Chaperone activity of cytosolic small heat shock proteins
from wheat
Eman Basha
1,
*, Garrett J. Lee
1,
†, Borries Demeler
2
and Elizabeth Vierling
1
1
Department of Biochemistry & Molecular Biophysics, University of Arizona, Tucson, AZ, USA;
2
Department of Biochemistry,
University of Texas, San Antonio, TX, USA
Small Hsps (sHsps) and the structurally related eye lens
a-crystallins are ubiquitous stress proteins that exhibit ATP-
independent molecular chaperone activity. We studied the
chaperone activity of dodecameric wheat TaHsp16.9C-I, a
class I cytosolic sHsp from plants and the only eukaryotic
sHsp for which a high resolution structure is available,
along with the related wheat protein TaHsp17.8C-II, which
represents the evolutionarily distinct class II plant cytosolic
sHsps. Despite the available structural information on
TaHsp16.9C-I, there is minimal data on its chaperone
activity, and likewise, data on activity of the class II pro-
teins is very limited. We prepared purified, recombinant
TaHsp16.9C-I and TaHsp17.8C-II and find that the class II
protein comprises a smaller oligomer than the dodecameric
TaHsp16.9C-I, suggesting class II proteins have a distinct
mode of oligomer assembly as compared to the class I

express both cytosolic sHsps and specific isoforms targeted
to intracellular organelles. There are at least two types of
sHsps in the cytosol, referred to as class I and class II
proteins, which share only  50% identity in the a-crystallin
domain and are estimated to have diverged over 400 million
years ago [6]. Five separate gene families encode mitochon-
drion, plastid, peroxisomal, nuclear and endoplasmic
reticulum-localized sHsps, each with appropriate organelle
targeting signals [3,4]. The evolutionary expansion of the
plant sHsp family may be the result of selection pressure
for tolerance to the many types of stresses encountered
by plants when they made the transition to growth on land.
In what way these sHsp families may serve specialized
functions is unknown.
High resolution structures of two sHsp oligomers are
now available: the class I plant sHsp, Triticum aestivum
(wheat) TaHsp16.9 C-I, and an sHsp from a prokaryotic
archeaon, Methanococcus jannaschii MjHsp16.5. Although
TaHsp16.9C-I is a dodecameric disk [7], and MjHsp16.5
forms a sphere composed of 24 subunits [8], both sHsp
oligomers are built from a conserved dimer structure, and
similar contacts between dimers stabilize the oligomer.
Although the oligomer is the dominant species at optimal
temperature for the organism, sHsp oligomers are in rapid
equilibrium with dissociated species as revealed by subunit
exchange [7,9–12], and some sHsps dissociate to a stable
suboligomeric species at the heat stress temperatures at
which they are predicted to be most active [7,13]. These
dynamic properties are likely to be important for sHsp
function.

To better define sHsp chaperone function and the
potential differences in function between the divergent
cytosolic class I and II plant sHsps, we initiated in vitro
studies of the chaperone activity of class I wheat
TaHsp16.9C-I in comparison to a wheat class II protein,
TaHsp17.8C-II. As mentioned above, TaHsp16.9C-I is the
only eukaryotic sHsp for which a high resolution structure is
available, but no significant characterization of its chaper-
one activity has been performed. Wheat TaHsp17.8C-II is
 33% identical overall to TaHsp16.9C-I. Only two previ-
ous studies have examined the chaperone activity of this
classofsHsp,andnoplantclassIIsHsphasbeentested
for the ability to support substrate refolding [21,22]. Both
TaHsp16.9C-I and TaHsp17.8C-II are undetectable in
vegetative plant tissues, but accumulate dramatically during
heat stress and are also expressed during seed development
(E. Basha & E. Vierling, unpublished observation). In vivo
studies indicate that plant class I and II sHsps, although
both present in the cytosol, do not coassemble into mixed
oligomers, suggesting they have distinct functions in the
cell [23].
We prepared purified, recombinant TaHsp16.9C-I and
TaHsp17.8C-II and found that the class II protein compri-
ses a smaller oligomer than the dodecameric TaHsp16.9C-I,
suggesting class II proteins have a distinct mode of oligomer
assembly as compared to the class I proteins. Using malate
dehydrogenase (MDH) as a substrate, TaHsp16.9C-I was
shown to be a much more effective chaperone than
TaHsp17.8C-II in preventing heat-induced MDH aggrega-
tion. Surprisingly, heat-denaturing firefly luciferase (Luc), a

TaHsp16.9C-I was enriched in the 55–90% (w/v) ammo-
nium sulfate fraction, while TaHsp17.8C-II was more
concentrated in the 40–70% (w/v) fraction. For
TaHsp17.8C-II, DEAE chromatography (diethylamino-
ethyl-Sepharose Fast Flow resin; Sigma) was performed in
3.2
M
urea (2.8
M
urea for TaHsp16.9C-I). After DEAE
chromatography, fractions containing sHsps were dialyzed
into 25 m
M
Tris/HCl, 1 m
M
EDTA, pH 7.5 (T25E1 buffer)
and applied to an hydroxyapatite column equilibrated in
10 m
M
Na/P
i
buffer, pH 7.5. The columns were eluted using
10–400 m
M
Na/P
i
buffer, pH 7.5. Fractions containing
sHsps were pooled and dialyzed against T25E1 and
concentrated, if necessary, to 1–2 mgÆmL
)1

dhoven, the Netherlands) and micrographs were taken at
82 000· magnification.
Sedimentation velocity experiments
Analytical ultracentrifugation was performed with a Beck-
man Optima XL-A ultracentrifuge. Samples (450 lL) were
centrifuged for 3.5 h at 4 °C and 40 000 r.p.m. in an AN 60
TI rotor using double sector epon centerpieces. Measure-
ments were taken at 230 and 280 nm using a 0.001 cm
radial step size in continuous measurement mode. Data
were analyzed with
ULTRA SCAN II
version 6.2 for Unix
( using the van Holde–
Weischet method [27] and finite element analysis as
described previously [28].
Ó FEBS 2004 Small Hsp chaperones (Eur. J. Biochem. 271) 1427
Thermal aggregation protection assays
Thermal aggregation protection assays were performed with
MDH essentially as described by Lee et al. [15] using 0.6 l
M
MDH and purified sHsps from 0.18 to 3.0 l
M
(monomer)
in 50 m
M
NA/P
i
buffer, pH 7.5. Samples were incubated in
1 mL quartz cuvettes in a thermostated water bath at 45 °C.
To quantify changes in light scattering, absorbance at

by size exclusion chromatography (SEC) on a Rainen
HPLC using a Toso-Haas TSK G4000 SWXL column, in a
mobile phase containing 250 m
M
Na/P
i
,pH7.3and
200 m
M
NaCl. For analysis of sHsp/MDH complexes,
6.0 l
M
of TaHsp16.9 C-I or TaHsp17.8C-II subunits were
incubated with different MDH concentrations in 50 m
M
Na/P
i
buffer, pH 7.5 for 30 or 90 min at 45 °C. After
incubation, samples were cooled on ice for 2 min and
centrifuged at 16 000 g for 15 min. NaCl was added to the
supernatant to a final concentration of 200 m
M
.Samples
were size-fractionated on the SEC column in a mobile phase
containing 250 m
M
Na/P
i
, pH 7.3 and 200 m
M

emission in a Turner 20/20 luminominer. Activity is
plotted as a percentage relative to that of an equivalent
amount of native Luc measured prior to the heating step.
As a negative control, 0.11 lgÆlL
)1
bovine IgG was
substituted for sHsp (equivalent weight) in the initial heat-
inactivation step. Data points and error bars reflect the
mean and standard deviation of three replicates.
Results
Comparison of TaHsp16.9 C-I and TaHsp17.8 C-II
To produce recombinant wheat class I and II sHSPs for
these studies, we utilized the wheat class I cDNA,
TaHsp16.9C-I (Accession number, S21600), corresponding
to the sHsp for which the high resolution structure (2.65 A
˚
)
has been described [7], and a new wheat class II cDNA,
TaHsp17.8 C-II (Accession number, AAK51797) [24].
Amino acid sequence alignment illustrates the conserved
and divergent regions of these two sHsps (Fig. 1).
TaHsp16.9C-I and TaHsp17.8C-II have an overall identity
of only  33%, but regions corresponding to secondary
Fig. 1. Amino acid sequence alignment of TaHsp16.9C-I (Accession number S21600) and TaHsp17.8C-II (Accession number AAK51797). Identical
residues are indicated with * and highly conservative replacements indicated with colons or periods under the alignment. Regions of secondary
structure in TaHsp16.9C-I [7] are indicated above the alignment. The a-helices are displayed as open bars; b-strands as lines. The conserved
a-crystallin domain extends from b-strand 2 to b-strand 9. Regions in gray shaded boxes correspond to consensus regions within the a-crystallin
domain that show particularly high conservation between plant sHsps. Residues in the N-terminal region shown in bold correspond to sequences
conserved in all class I or class II proteins, respectively. Residues in the C-terminus in bold correspond to the conserved Basic-X-I/V-Q-I/V motif
identified by de Jong et al. [29]. Underlined residues in the C-terminus of TaHsp17.8C-II correspond to a conserved motif of class II proteins [6].

estimated mass of 284 kDa, while TaHsp17.8C-II migrates
at 242 kDa. Similarly, on SEC the TaHsp16.9C-I peak
eluted at 10.32 min while TaHSP17.8C-II eluted later
at 10.65 min (Fig. 2C). Compared to TaHsp16.9C-I,
TaHsp17.8C-II always exhibited a fairly broad elution
profile, which could result from a variety of factors, including
oligomeric instability, nonuniformity of oligomer size or
interaction with the column matrix. As TaHsp16.9C-I
is a 12-subunit oligomer [7], these results suggest the
TaHsp17.8C-II oligomer is composed of fewer than 12
subunits.
Size of the recombinant sHsp oligomers
Although nondenaturing PAGE and SEC indicated the
class I and II sHsps have different oligomeric structures,
neither of these techniques are primary methods for size
determination. Therefore, to better understand the differ-
ence in subunit organization of these sHsps, we compared
them by EM using negative staining and by sedimentation
velocity centrifugation analysis.
As shown in Fig. 3, purified preparations of either sHsp
appear as mostly uniform, roughly spherical particles. The
TaHsp16.9C-I particles have a diameter of approximately
11 nm, consistent with the crystal structure [7]. They are
clearly larger than the TaHsp17.8C-II particles, which
have an estimated diameter of only 9 nm. Therefore, the
relative sizes of the two oligomers are consistent with
their behavior on nondenaturing PAGE and SEC. Their
appearance is also similar to what has been observed for
class I and II sHsps from Pisum sativum (pea) [21],
suggesting conservation of the subunit stoichiometry of

arising from buffer absorbance.
Ó FEBS 2004 Small Hsp chaperones (Eur. J. Biochem. 271) 1429
activity, in which it was shown to form complexes with the
model substrate MDH when the two proteins are heated
together [7]. We undertook a more detailed examination of
the activity of TaHsp16.9C-I and, in comparison,
TaHsp17.8C-II. A well-established assay for sHsp chaper-
one activity is the ability to prevent heat-induced protein
aggregation as measured by light scattering [25]. In this
assay, the ratio of sHsp to substrate that is required to
suppress light scattering can be used as a measure of
effectiveness of the chaperone. Using this assay we tested the
relative activity of the two wheat proteins in suppressing
aggregation of MDH. As shown in Fig. 5, both wheat
sHsps were effective in preventing MDH aggregation as
assessed by suppression of an increase in light scattering
over time at 45 °C. TaHsp16.9C-I achieved maximum
protection of MDH at a ratio of two to three subunits of
Fig. 3. Visualization of negatively stained
TaHsp16.9C-I (left) and Ta Hsp17.8C-II (right)
by electron microscopy. Both proteins appear
as homogenous, roughly spherical particles,
but TaHsp17.8C-II is smaller. Bar indicates
50 nm for both.
Fig. 4. Sedimentation velocity analysis of TaHsp16.9C-I and
TaHsp17.8C-II. Shown is the integral distribution plot from the van
Holde–Weischet analysis of the data obtained for TaHsp16.9C-I (s)
and TaHsp17.8C-II (d). For both proteins, the majority of the sample
sedimented between 8.6 s and 9.2 s, with a small amount of smaller
association states (less than 6% of the total concentration) sedimenting

M
TaHsp17.8C-II) the extent of
light scattering was actually higher than in the absence of
the sHsp. This may reflect the formation of very large
aggregates of MDH that also include the sHsp. At low sHsp
concentrations the sHsp could be bound to the MDH, but
not be abundant enough to prevent interaction of unfolded
MDH with itself.
Analysis of sHsp/MDH complexes
The differences in effectiveness of aggregation protection
between the two wheat sHsps suggests that the way in which
the sHsp and denatured substrate interact may be different.
To characterize sHsp/MDH complexes, SEC analysis
was performed after heating either TaHsp16.9C-I or
TaHsp17.8C-II (6.0 l
M
) with 1–4 l
M
MDH, yielding
sHsp/MDH ratios comparable to those used in the light-
scattering assays. MDH does not interact with either sHsp
when the proteins are incubated together at 22 °C (Fig. 6A);
the proteins elute at the predicted position based on their
individual native molecular masses. We have noted that
TaHsp17.8C-II consistently yields a lower absorbance
(A
220
)thanTaHsp16.9C-I on column chromatography.
We attribute this to either irreversible interaction of the
protein with the column, or presence of aggregates too large

complex peak does not increase after 30 min. This result is
consistent with the light-scattering data, which showed the
sHsp was unable to fully protect MDH at this ratio. As
the amount of sHsp to substrate is further decreased, most
of the MDH is no longer found in complexes, but rather
is aggregated and removed by centrifugation prior to
sample loading on the column (not shown). Interestingly,
the sHsp itself does not appear to be complexed with the
insoluble MDH at the 2 : 1, sHsp/MDH ratio; after
90 min the free sHsp is still all accounted for in the peak
at 10.65 min. However, some sHsp is clearly lost to the
insoluble fraction, which is not loaded on the column,
when the ratio is only 1.5 : 1. This is consistent with
the maximum light-scattering values observed for
TaHsp17.8C-II/MDH at a 1 : 1 ratio, which were higher
than those for MDH alone. A potential intermediate-sized
species of complex is also evident at  6.5 min in most of
the samples. In total, as observed by light scattering,
substrate denaturation and aggregation are time-depend-
ent, the sHsps can be saturated with substrate, and
TaHsp17.8C-II is less effective in protecting MDH
compared to TaHsp16.9C-I.
To visualize the sHsp substrate complexes directly,
samples incubated as for the SEC analysis at 45 °Cfor
30 min were observed by EM and negative staining
(Fig. 7). Note that because samples were centrifuged prior
to application to the grid, only soluble material was
observed. Two consistent observations arose from this
analysis. First, complexes formed at an sHsp to substrate
ratio that was sufficient, or higher, than that required for

Ó FEBS 2004 Small Hsp chaperones (Eur. J. Biochem. 271) 1431
the cuvette walls. As shown in Fig. 8A, Luc incubated
with TaHsp17.8C-II at 42 °C for 15 min was recovered
almost exclusively in the soluble fraction, indicating that
TaHsp17.8C-II was able to protect Luc from heat-induced
insolubilization. A similar weight of IgG gave no protec-
tion (not shown). Surprisingly, when Luc was incubated
with TaHsp16.9C-I, virtually all of the Luc was found in
the pellet fraction, while the sHsp remained soluble. Thus,
in contrast to results with MDH, TaHsp16.9C-I is a less
effective chaperone with Luc than is TaHsp17.8C-II. Note
that a higher ratio of sHsp to substrate is required to
protect Luc as compared to MDH. In parallel to these
observations, TaHsp17.8C-II formed a complex when
heated with Luc, which could be observed by SEC
(Fig. 8B). No such complex formed with TaHsp16.9C-I
(not shown). Thus, these two sHsps do not behave
equivalently with all substrates.
Denatured Luc bound to
Ta
Hsp17.8C-II can be
reactivated in a cell free lysate
The effectiveness of sHsp chaperone activity can also be
assessed by the ability of an sHsp to maintain substrate in
a state from which it can be refolded by ATP-dependent
Fig. 6. TaHsp16.9C-I is more effective in
forming complexes with MDH than is
TaHsp17.8C-II. All samples were separated by
SEC, and absorbance (220 nm) monitored
over elution time. Samples were centrifuged to

reactivation. Thus, formation of TaHsp17.8C-II/Luc com-
plexes is correlated with ability to support substrate
reactivation.
Discussion
Our data provide the first detailed analysis of the in vitro
chaperone activity of TaHsp16.9C-I, the only eukaryotic
sHsp for which a high resolution structure is available.
Surprisingly, although this sHsp effectively protects MDH
from insolubilization, it did not interact with a second
substrate, Luc, under the conditions tested. In parallel, we
analyzed a related wheat sHsp, TaHsp17.8C-II, which
proved to be less effective in protecting MDH, but
interacted well with Luc, both preventing aggregation and
supporting refolding. Thus, these results document the first
clear example of apparent substrate specificity for sHsps.
TaHsp16.9C-I and TaHsp17.8C-II represent two distinct
classesofcytosolicsHspsfromplants(classIandclassII),
Fig. 7. MDH/sHsp complexes visualized by
electron microscopy and negative staining.
TaHsp16.9C-I or TaHsp17.8C-II (6 l
M
sub-
units) heated with different concentrations of
MDH ranging from 6 to 1 subunit sHsp: 1
subunit substrate. Complexes were formed at
45 °C for 30 min then centrifuged and loaded
on the EM grids for visualization at magni-
fication of 820 000. Bar indicates 110 nm
for all.
Ó FEBS 2004 Small Hsp chaperones (Eur. J. Biochem. 271) 1433

features conserved in the class II proteins as well [1,6].
Regardless of absolute subunit numbers, class I and II sHsp
oligomers clearly have distinct modes of assembly, as also
reflected in the fact that these two classes of sHsps do not
coassemble into mixed oligomers in vivo or in vitro, although
class I or II sHsps will coassemble into normal oligomers
when mixed with class I or II sHsps, respectively, from
different plant species ([7,23], and E. Basha & E. Vierling,
unpublished observation). Distinct assemblies of different
sHsps in the same cell have also been observed in humans
and bacteria [30,31], suggesting there are different,
conserved roles for specific sHsps.
Both TaHsp16.9C-I and TaHsp17.8C-II were able to
suppress the heat-dependent aggregation of MDH. How-
ever, TaHsp16.9C-I suppresses MDH aggregation com-
pletely at a stoichiometry of 2–3 subunits sHsp to 1
subunit MDH. In contrast, complete suppression of
MDH aggregation by TaHsp17.8C-II required the higher
ratio of 4–5 sHsp subunits:1 MDH subunit. From
previous work, PsHsp18.1C-I was found to be somewhat
more effective in the aggregation protection of MDH than
either of the wheat sHsps, suppressing MDH aggregation
at a ratio of 2 : 1, sHsp subunit:MDH [15]. The pea class
II protein was not tested with MDH, but when tested with
citrate synthase, it was more than sixfold less effective
than the PsHsp18.1C-I [21]. Thus, using these in vitro
assays with two different substrates, class II proteins have
proven to be less effective as chaperones than class I
proteins, consistent with some type of substrate specificity
for these two classes of proteins.

M
Luc either before (22 °C) or after heating at 42 °Cfor15min(42°C). Approximate elution
times of molecular mass markers are indicated. (C) Time course of Luc reactivation in reticulocyte lysate. (d) TaHsp17.8C-II + ATP; (j)
TaHsp17.8 C-II – ATP; (m) TaHsp16.9C-I + ATP; (h)Hsp16.9C-I–ATP;(s)IgG+ATP.
1434 E. Basha et al. (Eur. J. Biochem. 271) Ó FEBS 2004
imitated the same effect of the R120G mutation in
aB-crystallin.
SEC analysis showed that the complexes formed between
the two wheat sHsps and MDH are quite large. Working
with PsHsp18.1C-I, Lee et al. [15] found complexes with
MDH were much smaller than those formed with the wheat
sHsps, although the size observed by SEC was dependent
on the substrate concentration as well as the denaturation
temperature. The less efficient aggregation protection
obtained with the wheat sHsps (on a molar basis of sHsp:
substrate) compared to PsHsp18.1C-I suggests that the
MDH aggregates more rapidly than it can form stabilizing
interactions with the wheat sHsps. It is interesting that there
isalwaysafreepeakofsHsponSEC,evenwhensome
of the substrate has precipitated. The free sHsps could
still have a role in protection, by cycling on and off the
aggregates, as suggested by both Lindner et al.[33]and
Friedrich et al. [12].
The decrease in SEC complex peak height and the
eventual loss of sHsp at the highest substrate concentrations
is due to the insolubility of the sHsps bound to excess
substrate (as indicated by SDS PAGE; not shown).
Transition of sHsps to an insoluble fraction is observed
in vivo in many organisms [15,34,35], and may also result
from overloading of the sHsp with substrates. Experiments

aggregation and formed high molecular mass complexes
with Luc. We also showed that TaHsp17.8C-II supported
Luc refolding using rabbit reticulocyte lysate as a source
of ATP-dependent eukaryotic chaperones. However, the
inability of TaHsp16.9C-I to protect Luc is not true for all
classIsHsps.PsHsp18.1C-I has been shown to protect
Luc with the same effectiveness as TaHsp17.8C-II and to
support Luc refolding [12,15,18]. This fact indicates that the
differences in sHsp–substrate interactions must be more
subtle than the differences between class I and II sHsps in
primary sequence or quaternary structure. TaHsp16.9C-I
and PsHsp18.1C-I show 80% identity and 86% similarity in
the conserved C-terminal a-crystallin domain. In contrast
they show only 41% identity and 50% similarity in the
N-terminal arm, suggesting substrate specificity is deter-
mined by the N-terminal arm. The N-terminus of
PsHsp18.1.C-I was also implicated in substrate interactions
in bis-ANS binding experiments [15]. As it is proposed that
substrate binding and protection involves oligomer dissoci-
ation and some type of reassociation to form the large sHsp/
substrate complexes [7,13], it must also be considered that
overall differences in oligomer stability and/or the kinetics
of oligomer dissociation, rather than specific sequence
differences, dramatically affect sHsp interactions with
different substrates.
Although to date sHsps have been ascribed little substrate
specificity, it is clear from this study and previous work
[21,37] that the effectiveness of substrate protection, on a
molar basis, by different sHsps can vary significantly under
the same conditions. A difference in effectiveness is obvious

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