The Dynamics of Hsp25 Quaternary Structure
STRUCTURE AND FUNCTION OF DIFFERENT OLIGOMERIC SPECIES*
Monika
Ehrnsperger
,
Hauke
Lilie§,
Matthias
Gaestel¶, and
Johannes
Buchner
From the
Institut für Biophysik und
Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, § Institut für Biotechnologie,
Martin-Luther Universität Halle-Wittenberg, 06120 Halle, and
¶ Martin-Luther-Universität Halle-Wittenberg,
Innovationskolleg Zellspezialisierung, Hoher Weg 8,
06120 Halle, Germany
 |
ABSTRACT |
Small heat shock proteins (sHsps), including
-crystallin, represent a conserved and ubiquitous family of
proteins. They form large oligomers, ranging in size from 140 to more
than 800 kDa, which seem to be important for the interaction with
non-native proteins as molecular chaperones. Here we analyzed the
stability and oligomeric structure of murine Hsp25 and its correlation
with function. Upon unfolding, the tertiary and quaternary structure of
Hsp25 is rapidly lost, whereas the secondary structure remains remarkably stable. Unfolding is completely reversible, leading to
native hexadecameric structures. These oligomers are in a
concentration-dependent equilibrium with tetramers and dimers,
indicating that tetramers assembled from dimers represent the basic
building blocks of Hsp25 oligomers. At high temperatures, the Hsp25
complexes increase in molecular mass, consistent with the appearance of
"heat shock granules" in vivo after heat treatment.
This high molecular mass "heat shock form" of Hsp25 is in a slow
equilibrium with hexadecameric Hsp25. Thus, it does not represent an
off-pathway reaction. Interestingly, the heat shock form exhibits
unchanged chaperone activity even after incubation at 80 °C. We
conclude that Hsp25 is a dynamic tetramer of tetramers with a unique
ability to refold and reassemble into its active quaternary structure
after denaturation. So-called heat shock granules, which have been
reported to appear in response to stress, seem to represent a novel
functional species of Hsp25.
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INTRODUCTION |
Small heat shock proteins
(sHsps),1 exhibiting a
monomeric molecular mass of 9-42 kDa, are expressed in all organisms
investigated so far. Because of functional as well as structural
homologies,
-crystallin, a major mammalian eye lens protein, which
is also expressed in non-lenticular tissue, is a member of this protein family (1-4). Although the overall homology between different sHsps is
rather low, they are grouped together based on conserved sequences in
the C-terminal half of the protein and short, conserved, phenylalanine-rich stretches near the N terminus of the protein (5, 6).
Mammalian sHsps are expressed constitutively even under physiological
conditions. However, stress factors such as heat shock induce a strong
up-regulation of protein levels by 10-20-fold to maximum
concentrations of 0.1% of the cellular protein (7, 8). Overexpression
of different mammalian sHsps increases cellular thermoresistance
significantly (9, 10). Furthermore, sHsps have been suggested to
function in different, seemingly unrelated processes like RNA
stabilization (11), interaction with the cytoskeleton (12, 13), or
apoptosis (14). Interestingly, sHsps are also overexpressed in several
cancers and neurodegenerative diseases like Alzheimer's disease or
multiple sclerosis (15-17). In plants, five different classes of sHsps
have been identified, which are partly localized in
organella (8, 18).
In vitro sHsps act as molecular chaperones in preventing
unfolded proteins from irreversible aggregation (3, 4, 19) and, in
cooperation with other factors, e.g. Hsp70 and ATP,
facilitate productive refolding of unfolded proteins (20, 21). In this context, sHsps are more efficient than the model chaperone GroEL, due
to their high binding capacity of up to one substrate molecule per sHsp
subunit (21-24).
Although sHsps are rather heterogeneous both in monomeric molecular
weight and amino acid sequence, they all share the striking feature of
forming high molecular weight oligomeric complexes of variable size.
Although the 16-kDa antigen of Mycobacterium tuberculosis
forms a nonamer assembled as a trimer of trimers (25), the well
characterized
-crystallin has been reported to adopt different
oligomeric structures with molecular masses ranging from 125 kDa to 2 MDa (26, 27). In electron micrographs,
-crystallin and sHsp
complexes show typically globular or spherical appearance of 10-25 nm
(20, 21, 28, 29). By cryo-electron microscopy and image analysis, Haley
et al. (30) have shown that human, recombinant
B-crystallin forms a hollow, globular shell with a molecular mass of
650 kDa (± 150 kDa) and asymmetric appearance. Several species were
observed differing in size and shape. This is interesting in the light
of data that demonstrate subunit exchange between
-crystallin
complexes as well as between Hsp25/27 and
-crystallin in
vivo and in vitro (19, 31, 32). It remains to be seen
whether this is an indicator of similar oligomeric association of
-crystallin and the mammalian Hsp25/27. These hetero-oligomeric
complexes dissociate during heat treatment (33), whereas
-crystallin
and homo-oligomeric sHsp complexes were repeatedly reported to increase
significantly in size upon heat shock as so-called "heat shock
granules" and to redistribute from the cytosol to the perinuclear
space or the nucleus itself (26, 28, 34). Furthermore, in heat-shocked
tomato cell cultures, sHsps were found as large heat shock granules in
the nuclear periphery (11). The chloroplast-localized sHsp, Hsp21,
stays soluble during an initial heat shock and insolubilizes during
additional or prolonged heat treatment (35). This increase in complex
size seems to be modulated by the metabolic state (36) and the degree
of thermoresistance of the cell (26), e.g. during heat
shock, Dm-Hsp27 forms insoluble superoligomers (>1 MDa), which are
redistributed to the nucleus (26), whereas pretreated, thermotolerant
cells do not show these effects (37). In contrast, chemical stress such
as arsenite treatment or stress caused by serum starvation leads to a
decreased level of Dm-Hsp27 oligomerization, shifting the molecular
mass to about 200 kDa (34, 38).
In all cases described so far, changes in sHsp oligomerization upon
stress were reversible, with the complexes relocalizing to their
physiological compartment during recovery.
As the quaternary structure of sHsps is essential for their function
and regulation of activity, but its basic properties are still rather
poorly understood, we investigated changes in the oligomeric structure
and stability of murine Hsp25 in detail.
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EXPERIMENTAL PROCEDURES |
Materials
Recombinant murine Hsp25 was expressed and purified as described
previously (4, 39). Mitochondrial citrate synthase (CS) from pig heart
(EC 4.1.3.7) was obtained from Roche Molecular Biochemicals. The
concentrations of CS and Hsp25 were determined using the extinction
coefficients of 1.78 and 1.87, respectively, for a 1 mg/ml solution at
280 nm. CS was stored in 50 mM Tris, 2 mM EDTA,
pH 8.0; Hsp25 in 40 mM Hepes-KOH, pH 7.5. The
concentrations for CS and Hsp25 given in the text refer to a dimer and
a hexadecamer, respectively.
Analysis of Hsp25 Activity: Aggregation Assay
15 µM CS was diluted 1:200 in 40 mM
Hepes-KOH, pH 7.5, equilibrated at 43 °C, in the presence and
absence of Hsp25 (concentrations given in the figure legends). To
monitor the kinetics of thermal aggregation, light scattering was
measured in a Perkin-Elmer MPF44A fluorescence spectrophotometer in
stirred and thermostated quartz cells. During the measurements, both
the excitation and emission wavelength were set to 500 nm with a
spectral bandwidth of 2 nm.
Analysis of Hsp25 Particles
Electron Microscopy--
To determine changes in oligomeric size
upon heating, Hsp25 (0.2 µM in 40 mM Hepes pH
7.5) was incubated either at 20 °C for 60 min or at 80 °C for 15 min, and then Hsp25 was cooled to 20 °C for another 60 min. Aliquots
were 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 operated at 120 kV.
Native Gel Elecrophoresis--
10 µg of Hsp25 at
concentrations of 2 and 0.25 mg/ml in the suggested native sample
buffer were applied to a precast 4-12% Tris-glycine gel (Novex, San
Diego). Buffers and conditions were chosen according to the
manufacturer's protocol. As controls, 10 µg of ferritin and GroEL
were applied according to the same protocol.
Size Exclusion Chromatography--
Size exclusion HPLC (SEC) was
performed using a TosoHaas TSK 4000 SW column (30 cm x 0, 75 cm;
separation range 10-7,000 kDa). If not otherwise specified,
chromatography was carried out in 100 mM Hepes-KOH, pH 7.5, with a flow rate of 0.75 ml/min (Figs. 1, 2, and 4) or 0.5 ml/min (Fig.
6). The sample volume was 100 µl. Hsp25 was detected by fluorescence
at an excitation wavelength of 280 nm and an emission wavelength of 335 nm, using a Jasco FP 920 fluorescence detector.
For cross-linked samples Hsp25, at a concentration of 0.3 mg/ml was
incubated with 10 mM glutaraldehyde for 2 min at 37 °C in 40 mM Hepes, pH 7.5. The reaction was stopped with 35 mM Tris/HCl, pH 8.0. The non-cross-linked Hsp25 control was
treated accordingly, with the exception that buffer was added instead
of glutaraldehyde. Samples were centrifuged for 5 min at 14,000 × g at 20 °C. Estimation of molecular masses was achieved
by comparison with established marker proteins.
Analytical Ultracentrifugation--
Sedimentation velocity
analysis was performed in an analytical ultracentrifuge (Beckman Optima
XL-A). Double sector cells were used at 20,000 rpm in a rotor AnTi 60 at 20 °C. The protein concentration was 0.28 mg/ml. The data were
analyzed using the sedimentation time derivative method (40).
Spectroscopy
Temperature-induced Structural Changes in Hsp25--
To monitor
thermal unfolding, the intrinsic fluorescence and light scattering of
the protein solution were measured from 25 to 80 °C in stirred
quartz cells. The measurements were carried out in a Perkin-Elmer
MPF44A fluorescence spectrometer with a thermostated cell holder
connected to a thermoprogrammer. Changes in protein structure upon
heating, resulting in exposure of tryptophans and tyrosines to the
solvent, led to changes in protein fluorescence. The tryptophan
fluorescence of Hsp25 was recorded at an excitation wavelength of 295 nm and an emission wavelength of 338 nm. The spectral bandwidth was 5 nm for both excitation and emission. The heating rate was 1 °C/min.
The Hsp25 concentration was 50 µg/ml in 40 mM Hepes, pH
7.5.
Light scattering measurements were performed to determine changes in
the size of Hsp25 particles at different temperatures or increasing
concentrations of the denaturant urea. Excitation and emission
wavelength were set to 360 nm. The spectral bandwidths were 5 and 2.5 nm, respectively. For thermal unfolding and refolding of Hsp25, the
heating or cooling rate was 0.5 °C/min. The Hsp25 concentration was
50 µg/ml in 40 mM Hepes, pH 7.5. To determine the light
scattering signal of Hsp25 in increasing concentrations of urea, the
protein (50 µg/ml) was incubated at the respective urea
concentrations overnight at 20 °C. Measurements were then performed
as described above.
The thermal unfolding transition of Hsp25 in the far UV CD range was
recorded in a Jasco J715 CD spectropolarimeter with a PTC 343 Peltier
heating unit. The protein concentration was 150 µg/ml in 40 mM Hepes, pH 7.5. The cuvette pathlength was 1 mm. The
heating rate was 1 °C/min, and the CD signal was measured at 225 nm.
Urea-induced Unfolding and Refolding of Hsp25--
Urea-induced
unfolding of Hsp25 was performed by diluting the protein into
increasing concentrations of urea (in 40 mM Hepes, pH 7.5)
ranging from 0 to 7 M. The samples were incubated for 20 h at 20 °C to achieve equilibrium.
For refolding experiments, Hsp25 was denatured at a concentration of 2 mg/ml by incubation in 8 M urea for 10 h at 20 °C. Refolding was started by diluting the protein in decreasing urea concentrations to a final protein concentration of 10 µg/ml before incubation at 20 °C for 20 h. To study the influence of salt on the stability of Hsp25, urea-induced unfolding and refolding was performed as described in the presence and absence of 1.15 M NaCl.
The unfolding and refolding transitions were monitored by measuring the
change in intrinsic fluorescence. Unfolding was furthermore monitored
by far UV CD spectroscopy.
The fluorescence measurements were carried out in a Spex Fluoromax
fluorospectrometer at 20 °C. The individual spectra at each urea
concentration were recorded from 295 to 400 nm in 1-cm quartz cells at
an excitation wavelength of 280 nm. The spectral bandwidth was 5 nm for
both excitation and emission. All spectra were baseline-corrected.
Protein fluorescence at 320 nm, the wavelength with the largest signal
difference between native and unfolded Hsp25, was plotted against urea concentrations.
To follow the development of secondary structure during urea-induced
unfolding, far UV CD spectra of Hsp25 (1 mg/ml) in various urea
concentrations, ranging from 0 to 7 M, were recorded in a Jasco J715 CD spectropolarimeter. Spectra were measured from 250 to 200 nm at a pathlength of 0.01 cm at 20 °C. All spectra were baseline-corrected. The CD signal at 213 nm was used to evaluate the
influence of urea on Hsp25 secondary structure.
 |
RESULTS |
Dynamic Hsp25 Oligomers Are Formed from Tetramers--
The
analysis of the oligomeric assembly of sHsps is important for
understanding the function of this family of chaperones. When applied
to a size exclusion chromatography (SEC) column, murine Hsp25 eluted
predominantly as a 400-kDa complex as judged by calibration of the
column with marker proteins. This peak was consistent with a
hexadecamer (Fig. 1A). A
second peak corresponding to a tetramer was present in smaller amounts.
Apart from a shoulder in the tetramer peak, which represents a dimer,
no further species were observed. It is therefore likely that the Hsp25
complex is formed from tetramers as basic building blocks. When the
Hsp25 complex was cross-linked with glutaraldehyde prior to
chromatography, smaller species were no longer detected, indicating
that Hsp25 in solution is solely hexadecameric (Fig. 1A).
The slight increase in size of cross-linked Hsp25 was probably due to
the addition of glutaraldehyde to the complex during cross-linking. The
notion that Hsp25 is present as a defined complex in solution was
further confirmed by native gel electrophoresis. Here again, similar to GroEL and ferritin, only one oligomeric species was detected at different Hsp25 concentrations (Fig. 1B). An estimation of
the molecular weight was not possible, as the running behavior of proteins on native gels is not only dependent on their molecular weight
but also on the isoelectric point of the proteins. As an independent
method to determine the molecular mass of Hsp25 complexes, we used
analytical ultracentrifugation. Analysis of sedimentation velocity
experiments at a Hsp25 concentration of 0.28 mg/ml resulted in a
s value of s = 11.9 S,
confirming a hexadecameric association of Hsp25 with a calculated
molecular mass of 398 kDa. As the data can be described by a fit for a
monodispersed system (Fig. 1C) under the given conditions,
the association of Hsp25 seems to be a defined process yielding only
one oligomeric species.

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Fig. 1.
Quaternary structure of Hsp25 in
solution. A, SEC was performed using a TosoHaas G 4000 SW column as described under "Experimental Procedures." Hsp25 at a
concentration of 0.3 mg/ml was incubated at 20 °C for 30 min, before
one half of the sample was cross-linked with 10 mM
glutaraldehyde (dotted line), whereas the other
half was applied to the column without cross-linking (solid
line). B, native gel electrophoresis was
performed with a precast 4-12% Tris-glycine gel. Lane
1, 10 µg of GroEL; lane 2, 10 µg
of ferritin; lanes 3 and 4, 10 µg of
Hsp25 were applied at concentrations of 2 and 0.25 mg/ml, respectively.
The gel was stained with Coomassie Blue. C, analytical
ultracentrifugation of Hsp25. The sedimentation velocity of Hsp25 was
determined at 20,000 rpm, 20 °C. The experimental data ( ... )
were fitted (------) according to the sedimentation time derivative
method, yielding s = 11.9 S.
sapp, apparent s value.
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To further investigate the dynamics of the Hsp25 complex, we applied
the protein at concentrations ranging from 37.5 µg/ml to 1.2 mg/ml to
a TSK4000 SEC column (Fig. 2). At all
concentrations, the protein appeared in three oligomeric forms
corresponding to a hexadecamer, a tetramer and a dimer (Fig. 2,
A-C). The relative ratios of the peaks, however, changed
significantly with increasing Hsp25 concentrations. Whereas at the
lowest concentration, mainly the dimer was present, at 0.2 mg/ml the
dimers shifted to tetramers and the hexadecamer became the predominant
species. This tendency carried on up to a concentration of 1.2 mg/ml,
where the hexadecamer was by far the predominant oligomeric species.
From these data we conclude that Hsp25 forms an oligomer of 16 subunits, which is in a concentration-dependent equilibrium
with a tetrameric form. The smaller amount of dimer present at all
concentrations and the fact that no monomer was detected suggest that
the tetramer is not formed from four monomers but rather consists of
two dimers. To further analyze the concentration-dependent
equilibrium between Hsp25 oligomers, we collected the hexadecamer and
tetramer peaks (see Fig. 2B, solid and
dotted lines) and directly reapplied them to the
same column. As expected, the 16-mer peak dissociated to the tetrameric
form, whereas the rechromatographed tetramer resisted further
dissociation, even at this lower concentration (Fig. 2D). This suggests that the tetramer is a rather stable building unit of the
high molecular weight Hsp25 oligomer.

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Fig. 2.
Concentration-dependent
equilibrium of Hsp25 oligomerization. Hsp25 at concentrations of
37.5 µg/ml (A), 0.2 mg/ml (B), and 1.2 mg/ml
(C) was incubated for 2 h at 25 °C before
centrifugation and application to the gel filtration column.
D, the maxima of the two major peaks (solid and
dotted line) from panel B
were collected and reapplied to the column without further
treatment.
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Unfolding and Inactivation of Hsp25 Is a Highly Reversible
Process--
After having established that Hsp25 forms dynamic
oligomeric complexes, we were interested in the stability of those
structures against chemical denaturation (Fig.
3A) and the changes in
quaternary structure involved in unfolding. Incubation of the protein
with increasing concentrations of urea led to a gradual decrease of the
fluorescence signal over a concentration range of 0.5-5 M urea. (Fig. 3A). As the maxima of the fluorescence spectra
changed in the same urea concentration range, the observed transition reflected a gradual loss of tertiary structure, with tryptophans being
successively exposed to the polar solvent (data not shown; see Ref.
19). These unfolding intermediates seemed to expose sticky surfaces, as
their analysis on SEC HPLC columns was not possible, due to interaction
with the resin (data not shown). To analyze changes in oligomeric
structure during the urea transition, the light scattering signal of
Hsp25 was measured. At urea concentrations above 1 M, the
signal decreased drastically indicating a reduction in particle size.
The secondary structure, however, as measured by far UV
CD-spectroscopy, did not change below 3.5 M urea, with denaturation being completed at 5 M urea (Fig.
3A). Thus, a first step during urea denaturation of Hsp25
seems to be complex dissociation. The following gradual exposure of
hydrophobic amino acids and decrease of fluorescence signal indicated
further loss of quaternary and/or tertiary structure, followed by
disintegration of secondary structure as a last step of unfolding. The
unfolding transition was completely reversible (Fig. 3A),
leading to correctly assembled Hsp25 oligomers that were
indistinguishable from native Hsp25 in gel filtration experiments (data
not shown). To analyze whether the renatured protein had regained its
full activity, we measured the ability of Hsp25 to suppress the thermal
aggregation of the model substrate CS. When compared with native Hsp25,
incubated at the same residual urea concentration, the renatured
protein showed unchanged activity in the chaperone assay (Fig.
3B). The same is true for Hsp25 renatured from GdnCl (data
not shown).

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Fig. 3.
Urea-induced unfolding, refolding, and
dissociation of Hsp25. A, samples were incubated in
urea concentrations ranging from 0 to 7 M for 20 h at
20 °C to achieve equilibrium. All experiments were performed in 40 mM Hepes, pH 7.5. Fluorescence, unfolding ( )
and refolding ( ) of Hsp25 at a concentration of 10 µg/ml in
various urea concentrations was followed by recording the fluorescence
signal at 320 nm. Far UV CD, changes in secondary structure
with increasing urea concentrations ( ) were determined by monitoring
the far UV CD signal of Hsp25 at 213 nm (1 mg/ml) at a pathlength of
0.01 cm. Light scattering, to monitor changes in particle
size with increasing urea concentrations light scattering of Hsp25 (50 µg/ml) was measured at an excitation and emission wavelength of 360 nm ( ). B, to determine the activity of refolded Hsp25,
light scattering of thermally aggregating CS (75 nM) was
measured at 43 °C. After 8 h of incubation at 20 °C in 7 M urea, denatured Hsp25 (1.45 mg/ml) was diluted 1:200 into
40 mM Hepes, pH 7.5. After another 22-h incubation at
20 °C, refolded Hsp25 (20 nM) was directly applied to
the CS aggregation assay ( ). As a control, native Hsp25 at the same
concentration was incubated accordingly in the residual urea
concentration (35 mM) before application to the assay
( ). Spontaneous aggregation of CS was measured in the absence ( )
and presence of 35 mM urea ( ).
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Using GdnCl, another common denaturant, to unfold Hsp25, we found that
the protein loses its oligomeric structure as well as its native
fluorescence and CD signal in a cooperative reaction with a midpoint at
1.15 M GdnCl (data not shown). We wondered whether the
pronounced difference between the two denaturants was due to the ionic
nature of GdnCl. To test this hypothesis, we performed urea transitions
in the presence of salt (Fig.
4A). NaCl was added to a
concentration of 1.15 M, corresponding to the midpoint of
transition of the GdnCl measurement. At this salt concentration, the
urea transition of Hsp25 changed markedly, exhibiting a plateau of
fluorescence intensity between 1.8 and 4.5 M urea.
Secondary structure, as measured by far UV CD, did only change above 3 M urea, coinciding with the decrease in fluorescence signal
after the plateau region.

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Fig. 4.
Influence of NaCl on the urea-induced
denaturation of Hsp25. Samples were incubated in urea
concentrations ranging from 0 to 7 M for 20 h at
20 °C to achieve equilibrium. All experiments were performed in 40 mM Hepes, pH 7.5 ± NaCl. A,
Fluorescence, unfolding of Hsp25 at a concentration of 10 µg/ml in various urea concentrations from 0 to 7 M in the
absence ( ) and presence ( ) of 1.15 M NaCl was
recorded by measuring the fluorescence signal at 320 nm; Far UV
CD, changes in secondary structure with increasing urea
concentrations in the presence of 1.15 M NaCl ( ) were
monitored by measuring the far UV CD signal of Hsp25 (1 mg/ml) at 213 nm and a pathlength of 0.01 cm. B, SEC in the plateau region
of the urea/NaCl transition. 100 µg/ml Hsp25 were incubated in 3 M urea, 1.15 M NaCl, 40 mM Hepes,
pH 7.5, for 15 h at 20 °C. The sample was then centrifuged to
remove aggregates and applied to a TSK 4000 SW gel filtration column
equilibrated in the same buffer.
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To analyze the quaternary structure of Hsp25 in the plateau region, we
performed SEC at 1.15 M NaCl and 3 M urea. The
elution profile showed two distinct peaks corresponding to a dimer and a tetramer of Hsp25 (Fig. 4B), the established building
units of the complex (see above). The stabilization of small oligomeric species by salt without loss of structure suggests that subunit contacts in these species are predominantly hydrophobic.
Hsp25 Temperature Transition--
Having established that Hsp25
shows the remarkable ability to refold completely to the native state
after urea and GdnCl denaturation, we wondered whether this
reversibility of unfolding and disassembly of the Hsp25 oligomers is
restricted to chemical denaturation. As sHsp overexpression and
function is often correlated with heat shock, we investigated the
influence of temperature on the structure and stability of Hsp25. Fig.
5A shows the thermal stability
of Hsp25 as measured by fluorescence and far UV CD spectroscopy. In
agreement with previous data on
-crystallin and Hsp27 (41, 42), the
far UV CD signal of Hsp25 changed drastically at temperatures above
62 °C in a highly cooperative thermal transition. The changes in
Hsp25 fluorescence above 60 °C are less pronounced than the increase
in CD signal, but a change in the slope of the linear decrease in
fluorescence confirms a folding transition starting at 60-63 °C. To
determine the consequences of the thermal transition on the size of the
oligomers, we measured light scattering of Hsp25 between 30 and
80 °C (Fig. 5B). The increase in signal around 64 °C
is indicative of a pronounced increase in Hsp25 particle size. Taken
together, between 60 °C and 65 °C, Hsp25 seems to first lose
secondary structure (CD), followed by changes in tertiary structure as
observed by fluorescence (Fig. 5A). The tendency of the
protein to form large particles increased drastically at temperatures
above 65 °C and reached a plateau at 75 °C. When the sample was
subsequently cooled down to room temperature, the light scattering
signal decreased only slowly, suggesting that the change in quaternary
structure of Hsp25 during heating was not readily reversed. A
fluorescence spectrum recorded at 75 °C showed an emission maximum
of 347 nm, indicative of partial unfolding during the heating period
(Fig. 5B, inset). However, the fluorescence spectra of Hsp25 before and after heating were identical, suggesting restoration of tertiary structure.

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Fig. 5.
Temperature-dependent structural
changes in Hsp25. A, thermal unfolding and refolding of
Hsp25. The intensity of Hsp25 tryptophan fluorescence at 338 nm
(50 µg/ml) was monitored at a heating rate of 1 °C/min
( ). Data points were corrected for the
temperature-dependent, linear decrease of tryptophan
fluorescence. To monitor changes in secondary structure, the far UV CD
signal of Hsp25 (150 µg/ml) was recorded at 225 nm with a heating
rate of 1 °C/min ( ). B, changes in light scattering of
Hsp25 with increasing temperatures. Hsp25 (50 µg/ml) was incubated in
a stirred quartz cuvette. Light scattering was monitored at 360 nm both
during heating to 78 °C ( ) and subsequent cooling to 25 °C
( ). The heating rate was 0.5 °C/min. Inset,
fluorescence spectra of Hsp25 (50 µg/ml; excitation wavelength: 295 nm) before heating (solid line) at 75 °C
(dotted) and after cooling at 25 °C (dashed).
To compensate for the decrease in fluorescence with temperature, the
amplitude of the 75 °C spectrum was multiplied by a factor of
2.5.
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To analyze changes in complex size during heating and cooling more
directly, we performed SEC with a sample that had been heated to
80 °C for 15 min and then kept at 20 °C for variable times (Fig.
6, A-C). When compared with
the elution profile of native, untreated Hsp25 (Fig. 6A),
the protein eluted as a prominent peak at a retention time
corresponding to about 1.3 MDa, whereas the amount of smaller
oligomeric species decreased but retained their sizes. As there were no
intermediate size complexes observed between the hexadecamer and the
larger complex, unspecific aggregation is unlikely. Rather,
hexadecamers seem to associate cooperatively to larger oligomers. As
the high molecular mass complexes shifted slowly back to native
oligomeric Hsp25 when incubated for extended periods at 20 °C, this
heat shock complex was in a slow equilibrium with the hexadecamer and
the tetramer (Fig. 6, A-C).

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Fig. 6.
Influence of elevated temperature on Hsp25
quaternary structure. A, SEC was performed using a
TosoHaas TSK 4000 SW column as described under "Experimental
Proccedures." Hsp25 in 40 mM Hepes, pH 7.5, at a
concentration of 0.2 mg/ml was incubated for 30 min at 20 °C,
centrifuged, and applied to the column. B, to test the
influence of temperature on the oligomeric organization of Hsp25, the
protein at the same concentrations was incubated at 75 °C for 15 min, cooled to 20 °C for 30 min, centrifuged, and applied.
C, the influence of prolonged incubation at 20 °C after
heat treatment was investigated by keeping the protein at 20 °C for
38 h after the 75 °C incubation before application
(I and II, see panel E).
D, analysis of temperature-induced changes in Hsp25 complex
size by electron microscopy. Before application to the grid, Hsp25 (0.2 µM, 75 µg/ml) was incubated for 60 min at 20 °C
(left panel) or was incubated at 75 °C for 15 min and then directly applied to the grid (middle
panel) or cooled to 20 °C for another 60 min
(right panel). Magnification was 149,000; 1 cm
represents 70 nm. E, activity of the different oligomeric
Hsp25 species after heat treatment. Hsp25 at a concentration of 1.2 mg/ml was heat-incubated and cooled as described under panel
C. The protein was subsequently applied to the TSK 4000 SW
column, and the two predominant peaks were collected. After estimating
the protein concentration of the samples according to Bradford (55),
the material was directly applied to the CS aggregation assay. The
activity of the collected peaks was compared with native, untreated
Hsp25 at the same concentrations. Thermal aggregation (43 °C) of CS
(75 nM) alone ( ) and in the presence of 10 µg/ml peak
I ( ) and native Hsp25 ( ) or in the presence of 28 µg/ml peak II
( ) and native Hsp25 ( ).
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Next, we analyzed the size and shape of the Hsp25 particles formed at
high temperature by negative stain electron microscopy. Hsp25 kept at
20 °C formed the already described spherical particles with a
diameter of about 20 nm (20). When the protein was applied to the grid
after 15 min of incubation at 75 °C, the particles associated into a
network-like structure and few isolated, large globular particles.
However, when Hsp25 was kept at 20 °C for 60 min after heating, the
appearance of the complexes changed. In accordance with the gel
filtration data, a population of larger Hsp25 particles appeared with
diameters ranging from 40 to 70 nm with the original 20-nm species
still present to some extend (Fig. 3D).
To investigate whether the observed in vitro "granules"
showed chaperone activity, we collected both the high molecular mass complex and the hexadecamer eluting from the SEC column (see Fig. 6C, I and II). The activity of the
samples in suppressing thermal aggregation of CS was measured in
comparison to untreated Hsp25. Hsp25 from both peaks showed
undiminished activity in suppressing protein aggregation during heat
shock conditions (Fig. 6E). Thus, similar to the situation
in vivo, large granular sHsp structures, formed after heat
stress, play an active role in protecting proteins from irreversible
unfolding processes.
 |
DISCUSSION |
The complex quaternary structure of sHsps is one of their most
striking features. Their oligomeric state seems to be a prerequisite for chaperone activity, as, e.g., deletion of a N-terminal
region of Caenorhabditis elegans Hsp16-2 led to a loss of
both oligomerization and chaperone properties (43). Changes in the
oligomeric structure of mammalian sHsp due to stress factors are often
correlated with phosphorylation of the proteins. In vitro
phosphorylated Hsp27 as well as mutants mimicking phosphorylation
appear only in their dissociated tetrameric form, which does not show
chaperone activity (44). In contrast to the chaperone properties, which
seem to be coupled to large oligomers of sHsps, the influence of Hsp27 on actin polymerization has been mainly connected to small oligomeric and even monomeric forms of Hsp27 (45). In the light of these data,
changes in the oligomeric structure of sHsps seem to be one means for
regulating their multiple functions.
Recently, the crystal structure of Hsp16.5 from the hyperthermophilic
archaeon Methanococcus jannaschii has been solved (46). The
monomeric folding unit is a composite
-sandwich in which one
-strand comes from a neighboring subunit, indicating that dimers are
involved in the assembly of the oligomer. This 24-mer forms a hollow
spherical complex with octahedral symmetry including eight trigonal and
six square "windows." In addition, using cryo-electron microscopy
and image analysis, a model for the quaternary structure of human
B-crystallin has been proposed (30).
-Crystallin 32-mers also
form hollow spheres with a large central cavity. An important
difference to the archaebacterial complex is that the structure is
variable in detail with numerous different conformations being
observed. Although the shell-like structure is retained, attachment or
dissociation of several subunits is tolerated.
In contrast, we show here that murine Hsp25 exists as a hexadecamer in
solution with no significant population of other oligomeric species.
Similarly, only one spherical, oligomeric species, in this case a
24-mer, was observed for Hsp16.5 from M. jannaschii, supporting the notion that sHsp quaternary structure can be achieved with different numbers of subunits (47). Behlke et al. (48) reported 32-mers as the predominant oligomeric species for Hsp25. This
might be due to the association of two hexadecamers. We did not observe
such a "dimer of the 16-mer" under various experimental conditions.
In our experiments, the 16-mer is in a
concentration-dependent equilibrium with tetramers,
suggesting tetramers as the basic building block of the hexadecamer.
The notion that the 16-mer is formed from four tetramers, which
themselves represent dimers of dimers, was confirmed by the finding
that tetrameric and dimeric forms appear during Hsp25 denaturation in
urea and can be strongly stabilized by the addition of salt. This is in
agreement with data showing that phosphorylation of mammalian sHsp
results in the formation of tetramers (45, 49) and findings that
-crystallin forms tetramers under a number of experimental
conditions (49-51). In addition, it was suggested that the minimum
cooperatively melting structure of Hsp25 is a dimer (41).
As sHsps are active at conditions where other proteins are denaturing
we investigated the thermal stability of the protein. The size of Hsp25
particles increased drastically at temperatures above 60 °C. This is
in agreement with data by Dudich et al. (41). Furthermore,
-crystallin seems to unfold in this temperature range (42, 52).
Defined "heat shock" complexes with a size of about 1.3 MDa were
detected both by SEC and EM. These were in a slow equilibrium with the
Hsp25 hexadecamer, indicating that they represent a defined form of the
sHsp rather than an off-pathway reaction. The emergence of high
molecular weight particles is reminiscent of several reports in which
sHsp were shown to form large complexes (granules) after heat shock
(11, 28, 35, 53), which disappeared after return of the cell to
permissive folding conditions. These in vivo heat shock
granules were reported to be effective in reactivation of
heat-denatured nuclear proteins in mammalian cell lines (53, 54). In
these studies, overexpression of Hsp27 led to a marked acceleration of
recovery after heat shock, which was interpreted to be due to the
refolding of thermally unfolded proteins. A similar picture arises for
the high molecular mass complex formed by Hsp25 after heat treatment
in vitro. The isolated 1.3-MDa peak and the 16-mer peak both
showed chaperone activity comparable to untreated Hsp25. However, it is
important to note that, although the similarities between the two
species are striking, it is not established that the in
vitro aggregates correspond directly, both functionally and
structurally, to the heat shock granules found in vivo. This
remains to be elucidated in future studies.
Interestingly, tetramers, which we defined here as important
intermediates in the assembly process of Hsp25, have also been shown to
bind unfolded protein (22), indicating that different oligomeric Hsp25
species can posses chaperone activity (see Fig. 7).

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|
Fig. 7.
Model of the structure-function relationship
of Hsp25. The native hexadecameric form of Hsp25 is in equilibrium
with a tetrameric and dimeric species, which represent the basic
building units of the native oligomer. Unfolding and dissociation of
Hsp25 is a completely reversible process. During incubation at elevated
temperatures ( T), Hsp25 forms a granular complex, which
is in slow equilibrium with the Hsp25 16-mer. This heat shock form of
Hsp25, as well as the tetrameric and hexadecameric species, are active
in preventing heat-induced aggregation of model substrates.
U, unfolded protein; M, monomer; D,
dimer; NS, substrate, native state;
IS, substrate, intermediate state;
AS, aggregates of substrate protein.
|
|
From the data presented, we suggest the following, comprehensive model
for Hsp25 structure and function (Fig. 7). We could show that unfolding
of Hsp25 both after thermal and chemical denaturation is a reversible
process, proceeding via dimers and tetramers to the native
hexadecameric form of the protein. As no monomer or additional
oligomeric form could be detected under any experimental conditions,
association/dissociation seems to be a highly ordered process including
only a few defined steps. During incubation at elevated temperatures,
Hsp25 assembles into even bigger oligomeric complexes, which are in a
slow equilibrium with the hexadecamer. Importantly, the large granular
form of Hsp25 as well as tetrameric and hexadecameric species are
active in preventing heat-induced aggregation of model substrates.
Taken together, our data show that Hsp25 oligomers are remarkably
dynamic in structure and function, performing chaperone activity at
different levels of quaternary structures.
 |
ACKNOWLEDGEMENTS |
We thank Reinhard Rachel for providing
equipment and Simone Gräber and Kerstin Rutkat for excellent
experimental help with electron microscopy.
 |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft, the Bundesministerium für Bildung,
Wissenschaft, Forschung und Technologie, 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.
To whom correspondence should be addressed. Tel.:
49-89-289-13341; Fax: 49-89-289-13345; E-mail:
johannes.buchner{at}ch.tum.de.
 |
ABBREVIATIONS |
The abbreviations used are:
sHsp, small heat
shock protein;
CD, circular dichroism;
CS, citrate synthase;
Dm, D. melanogaster;
GdnCl, guanidinium hydrochloride;
Hsp25, 25-kDa murine heat shock protein;
Hsp27, 27-kDa human heat shock
protein;
SEC, size exclusion chromatography;
Hsp, heat shock protein;
HPLC, high performance liquid chromatography.
 |
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