Some properties of human small heat shock protein Hsp20 (HspB6)
Olesya V. Bukach
1
, Alim S. Seit-Nebi
1
, Steven B. Marston
2
and Nikolai B. Gusev
1
1
Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia;
2
Imperial College School of Medicine
at National Heart and Lung Institute, London, UK
Human heat shock protein of apparent molecular mass
20 kDa (Hsp20) and its mutant, S16D, mimicking phos-
phorylation by cyclic nucleotide-dependent protein kinases,
were cloned and expressed in Escherichia coli. The proteins
were obtained in a homogeneous state without utilization
of urea or detergents. On size exclusion chromatography at
neutral pH, Hsp20 and its S16D mutant were eluted as
symmetrical peaks with an apparent molecular mass of
55–60 kDa. Chemical crosslinking resulted in the forma-
tion of dimers with an apparent molecular mass of 42 kDa.
At pH 6.0, Hsp20 and its S16D mutant dissociated, and
were eluted in the form of two peaks with apparent
molecular mass values of 45–50 and 28–30 kDa. At
pH 7.0–7.5, the chaperone activity of Hsp20 (measured by
its ability to prevent the reduction-induced aggregation of
insulin or heat-induced aggregation of yeast alcohol
dehydrogenase) was similar to or higher than that of

oligomers that vary in structure and number of monomers
[6,7]. These complexes can be formed by identical or
nonidentical subunits. Subunits of a-crystallin, Hsp20,
Hsp22, and Hsp27 seem to be involved in the formation
of different heterooligomeric complexes [8–12]. Hsp27 and
aB-crystallin have been analyzed in detail [2–5,13,14],
whereas other members of the large superfamily of sHsp
are less well characterized.
Hsp20 was described by Kato et al. [8] as a byproduct of
purification of human aB-crystallin and Hsp27. Hsp20 is
expressed in practically all tissues, reaching a maximal level
of 1.3% of total proteins in skeletal, heart and smooth
muscles [2,9,15]. Since 1997, the laboratory of Colleen
Brophy has performed detailed investigations of the role of
Hsp20 in the regulation of smooth muscle contraction. It
has been shown that cAMP- and cGMP-dependent protein
kinases phosphorylate Ser16 of Hsp20 and that phosphory-
lation of Hsp20 is associated with smooth muscle relaxation
that is independent of the level of phosphorylation of the
myosin light chain [16–20]. These findings have been
confirmed and extended [21–23]. Insulin induces phos-
phorylation of rat Hsp20 at Ser157 [24] and Hsp20
phosphorylated at two different sites (Ser16 and Ser157)
differently affects glucose transport [25,26]. Recently, Hsp20
was detected in blood and it has been shown that Hsp20
binds to and inhibits platelet aggregation [27]. Thus,
significant progress has been achieved in revealing a possible
physiological role of Hsp20. However, investigation of the
biochemical properties of isolated Hsp20 lag behind.
Indeed, the biochemical properties of rat Hsp20 were only

(XhoI restriction site underlined) primers, and Pwo DNA
polymerase (Roche). The 480 bp PCR product was purified
after electrophoresis in an agarose gel, then digested with the
restriction endonucleases NdeIandXhoI and inserted into
the plasmid vector pET23b (which had been predigested with
the same endonucleases). The resulting construct was verified
by DNA sequencing and used for expression and mutagen-
esis. A two step PCR-based ÔmegaprimerÕ method [28,29] was
used for the replacement of Ser16 of Hsp20 with Asp. In this
case, the primer S16D (5¢-GCCGCGCCGACGCCCCG
TTGC-3¢) was used for site-directed mutagenesis.
The human Hsp27 full-length cDNA (GenBank acces-
sion no.: NM001540) was amplified from Marathon-Ready
cDNA, Lung (Clontech) using the following forward
5¢-GAGATATA
CATATGGCCGAGCGC-3 and reverse
5¢-CC
GGATCCCTACTTCTTGGCTGG-3¢ primers con-
taining, respectively, NdeIandBamHI restriction sites
(underlined). The PCR product was purified and inserted
into the plasmid vector, pET11c (Novagen). The resulting
construct was verified by DNA sequencing and used for
expression and site-directed mutagenesis.
Three serine residues of Hsp27 (Ser15, Ser78 and
Ser82) were replaced with Asp. This was achieved by
using the following primers: 5¢-CGGGGCCCCGACTG
GGACCCC-3¢ for S15D and 5¢-GACCCCGCTGTC
GAGTTGCCGGTCGAGCGCGC-3¢ for the S78D and
S82D mutants. The two step PCR-based ÔmegaprimerÕ
method [28,29] permits creation of the so-called 3D

pH 8.0, 100 m
M
NaCl, 1 m
M
EDTA, 0.5 m
M
phenyl-
methanesulfonyl fluoride, 14 m
M
b-mercaptoethanol) was
fractionated with (NH
4
)
2
SO
4
(0–30% saturation) and
subjected to ion-exchange chromatography on a High-Trap
Q column (Amersham-Pharmacia) equilibrated with
buffer B (20 m
M
Tris/acetate, pH 7.6, 10 m
M
NaCl,
0.1 m
M
EDTA, 0.1 m
M
phenylmethanesulfonyl fluoride,
14 m

were similar to those described for Hsp20. Hsp27 and its 3D
mutant were fractionated by (NH
4
)
2
SO
4
(0–50% satura-
tion) and subjected to ion-exchange chromatography on a
High-Trap Q column (Amersham-Pharmacia), followed by
gel filtration on a Sephacryl S300 High-Prep 16/60 column
(Amersham-Pharmacia). If necessary, further purification
was achieved by hydrophobic chromatography on phenyl-
superose (Amersham-Pharmacia). Preparations of Hsp27
and its 3D mutant were concentrated by ultrafiltration and
stored frozen in buffer B containing 10% glycerol.
Denaturation and renaturation of sHsp. Denaturation
and renaturation of Hsp20 and commercial a-crystallin
(Sigma) was performed according to van de Klundert et al.
[15]. Recombinant wild-type Hsp20 in buffer B was freeze-
dried. The samples of freeze-dried Hsp20 or commercial
a-crystallin were dissolved in 50 m
M
phosphate (pH 7.5),
containing 100 m
M
Na
2
SO
4

The oligomeric state of sHsp was determined by size
exclusion chromatography on Superdex 200 HR 10/30
using the ACTA-FPLC system. The column was usually
equilibrated with buffer C (20 m
M
Tris/HCl, pH 7.5,
containing 150 m
M
NaCl and 15 m
M
b-mercaptoethanol).
292 O. V. Bukach et al. (Eur. J. Biochem. 271) Ó FEBS 2003
In the case of renaturation experiments, the same column
was equilibrated and developed with 50 m
M
phosphate
(pH 7.5) containing 100 m
M
Na
2
SO
4
.Thecolumnwas
calibrated using the following molecular mass markers:
thyroglobulin (669 kDa), ferritin (440 kDa), catalase
(240 kDa), aldolase (158 kDa), BSA (66 kDa) and chymo-
trypsinogen (25 kDa).
For investigating the exchange of subunits between
Hsp20 and Hsp27, equimolar quantities (0.4 mgÆmL
)1

Chemical crosslinking
Three different methods were used for crosslinking Hsp20.
In the first, Hsp20 (0.2 mgÆmL
)1
) was dialyzed overnight
against 50 m
M
phosphate buffer (pH 7.5), containing
100 m
M
Na
2
SO
4
. Before SDS gel electrophoresis, the
samples were either treated with an excess of b-mercapto-
ethanol or loaded onto the gel in the absence of
b-mercaptoethanol.
In the second method, Hsp20 (0.75 mgÆmL
)1
)in0.2
M
triethanolamine (pH 7.5) was incubated with dimethylsube-
rimidate (20 m
M
)for1hat20°C. The reaction was
stopped by the addition of SDS sample buffer. The protein
composition of the samples thus obtained was analyzed by
SDS gel electrophoresis.
In the third method, Hsp20 (1 mgÆmL

Determination of chaperone activity
The chaperone activity of Hsp20 and of bovine lens
a-crystallin (Sigma) was determined by their ability to
retard or to decrease aggregation of the insulin B-chain
(Sigma) [15]. All experiments were performed in buffer E
(50 m
M
phosphate, pH 7.5, 100 m
M
Na
2
SO
4
). Insulin (6.5
mgÆmL
)1
), dissolved in 2.5% acetic acid, was added to the
incubation mixture (270 lL) to a final concentration of
0.25 mgÆmL
)1
. The mixture was incubated at 40 °Candthe
reaction started by addition of a water solution of dithio-
threitol up to a final concentration of 20 m
M
. Reduction of
the disulfide bonds of insulin was accompanied by aggre-
gation of the B-chain and an increase of turbidity that was
measured at 360 nm on an Ultraspec 3100 Pro spectro-
photometer.
The chaperone activity of Hsp20 and of commercial

Isolation of human Hsp20 and its S16D mutant
As described in the Materials and methods, we developed
procedure for purification of recombinant wild-type human
Hsp20. All steps of extraction and purification were
performed in the absence of urea or detergents. The method
provided 5–7 mg of recombinant wild-type Hsp20 from 1 L
of the E. coli culture.
When the S16D mutant of Hsp20 was expressed in
E. coli, most of the protein was insoluble in the lysis buffer
and this buffer extracted less than 20% of the protein. The
S16D mutant that was extracted with lysis buffer was
subjected to the same steps of purification as the wild-type
protein and, according to the SDS gel electrophoresis, had
the same apparent molecular mass as the wild-type protein
(Fig. 1A). Most of the S16D mutant that was not soluble in
the lysis buffer could be dissolved in the same buffer
containing 6
M
urea and was subjected to ion-exchange
chromatography on a High-Trap Q column in buffer B,
containing 6
M
urea, at pH 8.5. According to SDS gel
electrophoresis, the apparent molecular mass of the protein
thus obtained was 2–3 kDa less than the corresponding
molecular mass of the wild-type Hsp20 or water-soluble
Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur. J. Biochem. 271) 293
S16D mutant of Hsp20 (Fig. 1A). Tandem MS analysis
performed by Dr R. Wait (Kennedy Institute of Rheuma-
tology Division, Faculty of Medicine, Imperial College,

exclusion chromatography, at neutral pH, on a Superdex
200 column under three different experimental conditions.
In the first we loaded the column with 240 lLofa
2.6 mgÆmL
)1
concentration of protein (curve 1 on Fig. 2A).
In the second, the column was loaded with the same volume
of 0.3 mgÆmL
)1
protein (curve 2 on Fig. 2A). In the third,
the column was loaded with 30 lLofproteinata
concentration of 2.6 mg mL
)1
(curve 3 on Fig. 2A). Under
these experimental conditions, the apparent molecular mass
of recombinant human wild-type Hsp20 was 58, 54 and
56 kDa for the first, second and third experimental condi-
tions, respectively. We also analyzed the effect of urea
induced denaturation, followed by renaturation, on the
oligomeric state of Hsp20. As shown in Fig. 2B (curves 3
and 4) denaturation–renaturation showed practically no
effect on the oligomeric state of Hsp20, and both intact and
Fig. 1. Characterization of recombinant human wild-type Hsp20 and its
S16D mutant. SDS gel electrophoresis (A) and IEF (B) of wild-type
Hsp20 (1), and of its S16D mutant that is soluble in the absence (2) and
in the presence (3) of urea. Arrows indicate the position of molecular
mass markers (14 and 25 kDa) and direction of pH gradient. (C)
Primary structure of wild-type Hsp20, and of its S16D mutant soluble
in the absence and in the presence of urea, as determined by HPLC/
tandem MS. The experimentally determined sequence is shown in

suggest that the 40 kDa band corresponds to Hsp20 dimer
crosslinked via single Cys46. Crosslinking of Hsp20 with
dimethylsuberimidate was also accompanied by the forma-
tion of an additional band with molecular mass 40 kDa
(Fig. 3B), that probably also corresponds to Hsp20 dimer.
Similar results were obtained if Hsp20 was subjected to
zero-length crosslinking by EDC and NHS (Fig. 3C). In
this case we observed two or three closely separated bands
with apparent molecular mass 38–40 kDa that probably
correspond to isomers of Hsp20 dimers. Thus, under the
experimental conditions used, Hsp20 predominantly forms
dimers of 40 kDa molecular mass, as judged by SDS gel
electrophoresis, and 54–58 kDa by size-exclusion chroma-
tography.
We considered that changes in pH might somehow affect
the quaternary structure of Hsp20. At pH 7.5–7.0 Hsp20
was eluted as a more or less symmetrical peak with apparent
molecular mass 54–58 kDa (Fig. 4). At pH 6.5, both wild-
type protein and its S16D mutant were eluted as broader
peaks with a slightly smaller apparent molecular mass
(46–47 kDa) (Fig. 4). When the pH was decreased to 6.0,
two peaks with apparent molecular masses of 47–50 and
28–30 kDa were observed on the chromatogram (Fig. 4).
At pH 5.5, the high molecular mass peak completely
disappeared and the small molecular mass peak became
broader and more asymmetric (Fig. 4). A decrease in pH
from 7.5 to 5.5 was accompanied not only by a decrease of
the apparent molecular mass of Hsp20, but also by a
decrease in the area under the protein peaks on the
chromatogram. Acidification probably results in the disso-

acidic pH is different from that at neutral pH values. Similar
results were obtained with the S16D mutant of Hsp20 (data
Fig. 3. Crosslinking of Hsp20. (A) Formation of disulfide crosslinked
Hsp20 dimers. A sample of oxidized Hsp20 treated with an excess of
b-mercaptoethanol (2), or loaded onto the gel without the addition of
reducing agents (3). (B) Crosslinking of Hsp20 with dimethylsube-
rimidate. Hsp20 before (2) or after (3) incubation with 20 m
M
dimethylsuberimidate. (C) Zero-length crosslinking of Hsp20. Hsp20
before (2) and after (3) incubation with 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (5 m
M
) and N-hydroxysuccinimide
(5 m
M
). In all cases the mixture of standards containing proteins with
molecular masses 94, 67, 43, 30, 20 and 14 kDa was loaded on the first
track.
Fig. 4. Effect of pH on the oligomeric state of recombinant human wild-
type Hsp20 and its S16D mutant. Three-hundred microliter samples
containing 90 lg of wild-type Hsp20 (solid lines) or its S16D mutant
(dotted lines) were loaded onto the column of Superdex 200 HR 10/30
equilibrated with buffer D (50 m
M
phosphate, 150 m
M
NaCl, 1 m
M
EDTA, 15 m
M

followed by renaturation had no effect on the chaperone
activity of the wild-type Hsp20, and complete prevention of
insulin aggregation was achieved at the same Hsp20/insulin
ratio as for intact protein (Fig. 6B). The S16D mutant of
Hsp20 also decreased the aggregation of insulin (Fig. 6C);
however, it was less effective than the wild-type protein.
Denaturation–renaturation of the S16D mutant only
weakly affected its chaperone properties (Fig. 6D). Com-
mercial a-crystallin that was not subjected to urea treatment
was very ineffective in preventing reduction-induced aggre-
gation of insulin. Even at a ratio of 1 : 1, a-crystallin only
slightly decreased the aggregation of insulin (Fig. 6E). Urea-
induced denaturation followed by renaturation significantly
improved the chaperone activity of a-crystallin (Fig. 6F).
This was probably caused by a change in the aggregation
state of a-crystallin that was induced by urea treatment and
identified by size-exclusion chromatography (see Fig. 2B).
However, even after treatment with urea, the chaperone
activity of a-crystallin was similar to that of the wild-type
Hsp20. Thus, at pH 7.5 and with reduced insulin as a model
substrate, the chaperone activity of the wild-type Hsp20
was comparable to or greater than that of commercial
a-crystallin.
At pH 7.0, the heating of isolated ADH in the absence of
divalent cations was accompanied by aggregation and a
large increase in the light scattering (Fig. 7, curve 1).
Addition of increasing quantities of the wild-type Hsp20
resulted in retardation of the onset of aggregation and a
decrease in the amplitude of light scattering (Fig. 7A, curves
2–5). At the ADH/Hsp20 ratio of 1 : 1 (wt/wt), aggregation

ADH (Fig. 7F). Hsp20 was more effective in preventing
Fig. 5. Far UV CD spectra of the wild-type Hsp20 (A) and commercial
a-crystallin (B). The spectra were recorded at pH 6.0 (1), 6.8 (2) or 7.5
(3).
296 O. V. Bukach et al. (Eur. J. Biochem. 271) Ó FEBS 2003
precipitation of denatured ADH (Fig. 7E, curve 3). Much
smaller quantities of Hsp20 were coprecipitated with dena-
tured ADH (Fig. 7F, curve 2). It is worthwhile mentioning
that isolated sHsp were not precipitated in the absence of
ADH, even after 60 min of incubation (Fig. 7F, curve 3).
Similar results were obtained with the S16D mutant of Hsp20
(data not shown). Thus, at pH 7.0, Hsp20 is a more potent
chaperone than a-crystallin, probably because complexes
formed by Hsp20 with denatured ADH are smaller or more
soluble than the corresponding complexes formed by dena-
tured ADH and a-crystallin.
As discussed above, a decrease in the pH to pH 6.0 may
induce partial unfolding and dissociation of small oligomers
formed by Hsp20 or its S16D mutant (Figs 4 and 5). As
unfolding and dissociation may affect the chaperone activity
of Hsp20, we analyzed the effect of different sHsps on the
aggregation of ADH at pH 6.0. Under these conditions,
heating also induced the aggregation of ADH (Fig. 8, curve
1). Addition of increasing quantities of wild-type Hsp20
increased the rate and amplitude of light scattering (Fig. 8A,
curves 2–5). Thus, the wild-type Hsp20, instead of prevent-
ing, promotes the aggregation of ADH. This is probably a
result of the formation of insoluble complexes of Hsp20 and
denatured ADH. Indeed, as shown in Figs 8E,F, incubation
of ADH with the wild-type Hsp20 resulted in the formation

Hsp27 [8,9]. Indirect data also indicate that Hsp20 may
interact with Hsp27 and aB-crystallin [10]. However, to our
knowledge, the hetero-oligomeric complexes formed by
Hsp20 with other sHsp have not been characterized and
reported in the literature. Therefore, we investigated the
Fig. 6. Influence of recombinant human Hsp20
(A and B), the S16D mutant of Hsp20 (C and
D) or commercial a-crystallin (E and F) before
(A, C and E) or after (B, D and F) urea treat-
ment followed by renaturation on the reduction
induced aggregation of insulin. The chaperone
activity was measured by the prevention of
dithiothreitol-induced aggregation of insulin
(0.25 mgÆmL
)1
)at40°C under conditions
described in the Materials and methods.
Insulin alone (1), or insulin in the presence of
0.06 mgÆmL
)1
(2), 0.12 mgÆmL
)1
(3) or
0.25 mgÆmL
)1
(4) small heat shock proteins.
Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur. J. Biochem. 271) 297
interaction of Hsp20 and its mutant mimicking phosphory-
lation with Hsp27.
Interaction of the wild-type Hsp20 with the wild-type

peaks, according to SDS/PAGE, contained almost identical
quantities of Hsp27 and Hsp20 (insert on Fig. 9A). Similar
results were obtained if the wild-type Hsp27 was mixed with
the S16D mutant of Hsp20. Thus, after mixing at 30–37 °C,
homo-oligomers of wild-type Hsp27 and Hsp20 (or the
S16D mutant of Hsp20) may rearrange, forming mixed
hetero-oligomers that contain similar quantities of these two
sHsp.
The isolated 3D mutant of Hsp27 produces a broad peak
with apparent molecular mass 96–106 kDa. A significant
decrease in molecular mass compared with the wild-type
Hsp27 is a result of the fact that mutations mimicking
phosphorylation induce dissociation of large oligomers of
Hsp27 [29–31]. As already mentioned, the wild-type Hsp20
and its S16D mutant are eluted as a single peak with an
apparent molecular mass of 56 kDa. A mathematical
summation of elution profiles obtained for the 3D mutant
of Hsp27 and the wild-type Hsp20 is presented on curve 1 of
Fig. 9B. Only one broad asymmetric peak, with an apparent
molecular mass of 100 kDa, was observed if, immediately
after mixing, the two proteins were loaded onto the column
(Fig. 9B, curve 2). The position and shape of this peak were
different from the sum of the two elution profiles obtained
for the isolated 3D mutant of Hsp27 and wild-type Hsp20
(compare curves 1 and 2 on Fig. 9B). If the mixture of the
Fig. 7. Effect of Hsp20, its S16D mutant and
a-crystallin on the heat-induced aggregation of
yeast alcohol dehydrogenase (ADH) at pH 7.0.
Aggregation of ADH (0.26 mgÆmL
)1

)(1),orADH
in the presence of either a-crystallin
(0.13 mgÆmL
)1
) (2) or wild-type Hsp20
(0.13 mgÆmL
)1
) (3). The percentage of ADH
in the pellet is plotted against the time of
incubation. (F) Co-precipitation of a-crystal-
lin (1) or wild-type Hsp20 (2) with heat
denatured ADH. The percentage of small heat
shock protein in the pellet is plotted against
the time of incubation. Lack of precipitation
of isolated small heat shock proteins is shown
on curve 3.
298 O. V. Bukach et al. (Eur. J. Biochem. 271) Ó FEBS 2003
3D mutant of Hsp27 and wild-type Hsp20 (or S16D mutant
of Hsp20) were incubated for 3 h at 30 °C, only one peak
with an apparent molecular mass 95 kDa was detected on
the chromatogram. Thus, homo-oligomers formed by the
3D mutant of Hsp27 and wild-type Hsp20 (or its S16D
mutant) rapidly rearrange, forming hetero-oligomeric
complexes.
Discussion
To our knowledge there are only two publications that
report a detailed investigation of the biochemical properties
of isolated Hsp20. Kato et al. [9] reported that Hsp20 is
presented in so-called aggregated and dissociated forms
with apparent molecular masses of 200–300 and 67 kDa,

present study human Hsp20 was used. Although rat and
human Hsp20 are highly homologous ( 90% identity of
the primary structure), the rat Hsp20 consists of 162
residues, whereas the human protein consists of 160 residues
and the dipeptide deletion is located at the very C-terminal
end (residues 154–155 of rat Hsp20). It is known that the
C-terminal extension affects the oligomerization and chap-
erone action of Hsp27 [37]. Therefore, we propose that the
difference in the C-terminal extension of human and rat
Hsp20 results in a different oligomeric state of these two
proteins. However, this suggestion is speculative and needs
experimental verification. Finally, as previously mentioned,
when expressing the S16D mutant we found that truncation
of 30–50 C-terminal amino acid residues results in the
formation of protein aggregates that were soluble only in
the presence of a high concentration of urea. Previously it
has been shown that the truncation of a short C-terminal
Fig. 8. Influence of Hsp20, its S16D mutant
and a-crystallin on the heat-induced aggrega-
tion of yeast alcohol dehydrogenase (ADH) at
pH 6.0. Aggregation of ADH (0.15 mgÆmL
)1
)
was measured either by light scattering (A–D)
or by centrifugation (E–F). A–C, ADH alone
(1), or ADH in the presence of 0.015 mgÆmL
)1
(2), 0.03 mgÆmL
)1
(3), 0.075 mgÆmL

age of ADH in the pellet is plotted against the
time of incubation. (F) Co-precipitation of
wild-type Hsp20 (1) or a-crystallin (2) with
heat denatured ADH. The percentage of small
heat shock proteins in the pellet is plotted
against the time of incubation. Lack of preci-
pitation of isolated small heat shock proteins
isshownoncurve3.
Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur. J. Biochem. 271) 299
peptide increases the hydrophobicity of Hsp27 and decrea-
ses its chaperone effect [37]. There were no signs of
proteolytic degradation in the samples of Hsp20 purified
by Kato et al. [9] and van de Klundert et al. [15]; however,
truncation of a short (2–4 kDa) fragment can be easily
overlooked. Therefore, we suggest that during expression
and/or purification, Hsp20 can undergo limited proteolysis,
and deletion of a short C-terminal fragment may result in
the formation of a mixture of small and large aggregates
that were reported in the previous publications.
Van de Klundert et al. [15] claimed that Hsp20 is a poor
chaperone. In our investigation we found that at neutral or
slightly alkaline pH, Hsp20 has comparable or even higher
chaperone activity than commercial a-crystallin (Figs 6 and
7). sHsp protect the cell against unfavorable conditions,
among them acidosis. For instance, the data of Wang [38]
indicate that a-crystallin prevents acidification-induced
aggregation of creatine kinase and luciferase. In our study,
at pH 6.0, a-crystallin partially prevented the aggregation of
yeast ADH, whereas the wild-type Hsp20 retained its ability
to interact with denatured substrates, but, instead of

high molecular mass peaks. Kato et al. [9] detected two
peaks with apparent molecular masses 200–300 and
68 kDa, whereas Pipkin et al. [19] detected only one peak
with an apparent molecular mass of 230 kDa. Brophy et al.
[17] postulated that cAMP-dependent phosphorylation
results in the change of macromolecular associations of
Hsp20. Finally, Hsp20 is usually copurified with Hsp27 and
a-crystallin [9,19]. Thus, all these data indirectly indicate the
formation of mixed oligomer complexes between Hsp20
and a-crystallin or Hsp27. To verify this, we analyzed the
chromatographic behavior of the mixture of Hsp20 and
Hsp27. In good agreement with Bova et al. [41], we found
that at low temperature the rate of subunit exchange
between the wild-type Hsp20 and Hsp27 was very slow.
However, at 30 or 37 °C the rate of exchange was
significantly increased and we detected two hetero-oligo-
meric complexes with apparent molecular masses 100 and
300 kDa that contained similar quantities of Hsp20 and
Hsp27 subunits (Fig. 9A). Mutation S16D, imitating phos-
phorylation of the Ser16 of Hsp20, had no significant effect
on the rate of subunits exchange or on the composition or
Fig. 9. Formation of hetero-oligomeric complexes between Hsp27 and
Hsp20. (A) Rearrangement of the complexes formed by the wild-type
Hsp27 and Hsp20. The wild-type Hsp27 and Hsp20 were loaded onto
the column immediately after mixing (1) or were incubated at 30 °C(2)
or at 37 °C (3) for 3 h. For clarity the profiles are shifted from each
other by 40 mAu. The protein composition of profile 3 fractions 25–33
is shown on the insert. The positions of Hsp27 and Hsp20 are marked
by arrows. (B) Rearrangement of the complexes formed by the 3D
mutant of Hsp27 and the wild-type Hsp20. A mathematical summa-

and a high concentration of Hsp20 and Hsp27 in certain
tissues [8,9], we may suppose that hetero-oligomeric com-
plexes are also formed in vivo. This will lead to a decrease in
the concentration of homo-oligomeric sHsp, affect oligomer
structure and result in the accumulation of hetero-oligo-
meric complexes with properties that might be different from
homo-oligomers. Isolation and detailed characterization of
hetero-oligomeric complexes will provide new, important
information on the functioning of sHsp in the cell.
Acknowledgements
The authors are grateful to Dr A. M. Arutunyan (Institute of Physico-
chemical Biology, Moscow State University) for his help in CD
measurements. This investigation was supported by the Russian
Foundation for Basic Research and by the Wellcome Trust.
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