Báo cáo khoa học: Effect of mutations in the b5–b7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11) pptx

Effect of mutations in the b5–b7 loop on the structure
and properties of human small heat shock protein HSP22
(HspB8, H11)
Alexei S. Kasakov
1,
*, 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, Russia
2 National Heart and Lung Institute, Imperial College London, UK
Small heat shock proteins (sHsp) form a large super-
family of ubiquitous proteins detected in all organ-
isms, except for some bacteria [1–3]. The members
of this family range in size from 12–42 kDa and
contain a conservative so-called a-crystallin domain
consisting of 80–100 residues that is located in the
C-terminal part of the polypeptide chain [1–3]. This
conservative domain is flanked by the N-terminal
domain and short C-terminal extension with a differ-
ent size and structure [4,5]. All sHsp tend to form
flexible oligomers, ranging from a dimer to more
than 40 subunits, exchanging their subunits [6,7], and
some sHsp are able to form mixed oligomers consist-
ing of subunits of different natures [8,9]. Crystal
Keywords

chaperone-like activity; intrinsically
disordered regions; oligomeric structure;
small heat shock proteins
Correspondence
N. B. Gusev, Department of Biochemistry,
School of Biology, Moscow State
University, Moscow 119991, Russia
Fax ⁄ Tel: +7 495 939 2747
E-mail:
*These authors contributed equally to this
work
(Received 17 June 2007, revised 30 July
2007, accepted 3 September 2007)
doi:10.1111/j.1742-4658.2007.06086.x
The human genome encodes ten different small heat shock proteins, each
of which contains the so-called a-crystallin domain consisting of 80–
100 residues and located in the C-terminal part of the molecule. The
a-crystallin domain consists of six or seven b-strands connected by different
size loops and combined in two b-sheets. Mutations in the loop connecting
the b5 and b7 strands and conservative residues of b7inaA-, aB-crystallin
and HSP27 correlate with the development of different congenital diseases.
To understand the role of this part of molecule in the structure and func-
tion of small heat shock proteins, we mutated two highly conservative resi-
dues (K137 and K141) of human HSP22 and investigated the properties of
the K137E and K137,141E mutants. These mutations lead to a decrease in
intrinsic Trp fluorescence and the double mutation decreased fluorescence
resonance energy transfer from Trp to bis-ANS bound to HSP22. Muta-
tions K137E and especially K137,141E lead to an increase in unordered
structure in HSP22 and increased susceptibility to trypsinolysis. Both
mutations decreased the probability of dissociation of small oligomers of

HSP22, and mutation K137E increased the probability of HSP22 crosslink-
ing. The wild-type HSP22 possessed higher chaperone-like activity than
their mutants when insulin or rhodanase were used as the model substrates.
Because conservative Lys residues located in the b5–b7 loop and in the b7
strand appear to play an important role in the structure and properties of
HSP22, mutations in this part of the small heat shock protein molecule
might have a deleterious effect and often correlate with the development of
different congenital diseases.
Abbreviations
bis-ANS, 4,4¢-bis(1-anilinonaphtalene-8-sulfonate); DMS, dimethylsuberimidate; FRET, fluorescent resonance energy transfer; GuCl,
guanidinium chloride; sHSP, small heat shock protein.
5628 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
structures are described in the literature for the
hyperthermophile Methanococcus jannaschii Hsp16.5
[10] and wheat (Triticum aestivum) Hsp16.9 [11], each
containing a single a-crystallin domain, and the par-
asitic flatworm Taenia saginata Tsp36, containing
two a-crystallin domains in the single polypeptide
chain [12].
Ten different sHsp are encoded in the human gen-
ome and are differently expressed in human tissues
[13,14]. None of these proteins has been crystallized;
however, different experimental approaches (cryo-
electron microscopy, electron spin resonance spectros-
copy, protein pin array, etc.) [15–17] and protein
modeling were used to reconstruct the structure of
mammalian aB-crystallin and Hsp27 (HspB1) [16–
18]. According to these models, the a-crystallin
domain of both proteins consists of seven b-strands
packed into two b-sheets [16–18]. The loop connect-

ing b5 and b7 and the N-terminal part of b7
appears to play an important role in the structure of
sHsp monomers [12,18] and intermonomer interac-
tions [12,18,19], as well as in the binding of protein
substrates to sHsp [17]. The importance of this part
of molecule of the sHsp is supported by the fact
that mutations in the loop connecting the b5 and b7
strands, or in the b7 strand of sHsp, often correlate
with the development of certain congenital diseases
(congenital cataract, desmin related myopathy, distal
hereditary motor neuropathy, amongst others)
[20,21].
A recently described protein with an apparent
molecular mass of 22 kDa, denoted as HSP22,
HspB8 or H11 kinase, shares structural properties
typical to all members of the family of sHsp [22].
HSP22 possesses chaperone-like activity [23–25] and
appears to be involved in the regulation of many
processes such as proliferation, myocardium hyper-
trophy and apoptosis [26]. Missense mutations of
K141 (K141E, K141N) located at the beginning
of b7 of HSP22 correlate with the development of
motor neuropathy and Charcot–Marie–Tooth disease
[27,28]. Another conservative residue of HSP22,
namely K137, presumably located in the b5–b7 loop,
is homologous to R136 of human HSP27 that is
mutated in the case of Charcot–Marie–Tooth type 2
disease [20,21]. Previously, we compared the structure
and properties of the wild-type HSP22 and its
K141E mutant [29]. The present study analyses the

structure and properties of K137E and the
K137,141E mutant of human HSP22, aiming to pro-
vide new information on the structure of sHsp and
to shed new light on their role in the development
of human congenital diseases.
Results
Peculiarities of HSP22 structure
Up to now, all attempts to crystallize mammalian
sHsp have been unsuccessful. Therefore, all structural
information derives from a comparison of human sHsp
with the crystal structures of M. jannaschii Hsp16.5
[10] and T. aestivum Hsp16.9 [11]. The 3D structure of
the monomer of T. aestivum Hsp16.9 is presented in
Fig. 1A (protein databank accession code 1GME) and,
as shown in Fig. 1B, we aligned the structures of
M. jannaschii Hsp16.5 and T. aestivum Hsp16.9 with
the corresponding structures of three human sHsp [30].
The elements of the secondary structure of M. janna-
schii Hsp16.5 and T. aestivum Hsp16.9, as determined
by X-ray crystallography, are indicated by solid blue
(a-helices) or solid red (b-strands) lines above and
below the corresponding sequences (Fig. 1A). Both
these proteins contain a large number of well preserved
b-strands that are predominantly (with the exception
of the b10 strand) located in the a-crystallin domain
[10–12].
The models built for two mammalian sHsp (aB-crys-
tallin [16] and HSP27 [18]) predict that both these pro-
teins contain short a-helices in the N-terminal part of
molecule (dashed blue lines denoted a1–a3 above the

aB-crystallin and below the HSP27 sequences in
Fig. 1B). According to these models, both aB-crystallin
and HSP27 contain seven b-strands (b2–b9) (dashed
red lines) located in positions homologous to the cor-
responding strands of two crystallized nonmetazoan
sHsp. Two predictions slightly differ with respect to
the location and length of specific b-strands. For
example, in the model of aB-crystallin, the b7 strand is
only four residues long [16] whereas, in the model of
HSP27, the same strand is ten residues long [18]. How-
ever, the overall structures of aB-crystallin and HSP27
predicted by these two models are very similar, and
the positions of the b-strands correlate well with the
corresponding positions of the b-strands in M. janna-
schii Hsp16.5 and T. aestivum Hsp16.9 (Fig. 1B).
Predictions of the secondary structure of HSP22 per-
formed with the jpred program (bio.
dundee.ac.uk) indicate that this protein contains very
small quantities of a-helices and is enriched in unor-
dered structure and b-strands. The residues of HSP22
that are predicted to form b-strands are indicated by
wide dashed red lines in Fig. 1B and are located in
positions corresponding to the b3, b
4, b5, b7 and b9
strands. jpred failed to predict the formation of a b2
strand in the HSP22 structure. According to this
prediction, residues 153–155 of HSP22 tend to form an
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5629
A

B
Fig. 1. Comparison of the structure of human HSP22 and other sHsp. (A) Ribbon diagram of T. aestivum Hsp16.9 monomer (protein data-
bank accession code 1GME). The N- and C-terminal domains are indicated by N and C correspondingly. All b-strands are numbered and
the b5 and b7 strands are shown in red and blue, respectively. G104 (equivalent to K137 of human HSP22) and R108 (equivalent to K141
of human HSP22) are shown in purple and grey, respectively. (B) Alignment of human HSP22 with human aB-crystallin and HSP27 and
M. jannaschii Hsp16.5 and T. aestivum Hsp16.9 made with
CLUSTALW [30] using the default settings. The residues shown in black are
identical in at least four sequences; residues in dark grey are conservative in at least four or identical in at least three sequences; resi-
dues in light grey are homologous at three or identical in at least two sequences. Solid blue and red lines above M. jannaschii Hsp16.5
and below T. aestivum Hsp16.9 sequences indicate a-helices and b-strands detected in the crystal structure of the corresponding proteins
[10,11]. Dashed blue and red lines above human aB-crystallin and below human HSP27 sequences indicate a-helices and b-strands pre-
dicted in the models of the corresponding proteins [16,18]. Residues of HSP22 predicted to form b-strands according to
JPRED are indi-
cated by wide dashed red lines and K137 and K141 are shown in red. Numbers in parenthesis correspond to NCBI-Entrez-Protein
database accession numbers.
Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.
5630 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
a-helix, whereas residues 156 and 157 tend to form a
very short b-strand that might correspond to the b8
strand of the other sHsp.
The primary structure of the a-crystallin domain of
human sHsp is very conservative and the loop connect-
ing the b5 and b7 strands is shorter than the corre-
sponding loop connecting the b5 and b7 strands of
nonmetazoan sHsp (Fig. 1B). Moreover, the structure
of human sHsp lacks the b6 strand that is involved in
dimer formation of nonmetazoan sHsp (Fig. 1A).
Although the b5–b7 loop is very short, it is not com-
pletely deleted in any human sHsp. This part of the
molecule has a very conservative primary structure and

appears to play a diverse and important role. For
example, mutation of a highly conservative positively
charged residue (R116 of aA-crystallin, R120 of aB-
crystallin or K141 of HSP22 located in homologous
position; Fig. 1B) correlates with the development of
congenital cataract and ⁄ or desmin related myopathy
[20,21], whereas mutations of R127, S135 and R136 of
human HSP27 are associated with distal hereditary
motor neuropathy and Charcot–Marie–Tooth disease
[20,21]. Therefore, it is advisable to analyze the effect
of a mutation in this part of the molecule on the struc-
ture and properties of human sHsp.
Oligomeric structure of HSP22 and its mutants
All samples of recombinant HSP22 and its mutants
purified by the method described previously [23] were
homogeneous according to SDS gel electrophoresis
(Fig. 2). HSP22 and its mutants are highly susceptible
to proteolysis [23,24,29] and occasionally contained
small quantities of proteolytic fragments. Under the
conditions used, the wild-type HSP22 and its K137E
and K141E mutants migrated on the SDS gel electro-
phoresis [31] as a band with an apparent molecular
mass of 25.4 kDa, whereas the apparent molecular
mass of the double mutant K137,141E was 30.4 kDa.
The calculated molecular mass of human wild-type
HSP22 is close to 21.6 kDa [22]. The unusually high
apparent molecular mass determined by SDS gel elec-
trophoresis can be due to anomalous binding of SDS
to acidic HSP22 and this effect is especially pro-
nounced in the case of the particularly acidic double

mutant K137,141E of HSP22. On native gel electro-
phoresis performed both at neutral [32] and alkaline
pH [33], the wild-type HSP22 and its mutants migrated
as a single band with an apparent molecular mass of
approximately 60 kDa (data not shown), thus indicat-
ing that, under these conditions, HSP22 and its
mutants form small oligomers.
Size-exclusion chromatography was used for further
investigation of the quaternary structure of HSP22 and
its mutants. When 200 lg of the wild-type HSP22 was
loaded on the column, a single peak was detected with
a Stokes radius equal to 26.2 A
˚
, corresponding to an
apparent molecular mass of 36.1 kDa (Fig. 3A). These
data agree well with the previously published data
[23,24,29]. On size-exclusion chromatography, both
K141E and K137,141E were eluted as symmetrical
peaks and the width at the respective half-height of
their peaks was similar to that of the wild-type HSP22.
The Stokes radii and apparent molecular masses of the
K141E and K137,141E mutants were similar: 26.7 A
˚
and 37.9 kDa (Fig. 3A) [29]. At the same time, the
K137E mutant of HSP22 was eluted as a broad peak
with a trailing end, with a Stokes radius and apparent
molecular mass of 28.2 A
˚
and 43.9 kDa, respectively
(Fig. 3A). Taking into account that the molecular mass

of HSP22 monomer is 21.6 kDa [22], it might be
assumed that, under conditions of size-exclusion chro-
matography, HSP22 and its mutants are either highly
asymmetric (or intrinsically unfolded) or presented in
the form of a mixture of monomers and dimers. The
data presented indicate that mutations in the b5–b7
loop (and especially K137E) affect either folding or
extension of oligomerization of HSP22.
To test this suggestion, we performed size-exclusion
chromatography on the Superdex 200 HR10 ⁄ 30 col-
umn in the presence of 6 m guanidinium chloride
(GuCl) and, under these conditions, calibrated the col-
umn with a set of protein standards (BSA, ovalbumin,
chymotrypsin A and RNAse) [34] (Fig. 3B). Under
denaturating conditions, all samples of HSP22 were
Fig. 2. SDS electrophoresis of the wild-type HSP22 (1) and its
K137E (2), K141E (3) and K137,141E (4) mutants. The positions of
the molecular mass standards (in kDa) are indicated by arrows.
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5631
eluted in the form of symmetrical peaks with an appar-
ent molecular mass of 22.8 kDa, which is close to the
calculated value of the HSP22 monomer (21.6 kDa).
The data presented agree with the suggestion that,
under native conditions, HSP22 and its mutants form
dimers that dissociate to monomers in the presence of
6 m GuCl.
If this suggestion is correct, we might assume that a
decrease in protein concentration will result in the dis-
sociation of small HSP22 oligomers and the formation

of protein species with smaller apparent molecular
mass. Indeed, if the quantity of the wild-type HSP22
loaded on the column was decreased from 200 lgto
10 lg, the elution volume of the protein peak was
increased from approximately 11.3 mL to 11.8 mL
(Fig. 3C). This increase in elution volume corresponds
to a decrease in the apparent molecular mass from
approximately 36.9 kDa to 29.3 kDa. A similar
decrease in the apparent molecular mass was observed
for the K137,141E mutant of HSP22; however, at all
concentrations, the apparent molecular mass of this
mutant was slightly larger than the molecular mass of
the wild-type protein (Fig. 3C). At high concentration,
the K137E mutant formed oligomers with an apparent
molecular mass of approximately 44 kDa whereas, at
very low concentration, the molecular mass of oligo-
mers formed by this mutant was close to 32 kDa
(Fig. 1C). The data presented mean that mutations of
K137 and K141 might affect either folding or dissocia-
tion of HSP22 oligomers.
There are many examples indicating that certain
point mutations do not dramatically affect the quater-
nary structure but, at the same time, induce destabili-
zation of the overall structure of the sHsp [35,36].
Therefore, we analyzed the effect of point mutations in
the linker connecting the b5 and b7 strands of HSP22
on its thermal stability. The wild-type protein or its
mutants were heated for 30 min at 70 °C and, after
Fig. 3. Size-exclusion chromatography of the wild-type HPS22 and
its point mutants. (A) Size-exclusion chromatography of the wild-

type HSP22 (1, 2) and its K137E (3, 4) and K137,141E (5, 6)
mutants on Superdex 75 column under native conditions. The sam-
ples were either kept on ice (solid curves 1, 3, 5) or heated for
30 min at 70 °C (dashed curves 2, 4, 6). Equal volumes (150 lL) of
each protein (210 lg) were subjected to chromatography on a Su-
perdex 75 HR10 ⁄ 30 column. For clarity, elution profiles of unheated
and heated proteins are shifted from each other by 10 mAu and
elution profiles between different proteins are shifted from each
other by 30 mAu. Arrows above the panel indicate the elution vol-
ume of protein standards and their apparent molecular masses. (B)
Size-exclusion chromatography of the wild-type HSP22 (1) and its
K137E (2), K141E (3) and K137,141E (4) mutants on the Super-
dex 200 HR10 ⁄ 30 column in the presence of 6
M GuCl. Equal
volumes (150 lL) of each protein (150 lg) were subjected to
chromatography. For clarity, elution profiles are shifted from each
other by 20 mAu. Arrows above the panel indicate the elution vol-
ume of protein standards and their apparent molecular masses. (C)
Dependence of elution volume on the quantity of protein loaded on
a Superdex 75 HR10 ⁄ 30 column. Equal volumes (150 lL) contain-
ing 10–200 lg of the wild-type protein (1) and its K137E (2) or
K137,141E (3) mutants were subjected to chromatography under
native conditions. The data are representative of three independent
experiments.
Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.
5632 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
cooling for 20 min and centrifugation, were subjected
to size-exclusion chromatography (Fig. 3A). Prolonged
heating at 70 °C did not affect the elution profile of
any of the proteins analyzed. The amplitude, position

and the width of the protein peaks were not dependent
on the transient heating. These data suggest that the
wild-type HSP22 and its mutants belong to the group
of the so-called intrinsically disordered proteins with
long stretches of unordered structure [37] and this
is one of the reasons for their unusual high thermal
stability.
To further investigate the oligomeric structure of
HSP22, we employed chemical crosslinking. HSP22
and its mutants at three different concentrations (0.1,
0.5 and 2.0 mgÆmL
)1
) were incubated in the presence
of 3.5 mm dimethylsuberimidate (DMS) for 1 h at
37 °C and the protein composition of the sample thus
obtained was analyzed by means of SDS gel electro-
phoresis. In good agreement with the previously pub-
lished data [23,29], we found that incubation of the
wild-type HSP22 with the bifunctional reagent resulted
in the formation of an additional protein band with an
apparent molecular mass of 50 kDa, which presumably
corresponds to the HSP22 dimer (Fig. 4A). Similar
results were observed in the case of the K137E mutant
of HSP22 (Fig. 4B); however, in this case, the intensity
of the band corresponding to the HSP22 dimer was
more intense than in the case of the wild-type protein.
Thus, although mutation K137E eliminates one poten-
tial site of chemical modification, the probability of
crosslinking of the K137E mutant by DMS is higher
than the probability of crosslinking of the wild-type

protein. This fact agrees well with the size-exclusion
chromatography data indicating that the K137E
mutant forms larger oligomers than the wild-type pro-
tein (Fig. 3C). If the double mutant K137,141E was
subjected to crosslinking, we detected only a very faint
band corresponding to dimer and this band was
detected only at a rather high protein concentration
(Fig. 4C). The decreased probability of crosslinking of
the K137,141E mutant might be due to replacements
of Lys residues being potential sites of crosslinking or,
more likely, to the overall changes in the structure of
HSP22 that are induced by replacing two closely sepa-
rated positively charged Lys residues by negatively
charged Glu (see below).
Effect of K137E and K137,141E mutations on the
structure of HSP22
The data presented might indicate that the analyzed
mutations affect the secondary and tertiary structure
of HSP22. To check this suggestion, we analyzed some
spectral properties of the wild-type protein and its two
mutants.
The maximum of intrinsic Trp fluorescence of the
wild-type HSP22 was located at 342 nm and the posi-
tion of this maximum was not changed by mutations
K137E or K137,141E (Fig. 5). Similar results were
obtained previously with the K141E mutant of HSP22
[29]. The fluorescence spectrum of HSP22 was decom-
posed into discrete components characteristic of Trp
located in different environments [38]. For this
A

B
C
Fig. 4. Crosslinking of the wild-type HSP22 (A) and its K137E (B)
and K137,141E (C) mutants by DMS. HSP22 was incubated either
in the absence of DMS (0), or in the presence of 3.5 m
M of DMS
(1–3). The protein concentration was equal to 0.10 (1), 0.50 (2) or
2.0 (3) mgÆmL
)1
and, after incubation, equal quantities (2.5 lg) of
protein were loaded onto the gel. The positions of the molecular
mass standards (in kDa) are indicated by arrows on the right.
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5633
purpose, the fluorescence spectra were fitted as a sum
of three polynomial distributions of the fourth of fifth
order, corresponding to three classes of Trp residues
differing in their environment, accessibility to solvent
and position of the fluorescent spectrum. Using this
approach, we estimated the portion of each class of
fluorophores in the protein spectrum and found that
HSP22 contains Trp residues belonging to the so-called
classes I, II and III. Class I corresponds to indole
located inside the protein globule, forming a 2 : 1 exci-
plex with neighboring polar groups and having maxi-
mum fluorescence at 330–332 nm. Class II corresponds
to Trp at the protein surface in contact with bound
water molecules (maximum fluorescence at 340–
342 nm). Finally, class III corresponds to indole
located at the protein surface in contact with free

water molecules (maximum fluorescence at 350–
355 nm). Approximately 44% of Trp residues of
HSP22 belong to class I, approximately 18% belong to
class II and approximately 38% belong to class III.
Point mutations K137E or K137,141E do not signifi-
cantly affect the distribution of Trp residues between
these classes (data not shown). This may be due to
the fact that three out of four Trp residues are located
in the N-terminal end (Trp48, Trp51, Trp60) and the
fourth Trp residue (Trp96) are located at the very
beginning of the a-crystallin domain, far apart from
the mutated Lys residues. Although the point muta-
tions do not affect the position of maximum
fluorescence, they slightly decrease the amplitude of
fluorescence and this decrease was more pronounced
for the K141E [29] and K137,141E mutants than for
the K137E mutant (Fig. 5). The small decrease in
the amplitude of fluorescence detected for the point
mutants of HSP22 might reflect small changes in
structure, leading to an altered Trp environment or
their accessibility to quencher or water molecules.
Hydrophobic interactions appear to play an impor-
tant role in oligomer formation and in the interaction
of sHsp with their protein substrates [1,10–12]. Hydro-
phobic surfaces of HSP22 and its mutants were probed
by using bis-ANS. In the isolated state, this hydropho-
bic probe has a very low quantum yield that is dramat-
ically increased after its binding to hydrophobic sites
on the protein molecules [24,29]. Titration of HSP22
with bis-ANS was accompanied by an increase in fluo-

rescence at 495 nm, indicating binding of the fluores-
cence probe to the protein [24,29]. In agreement with
the previously published data [29], we were unable to
achieve saturation and, in the range of 0–10 lm bis-
ANS, the fluorescence at 495 nm was approximately
proportional to the concentration of the fluorescent
probe added. These data indicate that HSP22 contains
many low affinity bis-ANS binding sites that cannot
be completely saturated in the range of bis-ANS con-
centrations used. This is to be expected if HSP22
belongs to the group of intrinsically disordered pro-
teins lacking well-organized hydrophobic sites. To
obtain more information on the structure, we analyzed
fluorescence resonance energy transfer (FRET) from
Trp residues of HSP22 and its mutants to the bound
bis-ANS. As indicated in Fig. 6, titration of the wild-
type HSP22 and its K137,141E mutant with bis-ANS
was accompanied by a decrease in intrinsic Trp fluo-
rescence at 342 nm and a concomitant increase in the
fluorescence of bis-ANS at 495 nm. Because, at any
bis-ANS concentration, the ratio of fluorescence at 342
to fluorescence at 495 nm (F
342
⁄ F
395
) was lower for the
wild-type protein than for its K137,141E mutant, we
conclude that the probability of FRET is higher for
the wild-type protein than for its mutant. This may
indicate that the mutation K137,141E affects the

mutual orientation, overall flexibility and ⁄ or distances
between Trp and bis-ANS bound to HSP22.
To obtain more detailed information on the struc-
ture of HSP22 mutants, we employed CD spectros-
copy. The far-UV CD spectra of the wild-type protein
has a negative maximum at 208 nm and its molar ellip-
ticity at this wavelength is rather low (Fig. 7). This
spectrum is characteristic for proteins with a low
a-helix content and a high content of unordered and
b-structures. Mutation K137E had no dramatic effect
on the far-UV CD spectra and a blue shift of only 2–
3 nm was observed in the position of the negative
maximum (Fig. 7). Previously, we have found that
mutation K141E induces a rather large increase in the
amplitude of the negative maximum on the far-UV
CD spectrum of HSP22 [29]. Even larger changes were
Fig. 5. Intrinsic Trp fluorescence of the wild-type HSP22 (1) and its
K137E (2) and K137,141E (3) mutants. Fluorescence was excited at
295 nm. The protein concentration was 0.1 mgÆmL
)1
.
Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.
5634 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
observed in the case of the double K137,141E mutant.
Indeed, the double mutation results in a blue shift of
5–6 nm with respect to the position of negative maxi-
mum and a significant increase in the amplitude of this
maximum. This change of the far-UV CD spectra can
reflect pronounced changes in the secondary structure.
Using the approach developed by Sreerama and

Woody [39], we attempted to estimate the changes
induced by the point mutations in the secondary struc-
ture of HSP22.
According to this estimation, the a-helix content is
equally low (approximately 5–6%) in the structure of
both the wild-type HSP22 and its two mutants. As
expected, the secondary structure of HSP22 and its
mutants was characterized by a high content of
b-strands (approximately 31–37%) and turns and
unordered structures (approximately 58–63%). Muta-
tion K137E induced only very moderate changes in the
secondary structure. At the same time, mutation
K141E [29] and especially double mutation K137,141E
were accompanied by a simultaneous decrease in the
content of b-structure (from 37% to 31%) and an
increase in the content of turns and unordered structure
(from 58% to 63%). These data might indicate that
mutations in the b5–b7 loop and in the N-terminal part
of the b7 strands destabilize the structure of HSP22.
Limited trypsinolysis of the wild-type HSP22 and
its K137E and K137,141E mutants
The method of limited trypsinolysis was used to check
the suggestion that the analyzed mutations affect the
stability of HSP22. The available literature [23,24,29]
indicate that HSP22 is highly susceptible to proteoly-
sis. Indeed, even at a weight ratio for HSP22 ⁄ trypsin
equal to 12 000 : 1, the sHsp was rapidly hydrolyzed
(Fig. 8A). Trypsinolysis of the wild-type HSP22 was
accompanied by disappearance of the band corre-
sponding to intact protein that migrated with an

apparent molecular mass of 25.4 kDa and accumula-
tion of peptides with apparent molecular masses equal
to 16.5, 18.0, 19.0, 22.0 and 23 kDa, respectively
(Fig. 8A). The same set of peptides was observed if
K137E and K137,141E mutants were subjected to
trypsinolysis. To compare the apparent rates of tryp-
sinolysis of the wild-type HSP22 and its mutants, we
plotted ln(A
t
⁄ A
o
) (where A
o
and A
t
are the intensities
of the band of intact protein at the beginning of tryp-
sinolysis and at the fixed time of trypsinolysis) against
the time of incubation (Fig. 8D). The apparent rate
constants of trypsinolysis under these conditions were
equal to 0.0496 ± 0.0027, 0.068 ± 0.026, 0.0863 ±
0.0039Æmin
)1
(n ¼ 7) for the wild-type HSP22, and its
K137E and K137,141E mutants, respectively. The data
Fig. 6. Fluorescence resonance energy transfer from Trp residues of the (A) wild-type HSP22 and (B) its K137,141E mutant to the bound
bis-ANS. All experiments were performed at a protein concentration of 0.03 mgÆmL
)1
(1.5 lM of HSP22 monomer) and bis-ANS (in lM) indi-
cated above each spectrum.

Fig. 7. Far-UV CD spectra of the wild-type HPS22 (1) and its K137E
(2) and K137,141E (3) mutants. The spectra were recorded at the
concentration 0.65 mgÆmL
)1
of each species with a cell path of
0.05 cm. The spectra reported are the average of eight determina-
tions.
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5635
presented mean that K137E and especially K137,141E
mutants were more susceptible to proteolysis than the
wild-type HSP22. Mutations K137E and K137,141E
should eliminate one or two potential sites of trypsin-
olysis and, in this way, were expected to decrease the
rate of proteolysis. Instead of decreasing susceptibility,
these mutations increased the susceptibility of HSP22
to trypsinolysis and this finding agrees well with
the data of far-UV CD indicating that the ana-
lyzed mutations induce destabilization of the HSP22
structure.
Chaperone-like activity of wild-type HSP22 and
its mutants
The data presented indicate that the point mutations
of residues 137 and 141 affect the structure and sta-
bility of HSP22. Therefore, it can be expected that
these mutations might change the chaperone-like
activity of HSP22. To investigate this idea, we used
two different model protein substrates. Reduction of
the disulfide bonds of insulin results in dissociation of
its peptide chains and aggregation of chain B. Addi-

tion of the wild-type HSP22 retarded the onset of
aggregation and decreased the amplitude of light scat-
tering induced by insulin aggregation (Fig. 9A,
curves 3 and 3¢). K137E (Fig. 9A, curves 1 and 1¢)
and K137,141E (Fig. 9A, curves 2 and 2¢) also
retarded the onset of insulin aggregation and
decreased the amplitude of light scattering; however,
their effects were less pronounced than the corre-
sponding effects of the wild-type protein. For exam-
ple, the aggregation curve in the presence of
0.2 mgÆmL
)1
of K137E was comparable to the aggre-
gation curve observed in the presence of 0.1 mgÆmL
)1
of the wild-type HSP22 (compare curves 1¢ and 3 in
Fig. 9). The double mutant (K137,141E) possessed
higher chaperone-like activity than the K137E
mutant. However, both at low and high concentra-
tions, the double mutant possessed slightly lower
chaperone-like activity than the wild-type protein
(curves 2 and 3 in Fig. 9).
Heating of rhodanase at 43 °C induces its denatur-
ation, which is followed by aggregation. The wild-
type HSP22 and its mutants decreased the rate of
rhodanase aggregation and the amplitude of light
scattering (Fig. 9B). In good agreement with the data
obtained for insulin, we found that the K137E
mutant was much less effective than the wild-type
protein in preventing rhodanase aggregation (com-

pare curves 1 and 3 in Fig. 9B). The chaperone-like
activity of K137,141E mutant was lower than, but
comparable to, the chaperone activity of the wild-
type protein. Thus, on two different protein sub-
strates, the chaperone-like activity of the wild-type
HSP22 was higher than the corresponding activity of
the two mutants analyzed and, among these mutants,
the chaperone activity of the K137E mutant was
especially low.
A
B
C
D
Fig. 8. Limited trypsinolysis of the wild-type HSP22 and its K137E
and K137,141E mutants. Kinetics of trypsinolysis of the wild-type
HSP22 (A) and its K137E (B) and K137,141E (C) mutants. The time
of incubation (in min) is indicated below each track and the arrows
show the positions of molecular mass markers. (D) Determination
of apparent rate constants of trypsinolysis of the wild-type HSP22
(1, squares), K137E mutant (2, circles) and K137,141E mutant (3,
triangles). The data are representative of three independent experi-
ments.
Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.
5636 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
Discussion
All sHsp are characterized by the presence of a highly
conservative a-crystallin domain consisting of six or
seven b-strands combined in two b-sheets [1,10–12,40].
The detailed location and orientation of these
b-strands is known only for Hsp16.5 of M. jannaschii

[10], Hsp16.9 of wheat [11] and Tsp36 of T. saginata
[12] that were all obtained in crystallized form. The
tertiary structure of other sHsp (including all human
proteins) is unknown; however, several models of their
structure have been proposed in the literature [16–
18,40]. These models are based on a comparison of the
primary structure of different sHsp, predictions of
their secondary structure and on superposition of the
mammalian protein sequence on the 3D structure of
already crystallized sHsp [16–18,40,41]. The a-crystallin
domain of human sHsp lacks the b6 strand detected in
the structure of Hsp16.5 of M. jannaschii and wheat
Hsp16.9 and the loop connecting b5 and b7 is much
shorter than the corresponding loop of bacterial or
plant sHsp (Fig. 1) [10–12,40,41]. This loop appears to
play an important role in the structure and properties
of the sHsp [10–12,17,19,40,41] and mutations inside
this loop or in the b7 strand correlate with the devel-
opment of different congenital diseases [20,21,27,28].
Because, at present, only two mammalian sHsp
(a-crystallin and HSP27) have been investigated in
detail, we were interested in analyzing the role of the
b5–b7 loop in the structure of recently described
human HSP22.
The data obtained with respect to far-UV CD
(Fig. 7), extra high susceptibility to proteolysis (Fig. 8)
and resistance to thermal denaturation (Fig. 3) indicate
that HSP22 has a predominantly unordered structure.
Therefore, we might assume that HSP22 belongs to
the group of intrinsically disordered proteins. Accord-

ing to the predictions, K137 is located either in the
C-terminal part of the b5–b7 loop or in the N-terminal
part of the b7 strand, whereas K141 is located inside
the b7 strand (Fig. 1A). Predictions of disordered
regions using two different programs (http://www.
strubi.ox.ac.uk/RONN and ) indi-
cate that residues 137–141 of HSP22 are located on
the border of the unordered and ordered regions of
HSP22 in the so-called downward spike [37] (Fig. 10).
Very often, these parts of the molecules are involved in
inter- or intramolecular interactions and play an
important role in recognition and cell signaling [37].
Fig. 9. Chaperone activity of the wild-type HSP22 and its K137E and
K137,141E mutants using insulin (A) and rhodanase (B) as a model
protein substrates. (A) Reduction induced aggregation of insulin
(0.2 mgÆmL
)1
) in the absence of HSP22 (curve 0) or in the presence
of 0.1 mgÆmL
)1
(empty symbols) or 0.2 mgÆmL
)1
(filled symbols) of
HSP22 (curves 3 and 3¢), or its K137E (curve 1 and 1¢) and
K137,141E mutant (curves 2 and 2¢). (B) Heat-induced aggregation of
rhodanase (0.14 mgÆmL
)1
) in the absence of HSP22 (curve 0) or in
the presence of 0.07 mgÆmL
)1

(empty symbols) or 0.14 mgÆmL
)1
(filled symbols) of HSP22 or its mutants. Curve numbers and
symbols are same as given in (A).
Fig. 10. RONN plot of disorder probability of the wild-type HSP22
(NCBI-Entrez-Protein database accession number Q9UKS3). The
horizontal line indicates the threshold for disorder prediction. Posi-
tions of K137 and K141 are marked by black dots.
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5637
Our results indicate that mutations K137E and
K137,141E affect the structure of HSP22. We found
that the four Trp residues of HSP22 belong to three
classes that differ in their accessibility to water
(Fig. 5). Our data agree well with the recently pub-
lished data of Chowdary et al. [42] who also detected
three classes of Trp residues in rat HSP22 with a dis-
tribution of Trp residues between these three classes
which was very similar to that determined in our inves-
tigation. Three out of four Trp residues of HSP22 are
located in the N-terminal part of the molecule. This
part of the molecule is assumed to be very flexible and
appears to interact with the a-crystallin domain [18]
through hydrogen bonds formed between certain resi-
dues of the N-terminal tail and Arg (or Lys) homolo-
gous to Lys141 of HSP22 [12]. We might suppose that
mutation of K141 or closely separated K137 makes
the interaction of the N-terminal tail with the a-crys-
tallin domain less probable. This might result in the
movement of the N-terminal tail away from the hydro-

phobic cluster that is formed by hydrophobic residues
belonging to the b2, b3 and b7 strands [12,18]. If this
suggestion is correct, then three Trp residues located in
the N-terminal tail will change their environment and
their distance from one of the hydrophobic clusters of
HSP22. This might explain the decrease in the intensity
of Trp fluorescence (Fig. 5) and the decrease in fluores-
cence resonance energy transfer (Fig. 6) observed for
K137E and K137,141E mutants of HSP22.
As previously mentioned, according to predictions,
K137 and K141 are located on the border of the unor-
dered loop and the b7 strand of HSP22. Mutations
K137E and especially K137,141E lead to an increase
in the proportion of unordered structure in HSP22
(Fig. 7) and increased susceptibility to trypsinolysis
(Fig. 8). These effects can be due to the overall
changes in the flexibility of HSP22 (e.g. the above-
mentioned movement of the N-terminal end) or to
changes in the flexibility of the b5–b7 loop itself. The
data available in the literature indicate that the b5–b7
loop is involved in a-crystallin intersubunit contacts
[19]. Data obtained via size-exclusion chromatography
(Fig. 3) and chemical crosslinking (Fig. 4) indicate that
HSP22 is presented in the form of an equilibrium
mixture of monomers and dimers. Mutation K137E
decreased the probability of the dissociation of dimers
(Fig. 3) and therefore the K137E mutant probably is
more easily crosslinked with DMS than the wild-type
protein (Fig. 4). Double mutation K137,141E also
decreased the probability of dissociation of HSP22

dimers (Fig. 2). However, because the double mutant
K137,141E has a very flexible structure (Figs 7 and 8),
the probability of its crosslinking is lower than the
probability of crosslinking of the wild-type protein
(Fig. 4).
The data available in the literature indicate that the
b5–b7 loop can be involved in the interaction of the
sHsp with different unfolded target proteins [17,40].
Therefore, it is desirable to analyze the effect of
K137E and K137,141E mutations on the chaperone-
like activity of HSP22. The data shown in Fig. 9 indi-
cate that the K137E mutant possesses lower chaperone
activity than the wild-type protein. The chaperone-like
activity of the K137,141E mutant was lower than, but
comparable to, the corresponding activity of the wild-
type protein (Fig. 9). The decreased chaperone-like
activity of K137E mutant can be due to its tendency
to form more stable oligomers than the wild-type pro-
tein (Figs 3 and 4) and ⁄ or to changes in its hydropho-
bic properties. The question arises as to why the
double mutant K137,141E has a higher chaperone
activity than the single K137E mutant. We propose
that this is explained by an increased flexibility of the
K137,141E mutant (Figs 7 and 8) and by its lower
ability to form high molecular mass oligomers com-
pared with the single K137E mutant (Fig. 4). It is pos-
tulated [37,43] that both these factors can increase the
chaperone-like activity of sHsp.
In summary, we conclude that mutations in the b5–
b7 loop located on the border of an intrinsically disor-

dered region and the b7 strand affect the structure of
HSP22 and its chaperone-like activity. This explains
why mutations in this part of different sHsp (aA-, aB-
crystallin, HSP27 and HSP22) induce deleterious
effects and are associated with different congenital dis-
eases.
Experimental procedures
Cloning, expression and purification of HSP22
and its mutants
BL21-DE3 cells were transformed with the pET23b con-
struct carrying the full sequence of the wild-type HSP22 or
its mutants and cultured in LB media containing
0.1 mgÆmL
)1
of ampicillin overnight at 25 °C. Two hundred
millilitres of the overnight culture were inoculated with 2 L
of LB media containing 0.1 mgÆmL
)1
of ampicillin and cul-
tured at 37 °C until an attenuance of 0.6 at D
600 nm
was
reached. Expression of HSP22 was induced by the addition
of isopropyl thio-b-d-galactoside up to a final concentration
0.5 mm and the culture was grown for a further 4 h. The
cells were collected by centrifugation, washed with the lysis
buffer (50 mm Tris ⁄ HCl pH 8.0, 0.1 m NaCl, 1 mm EDTA,
15 mm 2-mercaptoethanol, 0.5 mm phenylmethanesulfonyl
fluoride), suspended in 30–40 mL of this buffer and frozen.
Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.

5638 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS
After thawing, lysozyme was added up to a final concentra-
tion 0.05 mgÆmL
)1
and the suspension was incubated for
30 min at 4 °C. Subsequently, magnesium chloride and
DNAse I were added to the final concentration at 5 mm
and 1 lgÆmL
)1
, respectively, and the suspension was incu-
bated for another 15 min at 4 °C. The suspension thus
obtained was sonicated and subjected to centrifugation
(22 000 g, 20 min). The supernatant was collected and the
pellet was suspended in 20 mL of lysis buffer and subjected
to sonication and centrifugation. This operation was
repeated twice more and the supernatants obtained were
combined and subjected to ammonium sulfate fractionation
(0–30% saturation). The pellet obtained was dissolved and
dialyzed overnight against buffer A (20 mm Tris-acetate
pH 8.0, containing 10 mm NaCl, 0.1 mm EDTA, 15 mm
2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluo-
ride). The dialysate was subjected to ultracentrifugation
(105 000 g, 60 min) and the supernatant was used as the
starting material for HSP22 purification. Ammonium sul-
fate was added to the supernatant up to a final concentra-
tion of 0.3 m and the sample obtained was loaded onto a
5 mL phenyl-sepharose High Trap column (Amersham
Pharmacia, Helsinki, Finland) equilibrated with buffer A,
containing 0.3 m ammonium sulfate. After washing, the
column was developed with 15 column volumes of a

descending gradient of ammonium sulfate (0.3–0.005 m).
The fractions containing HSP22 were collected and concen-
trated by ultrafiltration. The samples obtained were sub-
jected to size-exclusion chromatography on a Sephacryl
S100 (16⁄ 60) column (Amersham Pharmacia) equilibrated
with buffer A containing 150 mm NaCl. The fractions con-
taining highly purified HSP22 were concentrated by ultrafil-
tration, dialyzed against buffer B (20 mm Tris-acetate
pH 7.6, containing 10 mm NaCl, 0.1 mm EDTA, 15 mm
2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluo-
ride) and kept frozen.
Size-exclusion chromatography
Variable quantities (from 10 lg to 200 lg) of the wild-type
HSP22 or its mutants in 150 l L of buffer C1 (20 mm Tris-
acetate pH 7.6, containing 150 mm NaCl, 0.1 mm EDTA,
15 mm 2-mercaptoethanol and 0.1 mm phenylmethanesulfo-
nyl fluoride) were loaded via a 500 lL loop onto Super-
dex 75 HR10 ⁄ 30 column connected to Acta FPLC
(Amersham Pharmacia) and eluted with the same buffer.
The following protein markers (with Stokes radii and
molecular masses given in parentheses) were used for cali-
bration of the column: BSA (33 A
˚
, 68 kDa), ovalbumin
(26.8 A
˚
, 43 kDa), chymotrypsinogen A (22.54 A
˚
, 25 kDa)
and ribonuclease (17.7 A

˚
, 13.7 kDa). To analyze thermal
stability, the wild-type HSP22 and its mutants were heated
in buffer C2 (20 mm Tris-acetate pH 8.0, containing
150 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol
and 0.1 mm phenylmethanesulfonyl fluoride) at 70 °C for
30 min, cooled on ice for 20 min, centrifuged (12 000 g,
10 min) and subjected to size-exclusion chromatography at
room temperature on a Superdex 75 HR10 ⁄ 30 column
equilibrated with buffer C2. Size-exclusion chromatography
in the presence of GuCl was performed on a Superdex 200
10 ⁄ 30 column according to Mann and Fish [34]. All sam-
ples were incubated overnight in 50 mm Tris-acetate
pH 7.6, containing 6 m GuCl and 200 mm 2-mercaptoetha-
nol and subjected to chromatography on a Superdex 200
10 ⁄ 30 column in the same buffer containing 15 mm 2-mer-
captoethanol. The column was calibrated with BSA
(68 kDa), ovalbumin (43 kDa), rabbit glyceraldehyde-3-
phosphate dehydrogenase (36 kDa) and ribonuclease
(13.7 kDa) that were subjected to chromatography in the
presence of GuCl under the above-mentioned conditions.
Electrophoretic methods
SDS gel electrophoresis on homogeneous (12.5% or 15%)
or gradient (5–20%) polyacrylamide gels was performed by
the method of Laemmli [31]. Native gel electrophoresis run
on gradient (5–20%) polyacrylamide gel using buffer sys-
tems with initial pH values equal to 7.0 and 8.9 was per-
formed according to Schagger et al. [32] or Davis [33],
respectively. Ferritin (880 kDa and 440 kDa), thyroglobulin
(670 kDa), catalase (230 kDa), BSA (136 kDa and

68 kDa), ovalbumin (43 kDa) and troponin C (18 kDa)
were used as standard markers.
Chemical crosslinking
Crosslinking was performed at three different protein con-
centrations (0.10, 0.50 and 2.00 mgÆmL
)1
) and a fixed con-
centration of DMS equal to 3.5 m m. Twenty microlitres of
protein solution in 20 mm Tris-acetate pH 7.6, containing
10 mm NaCl, 0.1 mm EDTA, 0.1 mm phenylmethanesulfo-
nyl fluoride and 30 mm 2-mercaptoethanol, were mixed
with an equal volume of 7 mm DMS in 400 mm triethanol-
amine ⁄ HCl (pH 8.0) and incubated at 37 °C for 60 min.
The reaction was stopped by the addition of SDS sample
buffer and the protein composition of samples thus
obtained was analyzed by means of SDS gel electrophoresis
on gradient (5–20%) polyacrylamide gel [31].
Fluorescence spectroscopy
Intrinsic protein fluorescence was measured on an Hitachi
F3000 spectrofluorometer (Hitachi Corp. Tokyo, Japan)
and fluorescence was excited at 295 nm (slit width ¼ 5 nm)
and recorded in the range 300–400 nm (slit width ¼
1.5 nm). All measurements were performed in buffer F
(50 mm phosphate buffer, pH 7.5, containing 150 mm NaCl
and 2 mm dithiothreitol) at a protein concentration of
0.08–0.12 mgÆmL
)1
at 25 °C. The fluorescence spectra were
A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22
FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5639

decomposed into components corresponding to discrete
states of Trp that differ in their environment and accessibil-
ity for the solvent [38].
Bis-ANS was used for probing hydrophobic sites of
HSP22 and its mutants. Proteins (0.03–0.04 mgÆmL
)1
)in
buffer F were titrated with the stock solution of bis-ANS
(200 lm in buffer F) so that the final concentration of the
fluorescence probe in the sample varied in the range
0–8 lm. All experiments were performed at room tempera-
ture (25 °C). Fluorescence of bis-ANS bound to HSP22
and its mutants was excited at 385 nm (slit width ¼ 5 nm)
and recorded in the range 400–600 nm (slit width ¼ 5 nm).
To estimate FRET, intrinsic Trp fluorescence of HSP22
was excited at 295 nm (slit width ¼ 5 nm) and fluorescence
was recorded in the range 300–600 nm (slit width ¼ 5 nm).
The decrease in fluorescence at 342 nm (intrinsic Trp fluo-
rescence) and the concomitant increase in fluorescence at
495 nm (fluorescence of bis-ANS bound to HSP22) indi-
cated fluorescence resonance transfer from excited Trp to
bis-ANS.
CD spectroscopy
Far-UV CD measurements were performed on Mark V
autodichrograph in the cells with an optical path of
0.05 cm in the range 200–250 nm. HSP22 and its mutants
were dissolved in buffer F and dialyzed against the same
buffer overnight. After dialysis, the samples were subjected
to centrifugation (12 000 g, 20 min) and the supernatant
was used for the optical measurements. All proteins were

used at identical concentrations of 0.6–0.8 mgÆmL
)1
. The
data presented are the average of 8–10 accumulations. The
method of Sreerama and Woody [39] was used to estimate
the secondary structure. All calculations were performed by
using the continll program with the reference set of 12
proteins containing a high proportion of b-strand (includ-
ing Hsp16.5 of M. jannaschii) and five denatured proteins.
Limited proteolysis
HSP22 and its mutants (0.6 mgÆmL
)1
) dissolved in buffer
containing 20 mm Tris-acetate pH 7.4, 10 mm NaCl and
30 mm 2-mercaptoethanol were mixed with N-tosyl- l -phen-
ylalanine chloromethyl ketone-treated trypsin (Sigma, St
Louis, MO, USA) at a weight ratio for Hsp20 ⁄ trypsin
equal to 12 000 : 1 and incubated at 37 °C for different
times. The reaction was stopped by the addition of phen-
ylmethanesulfonyl fluoride up to a final concentration
0.2 mm and the protein composition was determined by
SDS gel electrophoresis performed on gradient (5–20%)
polyacrylamide gel. The apparent rate constant of trypsin-
olysis was determined by plotting ln(A
t
⁄ A
o
) (where A
o
and

A
t
denote the intensity of protein bands of intact unhydro-
lized protein at the beginning and at the fixed time of incu-
bation) against the time of hydrolysis.
Chaperone-like activity
The chaperone-like activity of HSP22 and its mutants was
determined by their ability to retard or prevent aggrega-
tion of model protein substrates. In the first case, insulin
(Sigma) was dissolved in 2.5% acetic acid and, after over-
night incubation, was subjected to centrifugation
(12 000 g, 20 min). The pellet was discarded and the insu-
lin concentration in supernatant (usually 5–7 mgÆmL
)1
)
was determined spectrophotometrically using A
280
¼ 1.09
for 0.1% solution. Stock solution of insulin was added to
100 mm phosphate buffer containing 100 mm NaCl so that
the final pH of the mixture became equal to 7.1 and the
insulin concentration was equal to 0.40 mgÆmL
)1
. Some
120 lL of this insulin solution was mixed with 120 lL
of buffer B containing different quantities of HSP22 so
that the final concentration of HSP22 varied from
0–0.20 mgÆmL
)1
. After incubation for 10 min at 37 °C, the

reaction was started by the addition of 20 lL of 260 mm
solution of dithiothreitol. In the second case, 150 lLof
rhodanase (Sigma) (0.28 mg mL
)1
) in 100 mm phosphate
pH 7.0 containing 100 mm NaCl were mixed with 150 lL
of buffer B containing different quantities of HSP22 or its
mutants. The final concentration of HSP22 varied in the
range 0–0.14 mgÆmL
)1
. After addition of 20 lL of 320 mm
dithiothreitol, the incubation mixture was incubated for
10 min at 37 °C and the reaction was started by rapid
heating up to 43 °C. Aggregation of insulin and rhodanase
was followed by measuring attenuance at D
360 nm
on an
Ultraspec 3100 Pro (Amersham Pharmacia) spectropho-
tometer.
Acknowledgements
The authors are grateful to Dr A. M. Arutyunyan for
his assistance with the CD spectroscopy and to
D. A. Shavochkina and A. A. Shemetov for their help
with protein purification and the measurement of
chaperone-like activity. This investigation was sup-
ported by grants from Russian Foundation for Basic
Research and the Wellcome Trust.
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