From the Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany and § Medizinische Hochschule Hannover, Inst. für Biochemie, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
Received for publication, February 17, 2003, and in revised form, March 11, 2003
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ABSTRACT |
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The ubiquitous small heat shock proteins (sHsps)
are efficient molecular chaperones that interact with nonnative
proteins, prevent their aggregation, and support subsequent refolding.
No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, In response to environmental stress such as heat shock, which
leads to the accumulation of nonnative proteins, cells increase the
expression of several classes of proteins (1). The major conserved
families of these heat shock proteins
(Hsps)1 have been shown to be
involved in protein folding as molecular chaperones (2).
The most divergent of these chaperone classes are the small heat shock
proteins (sHsps). sHsps have been found in almost all organisms
investigated so far, with the number of members varying from species to
species. They share conserved regions mostly in the C-terminal part of
the protein, whereas the N-terminal part differs in sequence and
length, leading to molecular masses of 16-42 kDa for sHsps in
different organisms (3). The conserved C-terminal domain of ~100
amino acids shares sequence homology with the major eye lens protein
In vitro, sHsps act as molecular chaperones in preventing
unfolded proteins from irreversible aggregation and
insolubilization (19, 23-28). Because of their high binding capacity
of up to one substrate molecule/sHsp subunit, sHsps are more efficient than other chaperones in this respect (19, 25, 29-32). The nonnative
proteins seem to be surface-exposed in the complexes because bound
proteins and peptides are accessible to antibodies (33). The range of
substrates recognized covers peptides as well as oligomeric enzymes
(25, 33, 34). No substrate specificity has been observed for sHsps so
far, and complex formation with substrates has not been analyzed in
detail. Here, we set out to address this question using Hsp25 from
mouse and Hsp26 from yeast, two different well studied members of the
sHsp family.
Murine Hsp25 exists as a hexadecamer in solution. These oligomers are
in a concentration-dependent equilibrium with tetramers, suggesting tetramers as the basic building block of the hexadecamer (17). It was previously shown that Hsp25 is able to influence the
inactivation and subsequent aggregation of the model substrate citrate
synthase (CS). Hsp25 binds several nonnative CS molecules and protects
them from irreversible aggregation. Under permissive folding
conditions, the bound substrates can be released from Hsp25 and, in
cooperation with other ATP-dependent chaperones, regain
their native structures (34).
Under physiological conditions, yeast Hsp26 forms spherical 24-mers
with particle diameters of ~15 nm. Elevated temperatures induce the
dissociation of the oligomeric complex into dimers. These dimers
initiate the interaction with nonnative substrate proteins and
reassemble into larger defined sHsp-substrate complexes (19).
To gain insight into the general mode of the interaction of sHsps with
nonnative proteins, we used four different model substrate proteins. We
show that, for each substrate, morphologically distinct and defined
complexes are formed. The stoichiometry of the complexes between sHsps
and nonnative protein seems to be dependent on the substrate
investigated. Furthermore, we demonstrate that Hsp25 and Hsp26 are able
to form mixed complexes with CS and Materials
Recombinant murine Hsp25 and yeast Hsp26 were expressed and
purified as previously described (19, 24, 35). Purified isoenzyme P1 of
yeast CS was stored in 50 mM Tris-HCl and 2 mM EDTA,
pH 8.0; Analysis of Hsp25 and Hsp26 Activities
To induce aggregation, CS and The insulin aggregation assay in the presence of Hsp25 or Hsp26 was
performed in 40 mM Hepes-KOH, pH 7.5. Insulin (45 µM) was equilibrated in the absence and presence of
either Hsp25 or Hsp26 at 25 or 43 °C, respectively. Assays in the
presence of IgG served as a control for unspecific protein effects. The
aggregation reaction was started by the addition of dithioerythritol to
a final concentration of 20 mM, and the resulting turbidity
was monitored at 360 nm in an Amersham Biosciences Ultrospex 4060 UV-visible spectrophotometer equipped with a temperature control unit.
Analysis of sHsp-Substrate Complexes
Size-exclusion Chromatography (SEC)--
To analyze complex
formation between Hsp25 and the different substrates, SEC was performed
using a TosoHaas TSK4000SW column (30 × 0.75 cm, separation range
of 20-7000 kDa). Complex formation between Hsp26 and the
different substrates was analyzed on a TosoHaas TSK4000PW column
(30 × 0.75 cm; separation range of 10-1500 kDa). Hsp25 or Hsp26
in the absence and presence of substrate was incubated as described in
the figure legends. All samples were centrifuged at 14,000 × g for 5 min at 4 °C before application. Chromatography was carried out at 25 °C in running buffer (0.1 M
Hepes-KOH and 150 mM KCl, pH 7.5) at flow rates of 0.5 ml/min for samples with Hsp25 and 0.75 ml/min for those with Hsp26. The
sample volume was 100 µl. Hsp25 and Hsp26 were detected by
fluorescence at an excitation wavelength of 280 nm and emission
wavelengths of 330 and 323 nm, respectively, using a Jasco FP920
fluorescence detector.
Analysis of Binding Stoichiometries--
Hsp25-substrate
complexes were formed with 4.8 µM Hsp25 in the presence
of increasing concentrations of CS (0.15-5 µM) for 30 min at 43 °C. During SEC (described above), the respective complex
peaks were collected and precipitated with sodium
deoxycholate/trichloroacetic acid. Precipitates were then resuspended
in reducing SDS sample buffer (39), separated by SDS-PAGE (10-20%
precast Tricine gradient gels, Novex), and visualized by Coomassie Blue
staining. Scanned gels were analyzed using ImageMaster-1D software
(Amersham Biosciences). Protein amounts and ratios were calculated
for monomers and corrected for different staining intensities due to
differences in molecular mass.
Electron Microscopy--
Complex formation between Hsp25 or
Hsp26 and the different substrates was performed following the protocol
used for the SEC experiments. The samples were separated on a gel
filtration column. The complex peak was collected; and immediately
before application, samples were diluted to a protein concentration of
0.1 mg/ml in running buffer. As controls, substrates and Hsp25 or Hsp26
alone were also incubated under the respective experimental conditions. The samples were directly applied to glow-discharged carbon-coated copper grids and negatively stained with 3% uranyl acetate. Electron micrographs were recorded at a nominal magnification of ×60,000 using
a Philips CM12 electron microscope operating at 120 kV or at a nominal
magnification of ×33,000 using a Jeol 100CX electron microscope
operating at 100 kV.
sHsps Suppress the Aggregation of Different Model
Proteins--
The most commonly used experiment to investigate
chaperone activity is to assay for the ability to suppress aggregation
of nonnative proteins (2, 41, 42). It has been shown that murine
Hsp25 and yeast Hsp26 are able to interact with aggregation-sensitive proteins (19, 24, 33, 34). Here, we were interested in whether their
modes of interaction differ for various model substrates. The first
substrate investigated, dimeric CS (49 kDa/subunit), aggregates rapidly
when incubated at heat shock temperatures (>40 °C) (24). This
process can be monitored by light scattering (Fig.
1, A and E). The
addition of the control protein IgG, even at high excess, did not alter
the kinetics of CS aggregation significantly. However, in the presence
of increasing amounts of either Hsp25 or Hsp26, the thermal aggregation
of CS could be effectively reduced (Fig. 1, A and
E). This is in agreement with previous results (19, 24, 34).
Further addition of the respective sHsp (data not shown) did not
prevent the small amount of residual aggregation observed, indicating
that some CS folding intermediates could not be stably bound to Hsp25
or Hsp26.
Monomeric bovine rhodanese (33 kDa) is widely used as a model for
protein folding (38). In the absence and presence of IgG, the
enzyme aggregated completely within 25 min when incubated at 44 °C
(Fig. 1, B and F). Substoichiometric amounts of
Hsp25 were already able to suppress rhodanese aggregation significantly (Fig. 1B), whereas a 10-fold excess of Hsp26 was required to
obtain a similar effect (Fig. 1F). Similar to the situation
with CS, aggregation could not be completely suppressed even at high
Hsp25 and Hsp26 concentrations.
Another model substrate that shows the tendency to precipitate at
elevated temperatures is the cytoplasmic monomeric
To ensure that a broad molecular mass range of substrates was covered,
the effect of sHsps on the reduction-induced aggregation of insulin was
analyzed. Upon reduction of the interchain disulfide bond, the B-chain
of insulin precipitates, whereas the A-chain stays in solution (45).
With a molecular mass of 3.3 kDa, the insulin B-chain is significantly
smaller than the proteins studied above. After the addition of
dithioerythritol, the peptide aggregated completely within 60 min (Fig.
1, D and H). At substoichiometric concentrations
of either Hsp25 or Hsp26, however, a
concentration-dependent decrease in aggregation at 25 or
43 °C, respectively, could be observed (Fig. 1, D and
H). The addition of sHsps resulted in a marked reduction of
the light scattering signal. At an insulin/Hsp25 monomer ratio of
~6:1, the aggregation of insulin was completely suppressed at
25 °C. An insulin/Hsp26 monomer ratio of 4:1 and higher temperatures
(43 °C) were required for complete suppression of insulin
aggregation by Hsp26. At 25 °C, aggregation could not be completely
suppressed, even at high Hsp26 concentrations (data not shown; see Ref.
19).
The insulin assay also allowed us to address whether the chaperone
function of Hsp25 is temperature-dependent. For yeast
Hsp26, it has been shown previously that the chaperone activity
increases with temperature (19). When the insulin assay was performed at 25, 37, or 42 °C, the protective activity of Hsp25 did not change
(data not shown), whereas for Hsp26, complex dissociation is required
for full chaperone activity (19). Thus, the chaperone function of Hsp25
does not seem to be temperature-dependent.
Analysis of sHsp-Substrate Complexes by Electron
Microscopy--
After having established that mouse Hsp25 and
yeast Hsp26 interact with a variety of substrate proteins, we
investigated the structures and sizes of sHsp-substrate complexes by
electron microscopy. As a control for effects of high temperatures on
Hsp25 and Hsp26, the proteins were incubated at 43 °C for 15 min. In
comparison with a sample applied to the grid after incubation at
25 °C, no significant changes in the appearance of the globular
Hsp25 particles were observed. In both cases, globular Hsp25 particles
with a diameter of ~20 nm were detected (data not shown; see also
Ref. 34), whereas for Hsp26, significant changes in quaternary
structure were detectable after incubation at 43 °C. The well
defined oligomeric complex dissociated into stable dimers (data not
shown; see also Ref. 19). This dissociation seems to expose binding
sites for nonnative proteins.
sHsp-substrate complexes (Fig. 2) were
formed under experimental conditions comparable to the aggregation
experiments (Fig. 1). When the substrate proteins were incubated at
elevated temperatures in the absence of sHsps, in all cases, large
amorphous aggregates were detected (Fig. 2, A, D,
G, and J).
In the presence of Hsp25 and Hsp26, CS,
Globular, regularly shaped particles with a size of ~30 nm were found
in the case of Analysis of sHsp-Substrate Complexes by SEC--
After having
established that different sHsp-substrate complexes show distinct
morphologies, we used gel filtration experiments to gain further
insight into complex formation. As already described (34), in the
absence of substrate, Hsp25 eluted as a dominant peak of ~400 kDa,
consistent with a hexadecameric complex. Tetramers represented a minor
species. This elution behavior did not change after incubation at
elevated temperatures or under reducing conditions (data not shown). At
25 °C, Hsp26 eluted as a single distinct peak with an apparent
molecular mass of 550 kDa. After incubation under reducing conditions,
the elution behavior did not change (data not shown), whereas after
incubation at elevated temperatures, the 24-mer complex dissociates
reversibly into dimers (19). In agreement with the observation
that all of the substrates assayed formed large aggregates after
incubation at elevated temperatures (Fig. 2), hardly any soluble
protein could be detected in the absence of sHsps (data not shown).
SEC analysis of samples in which Hsp25 had been incubated together with
CS or rhodanese at elevated temperatures resulted in the appearance of
an additional peak in the void volume of the column consistent with a
molecular mass of several megadaltons (Fig.
3, A and B). No
intermediate-sized Hsp25-substrate complexes were detected even at low
substrate concentrations. At all ratios, Hsp25 was also present in its
uncomplexed form, indicating that either not all Hsp25 molecules were
involved in complex formation or that smaller substrate complexes
dissociated during chromatography.
After the incubation of Hsp26 with CS (Fig. 3E) or rhodanese
(Fig. 3F) at elevated temperatures, the samples were applied to a gel filtration column running at 25 °C. An additional peak appeared that eluted in the void volume of the column (Fig. 3, E and F). This indicates that binding of
denatured CS or rhodanese to Hsp26 at heat shock temperatures resulted
in the formation of particles of >1500 kDa. Similar to Hsp25,
full-sized Hsp26-substrate complexes were detected immediately, with no
intermediate-sized Hsp26-substrate complexes even at low substrate concentrations.
A different picture emerged for both
When Hsp26 was incubated with substoichiometric amounts of
In the case of insulin and Hsp26 (Fig. 3H), complexes larger
than Hsp26 alone were detected in the range of 550 kDa to 1 MDa. Unbound protein was still visible and also additional peaks, which may
have resulted from interactions of the Hsp26-insulin complexes with the column.
Stoichiometry of Hsp25-CS Complexes--
Haslbeck et
al. (19) described the formation of the large Hsp26-CS
complexes as a highly cooperative process with a maximum binding
capacity at a molar ratio of one CS monomer to Hsp26 dimer. To
determine the stoichiometry in Hsp25-CS complexes, we incubated Hsp25
at elevated temperatures in the presence of increasing amounts of CS.
The samples were separated by SEC, and the complex peaks running in the
void volume of the column were collected. Their composition was then
analyzed by SDS-PAGE and densitometry (Fig. 4). When the CS concentration was
gradually increased, full-sized complexes were present from the start.
No intermediate-sized complexes could be detected. These observations
were confirmed by electron microscopy.
A maximum of one substrate monomer/Hsp25 monomer was incorporated into
the complex (Fig. 4) and one CS monomer/Hsp26 dimer (19). Densitometric
analysis showed that CS was incorporated in a cooperative manner into
the substrate complexes.
Mixed Complexes with
The profiles from the SEC analysis were comparable for the different
incubation sequences (Fig. 5, A and B). Whereas
in the case of Hsp25, all of the substrate protein was drawn into the Hsp25-substrate complex (Fig. 5A), in the case of Hsp26,
free CS,
When all three proteins (either Hsp25 or Hsp26 with CS and The overexpression of sHsps has been shown to convey
thermotolerance in a number of organisms and cell types (20, 40, 46-51), indicating a general thermoprotective function of the protein family. The mechanism of sHsp chaperone activity is poorly
characterized. Previous work has clearly demonstrated that sHsps
recognize misfolded proteins and maintain them in a soluble but
inactive state. In contrast to other chaperones, the chaperone function
of sHsps appears to be limited to binding and maintaining the
solubility of unfolding proteins, without promoting refolding directly
(8, 19, 23, 24, 29, 34).
Haslbeck et al. (19) suggested that, for yeast Hsp26,
dissociation is a prerequisite of chaperone function and that the Hsp26
dimer may act as the primary substrate-binding species, followed by
reassembly into a larger complex together with the substrate. This
hypothesis was further supported by in vitro and in
vivo studies (16, 20, 26) demonstrating that dynamic changes in
oligomerization are a prerequisite for sHsp function in vivo
and chaperone activity in vitro. For Hsp25, detectable dissociation of the oligomer does not precede the binding of nonnative protein, but an exchange of subunits has been suggested (34). We used
these two members of the sHsp family, mouse Hsp25 and yeast Hsp26,
which differ in their quaternary structures and in the mode of
activation, to study the general principles of complex formation of
sHsps with nonnative proteins.
Formation of Large sHsp-Substrate Complexes--
Little is known
about the structure of sHsp-substrate complexes. A striking feature of
sHsps is that, upon substrate binding, they form very large complexes.
For the four different model substrates used in this study, effective
interactions with both Hsp25 and Hsp26 were observed. Both sHsps showed
comparable promiscuity in substrate binding, and the morphologies of
the sHsp-substrate complexes as visualized by negative-stain electron
microscopy were similar. Hsp26 and Hsp25 exhibited globular structures
with outer diameters of ~15 and 20 nm, respectively. These structures increased in size upon substrate binding. Complexes formed with CS
showed mean diameters of ~50 and ~70 nm for Hsp26 and Hsp25, respectively. Complexes formed with rhodanese were smaller, with mean
diameters of ~45 nm for both sHsps. The
SEC experiments indicated that the process of complex formation of
Hsp25 and Hsp26 with nonnative protein is influenced by the substrate
used. After incubation of the sHsp with CS or rhodanese, full-sized
sHsp-substrate complexes could be detected immediately. Only the amount
of particles (and not their size) increased with prolonged incubation.
With
Surface binding of substrate has been shown for plant Hsp18.1 (29), and
the binding of peptides and proteins to Hsp25 (33) also argues for
accessibility of bound protein. In the case of Hsp26, the Hsp26-CS
complex appears, however, to be a completely new assembly. The original
Hsp26 shell is no longer detectable. Instead, an enlarged outer shell
and an additional, internal shell of density become visible (19). As
the bound substrates are still accessible to proteases, they are at
least partially surface-exposed (data not shown). Due to the
similarities in complex formation and morphology, it is reasonable to
assume that, also in the case of Hsp25 and for sHsps in general, large
rearrangements of the complex occur upon binding of nonnative protein.
Taken together, our data suggest that the formation of large defined
sHsp-substrate complexes is a general feature of sHsps. The morphology
of these complexes is substrate-dependent, but independent
of the sHsp used.
Cooperativity of Substrate Protein Binding--
The large complex
structures observed by SEC and electron microscopy indicate binding of
several substrate molecules to Hsp25 or Hsp26 oligomers. A maximum
binding capacity of one protein/sHsp subunit has repeatedly been
suggested (29, 31, 43). Especially in the case of CS and rhodanese, the
particles formed with Hsp25 or Hsp26 showed little variance in size and
shape. Only spherical, regularly shaped particles appeared. Partially
loaded and therefore smaller sHsp complexes were not detected. This
observation suggests coordinated binding of substrate molecules to
sHsps. For CS and Hsp26 (19) and for CS and Hsp25 (Fig. 4), this
process was found to be highly cooperative.
The simultaneous binding of several nonnative proteins seems to be a
prerequisite for efficient and stable complex formation. Interestingly,
for all proteins studied, except insulin, binding could be saturated at
a defined ratio of nonnative protein to sHsp. The cooperativity and
morphology of the complexes indicate that the substrates direct the
process of complex formation.
Incorporation of Different Substrate Proteins into sHsp
Complexes--
The formation of mixed substrate complexes is important
for the function of sHsps in the crowded environment of the cell. Here,
upon stress, different polypeptides unfold and have to be bound
simultaneously. We were interested in whether sHsps are able to bind
different substrates in one complex. Interestingly, two different
substrates could be incorporated into the sHsp-substrate complex,
leading to the formation of morphologically uniform mixed complexes.
The analysis of the mixed complexes by electron microscopy revealed
significant differences in complex morphologies depending on the order
of addition. If either Hsp25 or Hsp26 was first incubated with CS, the
complexes showed marked similarity to the structures that were observed
upon incubation of only CS and Hsp25 or Hsp26. Preincubation of sHsp
with
In conclusion, the analysis of the interaction of different substrate
proteins with sHsps provides new insights into the mode of complex
formation between sHsps and nonnative substrate proteins. sHsps bind
substrates in a range of at least 3-100 kDa. The degree of
cooperativity seems to be substrate-dependent. Taken
together, our results show that the formation of large, morphologically distinct complexes with nonnative proteins is a conserved feature of
the sHsp family of chaperones.
-glucosidase, rhodanese, and
insulin, and analyzed their interaction with murine Hsp25 and yeast
Hsp26 upon thermal unfolding. The two sHsps differ in their modes
of activation. In contrast to Hsp25, Hsp26 undergoes a
temperature-dependent dissociation that is required for
efficient substrate binding. Our analysis shows that Hsp25 and Hsp26
reacted in a similar manner with the nonnative proteins. For all
substrates investigated, complexes of defined size and shape were
formed. Interestingly, several different nonnative proteins could be
incorporated into defined sHsp-substrate complexes. The first
substrate protein bound seems to determine the complex morphology.
Thus, despite the differences in quaternary structure and mode of
activation, the formation of large uniform sHsp-substrate complexes
seems to be a general feature of sHsps, and this unique chaperone
mechanism is conserved from yeast to mammals.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-crystallin (4). Almost all sHsps assemble into large oligomeric
complexes of 9 to >30 subunits, and complexes in the range of 125 kDa
to 2 MDa have been found (5-11). Some sHsps such as those from plants
form assemblies with well defined stoichiometries, whereas other sHsps,
including the mammalian proteins, form a range of oligomeric sizes (12,
13). This polydispersity has limited the amount of structural
information available. The crystal structure of an archaeal sHsp (14)
and the cryo-electron microscopy reconstruction of
B-crystallin (15) revealed that the overall organization is that of a hollow globular sphere. A variation of this scheme is the three-dimensional structure of wheat Hsp16.9, which assembles into a dodecameric double disk, where
each disk is organized as a trimer of dimers (16). Many sHsps have
dynamic and variable quaternary structures with subunits that can
freely and rapidly exchange between oligomers, such as
B-crystallin,
Hsp25, Hsp27, and sHsps from Bradyrhizobium japonicum (8,
13, 15, 17, 18). Changes in quaternary structure at temperatures that
are physiologically relevant for sHsps are important for their
chaperone activity (16, 19-22).
-glucosidase (
-Gluc) and that
the first substrate bound determines the morphology of the resulting
sHsp-substrate complexes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gluc (maltase, EC 3.2.1.20) (36) with a specific activity of
>130 units/mg was a gift from Dr. A. Grossmann (Roche Diagnostics).
Mitochondrial CS from pig heart (EC 4.1.3.7) was obtained from Roche
Diagnostics. Bovine insulin and bovine rhodanese were from Sigma.
-Gluc was stored in 0.1 M potassium phosphate,
pH 6.8; insulin was stored in 20 mM sodium phosphate and
0.1 M NaCl, pH 6.5; rhodanese was stored in 50 mM Tris-HCl, 20 mM dithioerythritol, 50 mM sodium thiosulfate, pH 7.7; Hsp25 was stored in 20 mM Tris-HCl, pH 7.6, 10 mM MgCl2,
20 mM NH4Cl, and 0.5 mM
dithioerythritol; and Hsp26 was stored in 40 mM Hepes-KOH,
50 mM NaCl, 1 mM EDTA, and 1 mM dithioerythritol, pH 7.5. All protein concentrations refer to monomers.
-Gluc were diluted in 40 mM Hepes-KOH, pH 7.5, equilibrated at 43 and 46 °C,
respectively (24, 36, 37). Rhodanese (30 µM) was diluted
1:100 in 40 mM sodium phosphate, pH 7.7, at 44 °C (38).
Assays were performed in the absence and presence of Hsp25 or Hsp26.
Assays in the presence of IgG served as a control for unspecific
protein effects (concentrations given in the figure legends). To
monitor the kinetics of thermal aggregation in the presence of Hsp25
and Hsp26, light scattering was measured in a FluoroMax I fluorescence
spectrophotometer in a stirred and thermostated quartz cell. During the
measurements, both the excitation and emission wavelengths were set to
360 nm with a spectral bandwidth of 4 nm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Influence of Hsp25 (A-D) or
Hsp26 (E-H) on the aggregation of different substrate
proteins. The kinetics of aggregation were monitored by measuring
the light scattering of the samples at 360 nm. A and
E, influence of Hsp25 and Hsp26, respectively, on the
thermal aggregation of CS at 43 °C. A, CS (150 nM) was incubated in the absence ( ) and presence of 0.05 µM (
), 0.15 µM (
), and 1.0 µM (
) Hsp25 or 1 mM IgG (
).
E, CS (150 nM) was diluted in a thermostatted
solution of 1 µM (
), 1.7 µM (
), and
3.4 µM (
) Hsp26 or 1 µM IgG (
). The
spontaneous aggregation of CS is shown (
). B and
F, influence of Hsp25 and Hsp26, respectively, on the
thermal aggregation of rhodanese at 44 °C. B, rhodanese
(0.3 µM) was incubated in the absence (
) and presence
of 96 nM (
), 0.96 µM (
), and 4.8 µM (
) Hsp25 or 3.3 mM IgG (
).
F, rhodanese (0.3 µM) was incubated in the
absence (
) and presence of 3 µM (
) and 6 µM (
) Hsp26 or 3 µM IgG (
).
C and G, influence of Hsp25 and Hsp26,
respectively, on the thermal aggregation of
-Gluc at 46 °C.
C,
-Gluc (0.15 µM) was incubated in the
absence (
) and presence of 0.24 µM (
), 0.48 µM (
), and 0.96 µM (
) Hsp25 or 1.3 mM IgG (
). G,
-Gluc (0.25 µM) was diluted in thermostatted solutions in the absence
(
) and presence of 1 µM (
), 2.5 µM
(
), and 4 µM (
) Hsp26 or 2.5 µM IgG
(
). D and H, influence of Hsp25 and Hsp26,
respectively, on reduction-induced aggregation of insulin at 25 or
43 °C. D, insulin (45 µM) was diluted in
assay buffer in the absence (
) and presence of 1.6 µM
(
) and 8 µM (
) Hsp25 or 1.8 mM IgG
(
). The aggregation reaction was started by the addition of 20 mM dithioerythritol. H, insulin (45 µM) was incubated in the absence (
) and presence of
0.42 µM (
), 1 µM (
), and 10.4 µM (
) Hsp26 or 1.8 µM IgG (
) at
43 °C. The aggregation reaction was started by the addition of
dithioerythritol to a final concentration of 20 mM.
arb., arbitrary.
-glucosidase P1
from Saccharomyces cerevisiae (68.5 kDa) (36, 44). In the absence and presence of IgG, the enzyme aggregated completely within 30 min when incubated at 46 °C (Fig. 1, C and G).
The addition of Hsp25 resulted in a marked reduction of the light
scattering signal. At an enzyme/Hsp25 monomer ratio of ~1:3, the
thermal aggregation of
-Gluc was suppressed to a large extent. In
contrast, Hsp26 was less effective in suppressing the thermal
aggregation of yeast
-Gluc. An excess of Hsp26 was required to
reduce the light scattering signal significantly (Fig. 1G).
Aggregation could not be completely suppressed, even at high sHsp
concentrations. Residual light scattering might indicate that some
unfolding intermediates cannot be stably bound or that the large
sHsp-substrate complexes scatter light.
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Fig. 2.
Analysis of sHsp-substrate complexes by
electron microscopy. 6 µM CS was incubated at
43 °C for 15 min in the absence (A) and presence
(B) of 16 µM Hsp25. 4.2 µM Hsp26
was incubated at 43 °C for 30 min in the presence of 1 µM CS (C). 1 µM rhodanese
(Rho) was incubated at 44 °C for 30 min in the absence
(D) and presence of 4.8 µM Hsp25
(E) or 3 µM Hsp26 (F). 2 µM -Gluc was incubated at 46 °C for 30 min in the
absence (G) and presence (H) of 4.8 µM Hsp25. 6 µM
-Gluc was incubated at
46 °C for 40 min in the presence of 3 µM Hsp26
(I). After the addition of 20 mM
dithioerythritol, 65 µM insulin was incubated at 25 °C
for 6 h in the absence (J) and presence (K)
of 16 µM Hsp25. After the addition of 20 mM
dithioerythritol, 10.8 µM insulin was incubated at
43 °C in the presence of 3.4 µM Hsp26 (L).
Scale bars = 100 nm.
-Gluc, rhodanese, and
insulin formed defined complexes with sHsps (Fig. 2). Although for a
particular substrate, except insulin, regular uniform complexes were
observed, comparison of the different sHsp-substrate complexes revealed
significant differences in size and shape. With CS (Fig. 2B)
and rhodanese (Fig. 2E), Hsp25 formed globular or oval
structures with mean diameters of up to ~70 and ~45 nm,
respectively. Relatively uniform, globular shaped complexes were
detected in the case of Hsp26 and CS with mean diameters of ~50 nm
(Fig. 2C). In the case of rhodanese and Hsp26, more
irregular, smaller, but still globular particles with mean diameters of
~45 nm were formed (Fig. 2F).
-Gluc and Hsp25 (Fig. 2H), whereas in the
case of
-Gluc and Hsp26, the complexes were more irregular, with
mean diameters of ~30 nm (Fig. 2I). In contrast, binding of insulin to Hsp25 or Hsp26 resulted in the formation of fibrous, network-like structures with diameters of ~15-20 nm, corresponding to the size of Hsp26 and Hsp25 oligomers (Fig. 2, K and
L).
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[in a new window]
Fig. 3.
Analysis of sHsp-substrate complexes by
SEC. SEC was carried out using a TosoHaas TSK4000SW column
(for Hsp25) or a TosoHaas TSK4000PW column (for Hsp26) as
described under "Experimental Procedures." A and
E, interaction with CS. A, 16 µM
Hsp25 was incubated in the presence of 1 µM ( ) or 8 µM (
) CS for 30 min at 43 °C before separation.
E, 3 µM Hsp26 was incubated in the presence of
0.75 µM (
) or 1.5 µM (
) CS for 15 min
at 43 °C before separation. B and F,
interaction with rhodanese. B, 4.8 µM Hsp25
was incubated in the presence of 0.6 µM (
) or 4 µM (
) rhodanese for 30 min at 44 °C before
separation. F, 3 µM Hsp26 was incubated in the
presence of 1 µM (
) or 6 µM (
)
rhodanese for 45 min at 44 °C before separation. C and
G, interaction with
-Gluc. C, 4.8 µM Hsp25 was incubated in the presence of 1 µM (
) or 4 µM (
)
-Gluc for 30 min
at 46 °C before separation. G, 3 µM Hsp26
was incubated in the presence of 3 µM (
) or 6 µM (
)
-Gluc for 30 min at 46 °C before
separation. D and H, interaction with insulin.
D, 16 µM Hsp25 was incubated in the presence
of 40 µM (
) or 80 µM (
) insulin after
supplementation with 20 mM dithioerythritol for 3 h at
25 °C before separation. H, 3 µM Hsp26 was
incubated in the presence of 10 µM (
) or 45 µM (
) insulin at 43 °C. The reaction was started by
the addition of 20 mM dithioerythritol. rel.,
relative.
-Gluc and insulin (Fig. 3,
C and D). When these substrates were applied to
the column after incubation in the presence of Hsp25, prominent
Hsp25-substrate peaks appeared, whereas free Hsp25 disappeared.
With increasing substrate concentrations, the complexes
increased in size until nearly all of the protein with
-Gluc and
part of the sample with insulin were drawn into the complex peaks that
eluted in the void volume of the column.
-Gluc and
applied to the column, only part of the protein was drawn into the
complexes that eluted in the void volume of the column (data not
shown). Most of the protein was found in small complexes that eluted as
peaks in the range of 550-1500 kDa. The incubation of equimolar
amounts of
-Gluc and Hsp26 (Fig. 3G) resulted in the
formation of a distinct complex peak, but still free Hsp26 and unbound
-Gluc could be detected. With increasing substrate concentrations,
the complexes increased in size until, at an Hsp26/
-Gluc ratio of
1:2, all of the protein was drawn into substrate complexes eluting in
the void volume (Fig. 3G).
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Fig. 4.
Stoichiometry of Hsp25-CS complexes.
Hsp25 was incubated with increasing amounts of CS for 30 min at
43 °C and applied to a TSK4000SW column. The resulting complex peaks
were collected, precipitated, and analyzed by SDS-PAGE. The bands were
densitometrically analyzed to determine molar ratios of substrate and
Hsp25 in the complex. Ratios refer to monomeric molar concentrations.
Band intensities were corrected for differences in staining intensities
due to different molecular masses. Inset, representative
sections of Coomassie Blue-stained SDS-polyacrylamide gels used in the
analysis.
-Gluc and CS--
Because complexes of
Hsp25 or Hsp26 with CS or
-Gluc showed distinct morphologies, we
next investigated whether mixed complexes between sHsps and different
substrate proteins can be formed. The two substrates were incubated
either sequentially or together with Hsp25 or Hsp26 at elevated
temperatures. The resulting complexes were analyzed by SEC/high
pressure liquid chromatography and electron microscopy. Representative
elution profiles and electron micrographs are shown in Fig.
5.
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[in a new window]
Fig. 5.
A and B, formation of
mixed complexes between CS, -Gluc, and sHsps. A, 4.8 µM Hsp25 was incubated in the presence of 3 µM
-Gluc for 15 min at 44 °C; 4 µM CS
was added; and the sample was further incubated for 15 min. The
resulting complex peak was analyzed by SEC (dashed line). In
a second experiment, the order of substrate addition was reversed:
Hsp25 and CS were incubated first (solid line). The
respective complex peaks were collected, precipitated, and separated by
SDS-PAGE, and the resulting bands were analyzed as described above.
Insets, representative sections of Coomassie Blue-stained
SDS-polyacrylamide gels used in the analysis. B, 3 µM Hsp26 was incubated in the presence of 1.5 µM
-Gluc for 15 min at 44 °C; 1.5 µM
CS was added; and the sample was further incubated for 15 min. The
resulting complex peak was analyzed by SEC (dashed line). In
a second experiment, the order of substrate addition was reversed:
Hsp26 and CS were incubated first (solid line). The
respective complex peaks were collected, precipitated, and separated by
SDS-PAGE. In a third experiment, both substrate proteins were incubated together with
either Hsp25 or Hsp26 for 30 min at 44 °C. C-H, electron
microscopy of mixed complexes. In parallel, the complex peaks were
directly applied to copper grids and visualized by electron microscopy
(see above). Hsp25 (C) or Hsp26 (D) and CS were
incubated first before
-Gluc was added. Hsp25 (E) or
Hsp26 (F) and
-Gluc were incubated first before CS was
added. Hsp25 (G) or Hsp26 (H), CS, and
-Gluc
were incubated together for 30 min at 44 °C. Scale
bars = 100 nm. rel., relative.
-Gluc, and Hsp26 were still detectable (Fig.
5B). Both substrate proteins were found in complex with the
sHsps as analyzed by SDS-PAGE. The analysis of these complexes by
electron microscopy (Fig. 5, C-F) revealed that the
complexes obtained under one set of conditions were uniform in size and
shape. However, if the sHsp was first incubated with CS, the complexes
showed marked similarity to the structures that were observed when only
CS was incubated with Hsp25 or Hsp26 (Fig. 2, B and
C; and Fig. 5, C and D). Preincubation of the sHsp with
-Gluc led to typical complexes between the sHsp and
-Gluc (Fig. 2, H and I; and Fig. 5,
E and F). Interestingly, the first substrate
Hsp25 or Hsp26 was preincubated with seems to determine the morphology
of the sHsp-substrate complex.
-Gluc)
were incubated together (Fig. 5, G and H),
uniform particles were again formed. Most of the particles showed
shapes similar to the sHsp-CS complexes to which
-Gluc was added
(Fig. 5, compare C and D). However, they were
significantly smaller, with diameters of ~35-40 nm, i.e.
between those observed for the two mixed complexes shown in Fig. 5
(C/E and D/F). These
experiments show that two different substrates were able to bind
simultaneously to sHsps. Binding resulted again in the formation of
large distinct complexes. The morphology seems to be determined by the
first substrate bound.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gluc complexes were
smaller (mean diameters of ~30 nm) and more irregularly shaped than
the CS and rhodanese complexes. The complexes formed with insulin
revealed net-like structures with mean diameters of about the same size
of the respective sHsp. The differences in complex sizes between Hsp26
and Hsp25 with CS might be due to different binding stoichiometries
(see below).
-Gluc and insulin, intermediate-sized particles could be
detected after incubation of substoichiometric amounts of
substrate with sHsps.
-Gluc led to typical complexes between sHsp and
-Gluc. When
all three proteins were incubated together, the resulting complexes
showed morphologies similar to the sHsp-CS complexes, but they were
significantly smaller. Thus, the first substrate bound seems to
determine the morphology of the complex.
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ACKNOWLEDGEMENTS |
---|
We thank Simone Gräber, Kerstin Rutkat,
and Benjamin Robach for excellent experimental help and Elke Fischer
for proofreading the manuscript. We acknowledge Lin Müller for
help in creating electronic files, Martin Haslbeck for useful
discussions, and Reinhard Rachel and Sevil Weinkauf for providing equipment.
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to J. B.), and the Fonds der Chemischen Industrie (to J. B. and M. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Kendle GmbH & Co., GMI KG, Stefan-George-Ring 6, D-81929 Munich, Germany.
¶ To whom correspondence should be addressed. Tel.: 49-89-289-13340; Fax: 49-89-289-13345; E-mail: johannes.buchner@ch.tum.de.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M301640200
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ABBREVIATIONS |
---|
The abbreviations used are:
Hsps, heat
shock proteins;
sHsps, small heat shock proteins;
CS, citrate
synthase;
-Gluc,
-glucosidase;
SEC, size-exclusion
chromatography;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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