteins rhodanese and citrate synthase, and
calorimetric and surface plasmon resonance measurements showed that ATP
destabilizes Hsc66·IscU complexes in a manner expected for
hsp70-substrate complexes. Studies on the interaction of IscU with
Hsc66 truncation mutants further showed that IscU does not bind the
isolated ATPase domain of Hsc66 but does bind and stimulate a mutant
containing the ATPase domain and substrate binding Hsp701 proteins comprise
a widespread family of molecular chaperones that are composed of an
N-terminal ATP-binding domain exhibiting weak intrinsic ATPase activity
and a C-terminal peptide-binding domain that reversibly binds peptide
substrates (1-4). Proteins from this family have been implicated in a
variety of processes including stress response, de novo
protein folding, protein degradation, protein trafficking, and
disassembly of protein complexes (for reviews, see Refs. 5-7). Central
to all hsp70 activities is their ability to undergo
association/dissociation cycles with peptide substrates and to couple
ATP binding and hydrolysis with conformational changes that modulate
their peptide binding affinity. ATP binding results in conformational
changes leading to destabilization of hsp70·peptide substrate
complexes, whereas subsequent ATP hydrolysis to ADP results in
conformational changes leading to complex stabilization (8-13).
Many bacteria contain a constitutively expressed hsp70 designated Hsc66
in addition to the prototypical DnaK (14, 15). No specific peptide
substrates have been identified for Hsc66, and its exact cellular
function(s) are not known. It is also unclear whether Hsc66 functions
as a general chaperone with broad substrate specificity as is the case
for DnaK (2, 6, 16), or if Hsc66 has evolved to function with a
specific substrate. Localization of the gene encoding Hsc66,
hscA, to a gene cluster (iscSUA-hscBA-fdx) encoding proteins thought to function in the biogenesis of iron-sulfur clusters suggests that Hsc66 may play a specialized role in the folding
of Fe/S proteins (17-19). The hscB gene product from this cluster encodes a 20-kDa protein designated Hsc20 that exhibits sequence similarities with the N-terminal J-domain of DnaJ-type auxiliary co-chaperones, and in vitro studies indicate that
Hsc20 stimulates the ATPase activity of Hsc66 and regulates the rate of
conversion of Hsc66 between its different peptide affinity states (20).
Hsc66 and Hsc20 appear to comprise a distinct chaperone system with
nonoverlapping functions with the DnaK/DnaJ/GrpE system since
physiologically relevant levels of DnaJ or GrpE do not affect Hsc66
ATPase activity, and Hsc20 does not stimulate DnaK ATPase activity
(18).
Recently, we have found that the iscU gene product, IscU,
also stimulates the ATPase activity of Hsc66 (21). IscU is a novel Fe/S-binding protein believed to function as a scaffold for Fe/S cluster assembly (21-23). The nature of the interaction of IscU with
Hsc66 and the mechanism by which it stimulates Hsc66, however, is
unclear. IscU stimulation could arise from actions as an auxiliary, regulatory co-chaperone in addition to Hsc20. Alternatively,
stimulation could result from interactions of IscU with the
peptide-binding domain of Hsc66, i.e. with IscU serving as a
substrate for Hsc66. To better understand IscU regulation of Hsc66, we
have investigated the ability of IscU to compete with model peptide
substrates for binding to Hsc66 and have characterized the effects of
nucleotides on Hsc66 and IscU binding. In addition, we investigated the
ability of IscU to bind Hsc66 truncation mutants lacking specific
subdomains of the C-terminal peptide-binding region.
Materials--
Escherichia coli DH5 Overexpression and Purification of Proteins--
Recombinant
Hsc66, Hsc20, and IscU were expressed and purified as described
previously (20, 21). Vectors for overexpressing truncated forms of
Hsc66, pTrc66(D383stop) for residues 2-382, and pTrc66(D506stop) for
residues 2-505, were made by introducing stop codons into the vector
encoding Hsc66 (pTrc66; see Ref. 20) using the Unique-Site Elimination
method (CLONTECH). Both truncation mutants were
overexpressed and purified using similar methods as reported for
full-length Hsc66 (20).
ATPase Assays--
Steady-state ATPase rates were determined at
23 °C in HKM buffer (50 mM Hepes, pH 7.3, 150 mM KCl, and 10 mM MgCl2) containing 1 mM dithiothreitol (DTT) and 0.4 mM ATP as
previously reported (18, 20, 21) by measuring phosphate released using
a coupled enzyme assay with the EnzCheck phosphate assay kit (Molecular Probes).
Rhodanese and Citrate Synthase Aggregation Assays--
The
aggregation of bovine rhodanese and porcine citrate synthase were
performed in HKM buffer containing 1 mM ADP as described previously (18).
Surface Plasmon Resonance (SPR) Analysis--
SPR methods were
carried out at 25 °C with a Biacore 3000 instrument (Piscataway, NJ)
using methods described previously (21). Hsc66 in the presence of MgADP
was randomly cross-linked to the surface of the sensor chip using amine
coupling as recommended by the manufacturer. IscU was injected over the
immobilized Hsc66 in HKM buffer containing 1 mM DTT and
either 1 mM ADP or 1 mM ATP. Data are reported
as changes in relative response units (RU). Similar results were
obtained when Hsc66 was immobilized to the surface of a sensor chip in
the presence of MgATP indicating that the differences in binding
kinetics observed were not the result of the conditions of Hsc66 immobilization.
Isothermal Titration Calorimetry--
A Microcal (Amherst, MA)
Omega titration calorimeter was used to investigate the binding of IscU
to Hsc66, Hsc:2-505, and Hsc:2-382 in HKM buffer containing 1 mM DTT and 1 mM ADP using procedures described
previously (21, 24).
IscU Effects on Hsc66 Chaperone Activity--
To investigate the
possible role of IscU as a specific substrate for Hsc66, we examined
the ability of IscU to compete with the model peptide substrates
rhodanese and citrate synthase (18). Assays were carried out by
measuring the effect of Hsc66·ADP on the extent of rhodanese or
citrate synthase aggregation in the presence of varying levels of IscU.
The effect of IscU on the ability of Hsc66 to suppress the aggregation
of chemically denatured rhodanese is shown in Fig.
1A. IscU inhibited Hsc66
suppression of rhodanese aggregation in a
concentration-dependent manner with a molar ratio of IscU
to Hsc66 of 1:1 resulting in ~75% inhibition of Hsc66 chaperone
activity (15 min), and a ratio of 5:1 giving >95% inhibition. IscU
alone had no effect on rhodanese aggregation suggesting that the
changes in turbidity caused by addition of IscU to reactions containing
Hsc66 are not due to IscU directly enhancing rhodanese aggregation.
The effect of IscU on the ability of Hsc66 to suppress the aggregation
of thermally denatured citrate synthase is shown in Fig. 1B.
IscU inhibited Hsc66 suppression of citrate synthase aggregation, and
this effect was dependent on the level of IscU with a molar ratio of
IscU to Hsc66 of 2.5:1 inhibiting Hsc66 chaperone activity ~85% (40 min). IscU alone had no effect on citrate synthase aggregation. The
competition of IscU with both rhodanese and citrate synthase for
binding to Hsc66 is consistent with interaction of IscU with the
peptide-binding domain of Hsc66 and suggests that IscU may form a
stable complex with the high peptide affinity ADP state of Hsc66.
Nucleotide Effects on Hsc66 and IscU Binding--
A hallmark of
hsp70 proteins is their ability to modulate substrate binding in a
nucleotide-dependent manner. The ADP complexes of hsp70
proteins display high affinity for peptide substrates and exhibit slow
on and off rates, and exchange of ADP for ATP results in conformational
changes leading to a low substrate affinity form with faster substrate
association and dissociation rates (8-13). Because IscU was found to
compete with model peptide substrates for binding to Hsc66, it was of
interest to determine whether IscU binding to Hsc66 is regulated in a
manner similar to that observed for other hsp70 substrates.
In initial experiments, SPR analysis was used to investigate nucleotide
effects on the interaction of IscU with Hsc66. Fig. 2 shows the results of titrations in
which Hsc66 was randomly cross-linked to the sensor chip and exposed to
different concentrations of IscU in the presence of ADP or ATP. The
extent of binding of IscU to Hsc66 was similar in the presence of
either nucleotide, but the apparent affinity of Hsc66 for IscU was
greater in the presence of ADP (KD(app)
Because of possible complications in the SPR binding studies arising
from surface and/or immobilization effects, we also used isothermal
titration calorimetry (ITC) to more accurately quantitate the
interaction of IscU with Hsc66. Fig. 3
shows the enthalpic changes observed in an experiment in which
successive additions of IscU were made to Hsc66 in the presence of ADP.
The data are plotted as the integrated heats of binding
(Qinj) versus the molar ratio of IscU
to Hsc66. The best fit curve to the data indicates the presence of a
single, high affinity binding site (KD Domain Requirements for IscU Binding to Hsc66--
Hsp70 proteins
are composed of two functionally distinct domains, a highly conserved
N-terminal ATPase domain ~44-kDa and a C-terminal peptide-binding
domain ~25 kDa (1-4). Structural studies on the C-terminal domain of
DnaK complexed with peptide substrate indicate that this domain can be
further divided into two subdomains: a
The effect of IscU on the ATPase activity of Hsc66, Hsc:2-505, and
Hsc:2-382 was compared to characterize the region(s) of Hsc66 required
for IscU binding (Fig. 4C). Both truncation mutants exhibited intrinsic ATPase activity that was slightly greater than that
of full-length Hsc66. The ATPase activities of full-length Hsc66 and
Hsc:2-505 were stimulated by IscU indicating the C-terminal helical
cap is not necessary for IscU binding and activation. The activity of
the ATPase domain fragment Hsc:2-382, however, was not affected by
IscU indicating that the peptide-binding
Two approaches were used to further investigate the role of the
C-terminal helical cap of Hsc66 in interactions with IscU. To
investigate interactions of IscU with the ATP-bound state of Hsc:2-505, we examined the concentration dependence of IscU
stimulation of Hsc:2-505 ATPase activity (see Fig.
5A). The concentration of IscU
required for half-maximal stimulation of Hsc:2-505 activity (Km
To investigate interactions of IscU with the ADP-bound state of
Hsc:2-505, we measured the thermodynamics of IscU binding to
Hsc:2-505 in the presence of ADP. Fig. 5B shows the results of an ITC experiment in which successive additions of IscU were made to
a cell containing the Hsc:2-505·ADP complex. The binding affinity
observed (KD In earlier studies we found that both Hsc20 and IscU stimulate the
ATPase activity of Hsc66 and regulate the rate of conversion of Hsc66
between its different peptide affinity states (20, 21). Whereas Hsc20
shares sequence similarities with DnaJ-type auxiliary co-chaperones and
is thought to function in a manner similar to these proteins (18), IscU
represents a novel ATPase stimulatory protein whose mechanism of action
was unclear. IscU was also found to bind to Hsc20, and in the presence
of Hsc20 the concentration of IscU required for half-maximal
stimulation of Hsc66 ATPase activity was reduced (21). The interaction
between IscU and Hsc20 resembles the interaction of peptide substrates with DnaJ, a hsp40 co-chaperone. DnaJ decreases the concentration of
peptide substrates required for half-maximal stimulation of DnaK ATPase
activity (26), and thereby serves to target these substrates to the
high peptide affinity ADP state of DnaK (27). Similarities in the
actions of Hsc20 and DnaJ suggested that the increased binding affinity
of Hsc66 for IscU observed in the presence of Hsc20 could reflect the
role of IscU as a substrate.
The results described herein provide direct evidence that IscU behaves
as a substrate for Hsc66. IscU was shown to compete with model peptide
substrates for binding to the Hsc66·ADP complex consistent with IscU
binding to the high peptide affinity ADP state of Hsc66. Affinity
sensor studies also showed that nucleotides regulate Hsc66 binding to
IscU in a manner consistent with that observed for nucleotide effects
on hsp70·peptide substrate interactions (8-13). ATP-bound Hsc66
exhibited faster association and dissociation rates for IscU compared
with ADP-bound Hsc66 indicating that ATP destabilizes Hsc66·IscU
complexes. In addition, calorimetric studies revealed that IscU binds
to Hsc66 at a single site with an affinity (KD Studies examining the interaction of IscU with truncation mutants of
Hsc66 provide further support that IscU behaves as a substrate for
Hsc66. IscU stimulated the ATPase activity of a mutant containing the
ATPase domain and the peptide-binding Although the Hsc66 C-terminal helical cap was not required for IscU
stimulation of Hsc66 ATPase activity, this subdomain was required for
coupling of ATP hydrolysis with conformational changes that increase
Hsc66 binding affinity for IscU. In contrast to full-length Hsc66 in
which Hsc66·ADP complexes exhibit ~20-fold higher affinity for IscU
compared with ATP complexes, ADP and ATP complexes of Hsc:2-505
displayed similar IscU affinities indicating that both the The finding that IscU behaves as a substrate for Hsc66 raises the
question of whether the Hsc66/Hsc20 chaperone system has evolved
specifically to interact with IscU in Fe/S-cluster assembly or whether
it serves a more general chaperone function in the cell. Like other
hsp70 chaperones Hsc66 is able to bind denatured proteins
(e.g. rhodanese and citrate synthase) and to prevent their
aggregation (18). These "model substrates," however, have no effect
on the ATPase activity of Hsc66. IscU binding, in contrast, is coupled
to the nucleotide state of Hsc66 and regulates the chaperone's ATPase
reaction cycle. IscU also exhibits specific interactions with the
co-chaperone Hsc20 that is not observed with other proteins or peptides
(18, 21). These results, together with genetic findings implicating
yeast homologs of Hsc66, Hsc20, and IscU in iron-sulfur protein
biogenesis (19), suggest that the primary cellular function of the
Hsc66/Hsc20 chaperone system may be to interact with IscU in
Fe/S-cluster generation.
While the studies described herein indicate that IscU binds as a
substrate to Hsc66, the specific structural features of IscU required
for interaction with Hsc66 are not known. Hsc66 may recognize a
structured motif within IscU or may bind an unstructured region of IscU
in an extended conformation similar to that recognized by DnaK (25).
Further studies are also needed to investigate how Hsc66 and Hsc20
affect the function of IscU in iron-sulfur cluster assembly. The exact
role the Hsc66/Hsc20 chaperone system plays in iron-sulfur cluster
biogenesis remains unclear. The chaperone system may function in
assembly or stabilization of Fe/S clusters on IscU or may facilitate
transfer of Fe/S clusters formed on IscU to an apo-acceptor protein.
-sandwich
subdomain. These results support a role for IscU as a substrate for
Hsc66 and suggest a specialized function for Hsc66 in the assembly, stabilization, or transfer of Fe/S clusters formed on IscU.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FIQ cells were
from Life Technologies, Inc. Enzymes for DNA manipulation were obtained
from Roche Molecular Biochemicals, New England Biolabs, Inc., or
U. S. Biochemical Corp. Synthetic nucleotides were obtained from
Genosys. Bacterial growth media components were from Difco, and other
reagents were from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
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Fig. 1.
IscU inhibition of Hsc66 chaperone
activity. Aggregation reactions were performed in HKM buffer
containing 1 mM ADP. A, aggregation of 2 µM rhodanese alone ( ) and in the presence of 15 µM Hsc66 with 0 (
), 5 (
), 10 (
), 15 (
), or 75 µM (
) IscU. Denatured rhodanese in 6 M
guanidine hydrochloride was diluted into reaction mixture, and
absorbance changes at 320 nm were used to monitor aggregation at
25 °C. B, aggregation of 1.6 µM citrate
synthase alone (
) and in the presence of 4 µM Hsc66
with 0 (
), 1 (
), 3 (
), 5 (
), or 10 µM (
)
IscU. Native citrate synthase was diluted into reaction mixtures at
43 °C, and absorbance changes at 320 nm were used to monitor
aggregation resulting from thermal denaturation.
9 µM) compared with that observed in the presence of
ATP (KD(app)
37 µM).
Examination of the sensorgrams (insets Fig. 2) also reveals
differences in the binding kinetics as a function of nucleotide. IscU
association in the presence of ATP is faster than that observed in the
presence of ADP, and the half-time for IscU dissociation in the
presence of ATP (t1/2
2 s) is ~30-fold
faster than that observed in the presence of ADP
(t1/2
60 s). These results indicate ATP
destabilizes Hsc66·IscU complexes in a manner similar to that
observed for other hsp70-substrate interactions (8-13) and are
consistent with interaction of IscU with the peptide-binding site of
Hsc66.
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Fig. 2.
SPR analysis of nucleotide effects on IscU
binding to Hsc66. IscU was injected into solutions passing over a
sensor chip containing immobilized Hsc66 (~3000 RU) at 25 °C, and
maximum response signals observed are plotted as a function of the
concentration of IscU. The equilibration solution contained HKM buffer,
1 mM DTT, and either 1 mM ATP ( ) or 1 mM ADP (
). Curves shown represent fits of the
data to hyperbolic saturation functions with RUmax = 240 and KD = 9.3 µM in the presence of ADP
and RUmax = 232 and KD = 37.2 µM in the presence of ATP. The insets show
overlays of individual sensorgrams recorded for successive injections
of IscU in the presence of either ATP or ADP.
1.6 µM) consistent with specific binding to the Hsc66·ADP
complex in a manner expected for a substrate.
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Fig. 3.
Calorimetric analysis of IscU binding to
Hsc66. A series of 35 equivalent 8-µl aliquots of 1.0 mM IscU were injected into a cell containing 1.348 ml of
100 µM Hsc66 at 25 °C in HKM buffer containing 1 mM DTT and 1 mM ADP. Integrated heats due to
binding, Qinj (corrected for the heats of
dilution and divided by the moles of IscU injected), are plotted
versus the molar ratio of IscU to Hsc66 in the titration
cell. The solid line represents a best-fit curve assuming
0.969 binding sites, KD = 1.6 µM,
H = 12.3 kcal/mol, and
S = 67.6 e.u.
-sandwich region that
directly binds model peptide substrates, and a C-terminal helical cap
that lies above the peptide binding pocket of the
-sandwich (25). To
investigate which domain(s) of Hsc66 are required for IscU binding, we
constructed two truncation mutants (Fig.
4, A and B). The
first mutant, designated Hsc:2-505, lacked the C-terminal helical cap
but contained both the N-terminal ATPase domain and the peptide-binding
-sandwich subdomain (residues 2-505). The second mutant, designated
Hsc:2-382, contained only the ATPase domain (residues 2-382).
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Fig. 4.
Effect of IscU on the ATPase activity of
Hsc66 truncation mutants. A, diagrams of the domain
composition of Hsc66 constructs. B, SDS-PAGE analysis of 10 µg each of purified Hsc66 (lane 1), Hsc:2-505 (lane
2), Hsc:2-382 (lane 3). C, steady-state
ATPase activity of Hsc66, Hsc:2-505, and Hsc:2-382 were determined in
HKM buffer containing 400 µM ATP at 23 °C in the
absence (solid bars) and presence of 100 µM
IscU (open bars).
-sandwich subdomain is
required for IscU stimulation of ATPase activity as expected if IscU is
a substrate for Hsc66. To determine whether IscU interacts with the
isolated ATPase domain Hsc:2-382 in a manner not manifested as effects
on ATPase activity, we also used ITC to measure enthalpic changes that
might arise as a result of binding. Injection of IscU into a cell
containing Hsc: 2-382 and ADP did not result in any detectable
enthalpic changes suggesting that IscU does not interact directly with
the isolated ATPase domain (data not shown).
31 µM) is similar to that
previously reported for full-length Hsc66 (Km
34 µM; Ref. 21), although the maximal stimulation observed
for Hsc:2-505 (~14-fold) is slightly greater than that of
full-length Hsc66 (~8-fold; Ref. 21). These findings indicate that
removal of the C-terminal helical cap of Hsc66 has little effect on
either the affinity of ATP-bound Hsc66 for IscU or its ability to
couple IscU binding with increased ATPase activity.
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Fig. 5.
Nucleotide effects on IscU and Hsc:2-505
interactions. A, effect of IscU on the ATPase activity
of Hsc:2-505. Results are reported as the increase in basal ATPase
rates at 23 °C. The curve shown represents a best fit to
the data for a maximal stimulation of 13.8-fold and half-maximal
stimulation at 31 µM. B, ITC analysis of IscU
binding to Hsc:2-505. A series of 35 equivalent 8-µl aliquots of 1.5 mM IscU were injected into a cell containing 1.348 ml of
100 µM Hsc:2-505 at 25 °C in HKM buffer containing 1 mM DTT and 1 mM ADP. Integrated heats due to
binding, Qinj, are plotted versus the
molar ratio of IscU to Hsc:2-505 in the titration cell. The
solid line represents a best-fit curve assuming 0.83-binding
sites, KD = 26 µM, H = 12.85 kcal/mol, and
S = 64.1 e.u.
26 µM) is ~16-fold
weaker than that of full-length Hsc66 under similar conditions
(KD
1.6 µM; Fig. 3). Instead, it
is similar to the apparent affinity observed for the ATP-bound state of
Hsc:2-505 (31 µM; Fig. 5A). Thus, the
C-terminal helical cap of Hsc66 is required for the increased binding
affinity for IscU that occurs following ATP hydrolysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.6 µM) ~20-fold greater than the concentration of IscU
required for half-maximal stimulation of Hsc66 ATPase activity
(Km
34 µM; Ref. 21). Assuming that
the Km observed in ATPase stimulation assays
reflects the binding affinity of IscU for the ATP state of
Hsc66,2 the differences in
affinities of the ADP and ATP-bound states is similar to that observed
with other hsp70·substrate complexes (9, 11, 12).
-sandwich region (Hsc:2-505),
but no interactions were observed with a mutant composed solely of the
ATPase domain (Hsc:2-382). These results establish that the
-sandwich region of Hsc66 is essential for IscU binding as is the
case for substrate binding to other hsp70 proteins (25).
-sandwich
and helical cap subdomains are required for high affinity binding. The
C-terminal helical cap does not, however, appear to be required for all
hsp70-substrate interactions. A recent report showed that DnaK mutants
lacking the cap retain efficient coupling of ATP hydrolysis with
increased substrate binding affinity (28). This may reflect structural
differences between Hsc66 and DnaK or may arise from differences in the
types of substrates recognized. The short peptide used in the DnaK
studies (28) would not be expected to make extensive contacts with the C-terminal helical cap (25). The larger IscU protein, in contrast, may
interact with both the
-sandwich and helical cap subdomains of
Hsc66, and interactions of IscU with both subdomains may be required
for high affinity binding. There is evidence that the helical cap may
be important for regulatory interactions of the eukaryotic homologs of
Hsc66. Mutations in the corresponding C-terminal subdomain of the yeast
mitochondrial homolog of Hsc66, Ssq1, gives rise to phenotypic effects
indicative of reduced chaperone activity (19).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM54264 and Training Grant GM07311.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 Physiology
and Biophysics, University of California, Irvine, CA 92697. Tel.:
949-824-6580; Fax: 949-824-8540; E-mail: lvickery@uci.edu.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009542200
2 The Km obtained in ATPase assays is only a good estimate of the equilibrium dissociation constant of ATP-bound Hsc66 for IscU if binding of IscU is rapid relative to ATP hydrolysis (29).
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ABBREVIATIONS |
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The abbreviations used are: hsp, heat shock protein; DTT, dithiothreitol; Hsc:2-382, mutant containing residues 2-382 of Hsc66; Hsc:2-505, mutant containing residues 2-505 of Hsc66; SPR, surface plasmon resonance; RU, response units; ITC, isothermal titration calorimetry.
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