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

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-

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

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

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 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-

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

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

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

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

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-

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

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

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

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.

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

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

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

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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