Tài liệu Báo cáo khoa học: Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin doc

Interaction of the small heat shock protein with molecular mass
25 kDa (hsp25) with actin
Olesya O. Panasenko
1
, Maria V. Kim
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, Dovehose Street, London, UK
The interaction of heat shock protein with molecular mass
25 kDa (HSP25) and its point mutants S77D + S81D (2D
mutant) and S15D + S77D + S81D (3D mutant) with
intact and thermally denatured actin was analyzed by means
of fluorescence spectroscopy and ultracentrifugation. Wild
type HSP25 did not affect the polymerization of intact actin.
The HSP25 3D mutant decreased the initial rate without
affecting the maximal extent of intact actin polymerization.
G-actin heated at 40–45 °C was partially denatured, but
retained its ability to polymerize. The wild type HSP25 did
not affect polymerization of this partially denatured actin.
The 3D mutant of HSP25 increased the initial rate of poly-
merization of partially denatured actin. Heating at more
than 55 °C induced complete denaturation of G-actin.
Completely denatured G-actin cannot polymerize, but it
aggregates at increased ionic strength. HSP25 and especially

its 2D and 3D mutants effectively prevent salt-induced
aggregation of completely denatured actin. It is concluded
that the interaction of HSP25 with actin depends on the state
of both actin and HSP25. HSP25 predominantly acts as a
chaperone and preferentially interacts with thermally
unfolded actin, preventing the formation of insoluble
aggregates.
Keywords: small heat shock protein; actin; thermal
denaturation.
Actin is the major component of the thin filaments of
muscle cells and of the cytoskeleton system of nonmuscle
cells. It is therefore a very abundant protein, and its
concentration in smooth muscle is close to 800–900 l
M
[1].
Actin has a rather complex and labile tertiary structure [2,3].
Different types of stress can induce actin unfolding [4,5],
aggregation of partially folded actin [5,6] and redistribution
of actin inside the cell [7–9]. Accumulation of partially
folded or aggregated proteins can induce significant damage
to cells. This is especially important in the case of abundant
proteins, such as actin. Therefore the cell has evolved
different mechanisms to prevent the formation of insoluble
aggregates, and heat shock proteins (HSPs) play an
important role in this process.
The data in the literature indicate that the small heat
shock protein with molecular mass 25–27 kDa (HSP25)
plays an important role in actin remodeling, contractility of
different cell types and protection of the cytoskeleton under
different unfavorable conditions [7,8,10]. Miron et al.

[11,12] showed that avian HSP25 effectively inhibits actin
polymerization and prevents gelation of actin induced by
filamin and/or a-actinin. These observations were confirmed
by Benndorf et al. [13], who showed that nonphosphoryl-
ated monomers of HSP25 effectively inhibit actin
polymerization, whereas phosphorylated monomers and
nonphosphorylated multimers of HSP25 are ineffective in
the regulation of actin polymerization. The protein seg-
ments of monomeric HSP25 involved in the inhibition of
actin polymerization were determined recently [14].
Although these data are of great interest, their application
to cell physiology is questionable as under physiological
conditions HSP25 forms high molecular mass oligomers
that are in equilibrium with low molecular mass oligomers
[15,16], but practically do not dissociate to monomers. The
actin depolymerizing effect ascribed to HSP25 [11–14]
contrasts with the stabilizing of microfilaments induced by
HSP25 or its phosphorylated forms [7,17]. Moreover,
recently Butt et al. [18] have shown that under in vitro
conditions HSP25 either does not affect or even activates the
polymerization of actin.
To explain the contradictory results described in the
literature we assumed that the mode of interaction is
dependent both on the state of HSP25 and actin. In this
paper we analyze the effect of recombinant avian HSP25
and its mutants mimicking phosphorylation on the heat-
induced aggregation and polymerization of intact and
partially denatured actin.
Materials and methods
Proteins

HSP25 from chicken gizzard was purified by the procedure
described previously [19]. Chicken HSP25 was cloned,
Correspondence to N. B. Gusev, Department of Biochemistry,
School of Biology, Moscow State University,
Moscow 119992, Russia. Tel./Fax: + 7 095 939 2747,
E-mail:
Abbreviations: ANS, 8-anilinonaphtalene-1-sulfonic acid; HSP, heat
shock proteins; 1D mutant, chicken HSP25 with mutation S15D;
2D mutant, chicken HSP25 with mutation S77D + S81D;
3D mutant, chicken HSP25 with mutation S15D + S77D + S81D;
MAPKAP-2, mitogen-activated protein kinase-activated protein
kinase-2.
(Received 15 October 2002, revised 25 December 2002,
accepted 7 January 2003)
Eur. J. Biochem. 270, 892–901 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03449.x
expressed and purified as described by Bukach et al.[19],
and Panasenko et al. [20]. Three point mutants of HSP25
with replacements S15D (1D mutant), S77D + S81D (2D
mutant), and S15D + S77D + S81D (3D mutant) were
obtained by the procedures published earlier [19]. Introduc-
tion of extra negative charges in positions 15, 77 and 81
mimics phosphorylation of HSP25 by MAPKAP-2 kinase
that may occur in vivo [16]. These mutations induce changes
in the quaternary structure of avian HSP25 similar to those
evoked by the corresponding mutations in mammalian
HSP27 [16,20]. HSP25 may form oligomers of different sizes
[10,15,16], therefore for all calculations we used a molecular
mass for the monomeric proteins of 25 kDa.
Rabbit skeletal actin was purified according to Pardee
and Spudich [21]. G-actin in buffer G (5 m

M
Tris/HCl,
pH 8.2, 0.2 m
M
ATP, 0.1 m
M
CaCl
2
,0.5m
M
b-mercapto-
ethanol, 1 m
M
NaN
3
) was stored on ice and used within
10 days of purification. The purity of all proteins was
checked by SDS gel electrophoresis [22].
Thermal denaturation was achieved by incubation of
G-actin (usually 15 l
M
) in buffer G for the required period
of time at 43, 60 or 80 °C.
Limited proteolysis of intact and heated actin was
performed in buffer G at the weight ratio of actin/N-tosyl-
L
-phenylalanine chloromethyl ketone-trypsin (Sigma) equal
to 100 : 1 or 50 : 1. After incubation at 25 °C for 0.25–
20 min phenylmethanesulfonyl fluoride was added to a final
concentration of 0.5 m

M
. The samples were mixed with the
sample buffer and after boiling were subjected to SDS gel
electrophoresis [22]. After staining, the gels were scanned
and evaluated by the
ONEDSCAN
program. The intensity of
the band of unhydrolyzed actin was plotted against the time
of incubation.
Characterization of actin preparation quality
Fluorescence parameters were used for estimation of
nativity of actin preparations. Corrected spectra of actin
fluorescence excited at 297 nm were recorded in the range
300–400 nm on the Hitachi F-3000 fluorescence spectro-
photometer. Parameter A, introduced by Turoverov et al.
[23] and equal to the ratio of intensities of fluorescence at
320 and 365 nm (I
320
/I
365
) was determined for different
preparations of actin. Parameter A reflects the polarity of
the tryptophan environment. Any changes in actin structure
influence this environment and affect parameter A. Prepa-
rations of actin with A > 2.56 contain less than 2% of
inactivated actin, whereas parameter A for inactivated and
completely unfolded actin is equal to 1.3 and 0.4, respect-
ively [5].
The interaction of intact and partially folded actin with
the hydrophobic probe 8-anilinonaphtalene-1-sulfonic acid

(ANS) (Sigma) was measured at 25 °C in buffer G. Samples
containing 2.3 l
M
of intact or heated actin and 140 l
M
of
ANS were excited at 390 nm, and spectra of fluorescence
were recorded in the range 400–600 nm as before.
Actin aggregation
Light scattering, ultracentrifugation and size-exclusion
chromatography were used to follow the process of actin
aggregation. After heating in buffer G under different
conditions G-actin (10–15 l
M
) was cooled to 25 °C, and
aggregation was initiated by the addition of KCl and MgCl
2
up to 50 m
M
and 2 m
M
, respectively. The increase of ionic
strength promotes aggregation of thermally denatured actin
[5]. Aggregation was followed by light scattering, measured
at 560 nm, again using the Hitachi F-3000 fluorescence
spectrophotometer.
Ultracentrifugation was also used to follow aggregation
of partially unfolded actin. In this case G-actin (15 l
M
)in

bufferGwasheatedat60°C for 1 h. The samples were
diluted with cold buffer G, cooled to 25 °C, and mixed
either with buffer H (20 m
M
Tris/acetate pH 7.6, 10 m
M
NaCl, 0.1 m
M
EDTA, 15 m
M
2-mercaptoethanol, 10%
glycerol) or with different quantities of HSP25 in buffer H
and incubation for 20 min at 25 °C. Buffer S (50 m
M
imidazole pH 7.6, 750 m
M
KCl, 10 m
M
MgCl
2
,1m
M
ATP and 50 m
M
2-mercaptoethanol) (1/5 of the sample
volume) was added, and incubation was continued for
60 min at 25 °C. The samples obtained were then subjected
to ultracentrifugation at 100 000 g for 1 h. The protein
composition of the supernatant and pellet was determined
by quantitative SDS gel electrophoresis [22].

Size exclusion chromatography of intact and heated actin
was performed on Acta-FPLC (Amersham-Pharmacia
Biotech.) using Superdex 200 10/30 column. The column
was equilibrated and developed in buffer G. The samples
(90 lL) of intact or heated actin (15 l
M
) were loaded on the
column and eluted with buffer G at the rate of 0.5 mLÆmin
)1
.
Actin polymerization
The methods of fluorescent spectroscopy and ultracentri-
fugation were used for registration of actin polymerization.
In the first case F-actin was labeled by N-(1-pyrenyl)iodo-
acetamide according to Kouyama and Mihashi [24]. After
the removal of insoluble N-(1-pyrenyl)iodoacetamide by
low-speed centrifugation (10 min, 10 000 g), the modified
F-actin was collected by ultracentrifugation (1 h, 100 000 g).
The pellet of modified F-actin was dissolved in buffer G,
dialyzed against buffer G for 48 h and subjected to ultra-
centrifugation. The supernatant contained G-actin with an
extent of modification equal to 0.6–0.7 mol of N-(1-pyrenyl)
iodoacetamide per mole of actin. Size exclusion chroma-
tography of modified G-actin revealed that the sample does
not contain high molecular mass aggregates and is free of
fluorescent label, unattached to the protein. The sample of
modifiedG-actinwasstoredoniceandusedwithin1week
of purification. Polymerization of pyrene-labeled actin was
measured according to Pollard [25] and Miron et al.[12].
Briefly, polymerization was performed in buffer G and was

initiated by the addition of KCl and MgCl
2
up to 50 m
M
and 2 m
M
, respectively. Different quantities of actin nuclei
(short fragments of F-actin) were added simultaneously with
KCl and MgCl
2
if the initial rate of polymerization was
measured. In the series of preliminary experiments we have
shown that under the conditions used (1–4 l
M
of actin,
containing 10–15% of pyrenyl-actin) there was no self-
assembly of G-actin. We also observed proportionality of
the initial rate of polymerization to nucleus concentration.
In addition, the increase in fluorescence induced by actin
polymerization was hyperbolic in time, and that above the
critical concentration the rate of polymerization was linearly
Ó FEBS 2003 Actin and small heat shock protein (Eur. J. Biochem. 270) 893
dependent on actin concentration. According to Pollard [25]
fulfilment of all these criteria is desirable for proper
measurement of the initial rate of actin polymerization.
Polymerization of actin was performed at different actin
concentrations both in the presence and in the absence of
HSP25 or its mutant mimicking phosphorylation. In the
series of separate experiments we measured the extent of
actin polymerization. In this case, polymerization was

initiated by the addition of only KCl and MgCl
2
(50 m
M
and 2 m
M
, respectively). In this case increase in fluorescence
was sigmoidal in time and was followed for 60–90 min until
it reached its maximal value.
When ultracentrifugation was used for the measurement
of actin polymerization, G-actin (2–8 l
M
in buffer G) was
mixed with buffer H or with different quantities of HSP25
(or its mutants mimicking phosphorylation) in buffer H.
The samples were incubated for 20 min at 25 °Cand
polymerization was initiated by addition of buffer S (1/5 of
the sample volume). After mixing, the samples were
subjected to ultracentrifugation (1 h, 100 000 g), and the
protein composition of both supernatant and pellet was
determined by quantitative SDS gel electrophoresis. The
quantity of actin in the pellet was plotted against the total
quantity of actin in the sample.
Results
Heat denaturation of actin
Before starting the investigation of the HSP25–actin inter-
action it was desirable to characterize the properties of intact
and heated actin. We were mainly interested in the irrever-
sible changes in the structure of G-actin that were induced by
heating. Therefore in all experiments the samples of actin in

G-buffer were heated for 1 h at the appropriate temperature
and after cooling different properties of actin were measured
at 25 °C. In the first series of experiments we analyzed the
effect of heating on intrinsic W fluorescence of actin. After
recording corrected spectra of fluorescence, parameter A
(equal to I
320
/I
365
) was plotted against the temperature of
incubation (Fig. 1). Parameter A was 2.65 for intact actin
and decreased to 1.3 for actin heated at temperatures higher
than 55 °C. Further increase of the temperature up to 80 °C
had no effect on parameter A. The data presented agree with
the results of Kuznetsova et al. [6] and Turoverov et al.[5]
who showed that removal of Ca
2+
or addition of urea up to
4
M
results in partial unfolding of actin that is accompanied
by a decrease of parameter A from 2.5–2.6 to 1.2–1.3. Even
prolonged heating at 80 °C does not induce complete
unfolding, which is achieved only in the presence of 6–8
M
urea or 4–5
M
guanidine hydrochloride, and is characterized
by parameter A equal to 0.4 [5,6].
As already mentioned, for many proteins partial unfold-

ing is accompanied by self-aggregation. We used
size-exclusion chromatography to follow heat-induced
aggregation of actin. Under the conditions used, intact actin
was eluted as a symmetrical peak with the maximum at
8.65 mL (Fig. 2). Heating of actin for 1 h at 43, 50 or 60 °C
resulted in the appearance of an additional peak at 7.65 mL
on the elution profile. Increase of the temperature of
incubation was accompanied by the simultaneous increase
of the peak eluted at 7.65 mL and decrease of the peak eluted
at 8.65 mL. We were unable to determine the exact
molecular mass of the protein species eluted in these two
peaks because at low ionic strength of buffer G strongly
acidic actin was partially excluded from Superdex 200.
However, ovalbumin, having pI and molecular mass similar
to that of actin, was eluted as a symmetrical peak with a
maximum at 8.65 mL (data not shown). Therefore, we may
conclude that intact, unheated actin is eluted as a monomer.
Heating induces self-aggregation and the formation of actin
oligomers that are eluted from the Superdex 200 column at
7.65 mL.
Self-aggregation of actin can be due to the exposing of
hydrophobic regions upon heating. To check this suggestion
we analyzed the interaction of the hydrophobic probe ANS
with intact and heated actin (Fig. 3). Under the conditions
used, free ANS had a rather low intensity of fluorescence
with a broad maximum at 510–530 nm (Fig. 3, curve 1).
The addition of intact actin induced only a small increase in
the fluorescence at 440–500 nm (Fig. 3, curve 2). Much
Fig. 1. Effect of heating on the parameter A (I
320

/I
365
)ofactin.Actin
(15 l
M
) in buffer G was heated at different temperatures for 1 h. The
intensity of fluorescence at 320 nm (I
320
) and 365 nm (I
360
)excitedat
297 nm was used for the determination of parameter A.
Fig. 2. Size-exclusion chromatography of actin on a Superdex 200 col-
umn. Actin (15 l
M
) was kept at 4 °C (1) or heated for 1 h at 43 (2), 50
(3) or 60 °C (4) in buffer G. After cooling, 90 lLofsamplewere
loaded on the Superdex 200 column and eluted with buffer G at the
rate of 0.5 mLÆmin
)1
. For clearance the elution profiles are shifted
from each other by 8 mAu.
894 O. O. Panasenko et al.(Eur. J. Biochem. 270) Ó FEBS 2003
higher fluorescence was observed if ANS was mixed with
actin heated at 43 °C (Fig. 3, curve 3), and in this case the
maximum fluorescence was shifted to 490–510 nm.
The highest intensity of fluorescence, with a maximum at
465–475 nm, was observed when ANS was mixed with actin
heated at 60 or 80 °C (Fig. 3, curves 4,5).
The data presented indicate that heating is accompanied

by the change in the hydrophobic environment of W,
exposure of hydrophobic regions and self-aggregation of
actin. Heating at any temperature higher than 55 °C
induced similar effects on actin structure.
Actin is fairly stable to trypsinolysis. The main fragment
accumulated during incubation had an apparent molecular
mass of 33 kDa (Fig. 4A) but even after prolonged incu-
bation with trypsin more than 40% of the actin remained
uncleaved (Fig. 4B, curve 1). If actin heated at 60 °Cwas
subjected to trypsinolysis under the same conditions, the
band of actin disappeared in the first 15–30 s (Fig. 4B, curve
2) and a number of faint bands with different molecular
masses were accumulated in the incubation mixture
(Fig. 4A). These results agree with other data in the
literature [6] and indicate that heat-induced unfolding
increased the susceptibility of actin to trypsinolysis. In
contrast, if actin was heated for 1 h at 43 °C, it becomes
more resistant to trypsinolysis than intact actin (Fig. 4B,
curve 3). Three peptide bands with apparent molecular mass
of 29, 31 and 33 kDa were accumulated during the early
stages of trypsinolysis of actin heated at 43 °C (Fig. 4A).
During the late stages of trypsinolysis, predominantly one
major band with an apparent molecular mass of 33 kDa
was detected in the incubation mixture (Fig. 4A). The data
for limited trypsinolysis indicate that after heating at 43 °C
actin acquires a structure different from that of the intact
and thermally inactivated protein. In this state, the envi-
ronment of W residues remains comparatively hydrophobic,
actin weakly interacts with ANS and only a small portion of
protein forms high molecular mass aggregates.

Thermal unfolding can also affect actin polymerization.
To investigate this possibility we heated actin containing
10% of pyrene-labeled protein for 1 h at 43 and 60 °Cand
analyzed polymerization and aggregation induced by the
addition of KCl and MgCl
2
(Fig. 5). As expected, salt
addition induced rapid polymerization of control unheated
actin (Fig. 5A, curve 1), that was accompanied by 14–16-
fold increase in the fluorescence of the pyrene label attached
to C373. Actin heated at 43 °C was also able to polymerize,
although the rate and the extent of polymerization was
slightly lower than in the case of unheated actin (Fig. 5A,
curve 2). Heating at 60 °C completely prevented any
increase in fluorescence induced by salt addition (Fig. 5A,
curve 3). This indicates that actin heated at temperatures
higher than 60 °C was not able to polymerize. Addition of
salt can induce not only polymerization of intact actin, but
can also promote aggregation of partially unfolded actin [5].
This process was followed by light scattering (Fig. 5B).
Fig. 3. Fluorescence spectra of 140 l
M
ANS in the absence of added
proteins (1) and in the presence of 2.25 l
M
of intact actin (2) or actin
heated for 1 h at 43 (3), 60 (4) or 80 °C(5).All measurements were
performed in G buffer at 25 °C and the fluorescence was excited at
350 nm.
Fig. 4. Proteolytic fragmentation of intact actin and actin heated at 43

or 60 °C. (A) Limited proteolysis of actin by trypsin (weight ratio
actin/N-tosyl-
L
-phenylalanine chloromethyl ketone–trypsin, 50 : 1).
Actin (15 l
M
)inG-bufferwaskeptat4°C(gelsmarked4°C) or
heated at 60 or 43 °C for 1 h (gels marked 60 °Cand43°C, respect-
ively). After dilution up to 2 l
M
, actin was subjected to trypsinolysis
that was performed under identical conditions at 25 °C. Aliquots were
removed at the time indicated and the reaction was stopped by the
addition of phenylmethanesulfonyl fluoride, followed by boiling in
the sample buffer. The samples obtained were subjected to quantitative
SDS gel electrophoresis. (B) Time course of the disappearance of the
band of unhydrolyzed actin during limited trypsinolysis of intact actin
(1) or actin preincubated at 60 °C (2) or 43 °C(3).
Ó FEBS 2003 Actin and small heat shock protein (Eur. J. Biochem. 270) 895
Addition of the salts induced slow increase of the light
scattering of actin heated at 60 °C (Fig. 5B, curve 3). As this
sample of actin was unable to polymerize (Fig. 5A), the
observed increase in light scattering can be due only to the
slow aggregation of partially folded protein. In contrast,
addition of the salts to intact actin or to actin heated at
43 °C induced rapid increase of the light scattering that
coincides in time with polymerization (Fig. 5, curves 1,2).
Summing up, we may conclude that heating of actin at
43 °C induced rather mild changes in the structure of actin.
The samples of actin heated at 43 °Chaveamore

hydrophilic environment of W residues than the intact
protein, weakly bind ANS, contain rather small quantities
of high molecular mass aggregates, are more resistant to
trypsinolysis than intact actin and retain the ability to
polymerize. Actin samples heated at temperatures higher
than 55 °C are partially unfolded, strongly interact with
ANS, are highly susceptible to trypsinolysis, contain high
quantities of high molecular mass aggregates and are unable
to polymerize. We assumed that intact and heated actin
would interact differently with HSP25 so we analyzed the
effect of HSP25 on the polymerization of intact actin and
aggregation of heated actin.
Interaction of HSP25 with intact actin
In the first series of experiments we analyzed the effect of
HSP25 on the apparent critical concentration and initial
rate of actin polymerization (Fig. 6A). In this case, G-actin
containing 10% of pyrene-labeled protein was preincubated
with HSP25 or its 3D mutant, and polymerization was
initiated by the simultaneous addition of actin nuclei and
salts. Neither type of HSP25 had any significant effect on
the critical concentration of actin (Fig. 6A). Indeed the
critical concentration of actin was equal to 0.25 ± 0.07;
0.21 ± 0.05, and 0.15 ± 0.06 l
M
in the absence of HSP25,
and in the presence of the wild type HSP25 and its 3D
mutant, respectively. At the same time, both wild type and
especially 3D mutant of HSP25 significantly decreased the
initial rate of actin polymerization. The extent of polymeri-
zation was followed by means of fluorescence spectroscopy

and ultracentrifugation. In the first case, after preincubation
with HSP25, polymerization of actin was initiated by salt
addition. As can be seen from Fig. 6B, during the first
40 min the wild type recombinant HSP25 hardly affects
actin polymerization, whereas the 3D mutant decreased the
extent of polymerization by 20–25%. Similar results were
obtained by ultracentrifugation. In this case, different
quantities of actin were preincubated with HSP25 or its
mutant, and immediately after salt addition the samples
were subjected to ultracentrifugation at 100 000 g for 1 h.
The quantity of actin in the pellet was plotted against the
total quantity of actin in the probe (Fig. 6C). The wild type
HSP25 has little effect on the extent of polymerization of
intact actin, whereas both 2D and 3D mutants decreased the
quantity of polymerized actin in the pellet. It is worthwhile
to mention that the mutants of HSP25 affect the rate but not
the maximal extent of intact actin polymerization. If the
samples before ultracentrifugation were incubated for 4 h at
room temperature, the quantity of polymerized actin in the
pellet was almost independent of the presence of HSP25 or
its mutants (data not presented).
Summing up, we may conclude that the recombinant wild
type HSP25 has almost no effect on the polymerization of
intact actin. 2D and 3D mutants mimic the phosphorylation
of HSP25 by MAPKAP-2 kinase and form oligomers with
smaller molecular mass than the wild type HSP25 [20].
These mutants decrease the initial rate of actin polymeriza-
tion without affecting its critical concentration (Fig. 6A).
Decrease of the initial rate results in decreased extent of
polymerisation, measured during the first 40–60 min after

initiation of polymerization (Figs 6B,C), but does not affect
the final maximal extent of polymerisation, measured 4 h
after initiation of polymerization.
Effect of HSP25 on polymerization of actin heated
at 43 °C
As mentioned earlier, mild heating at 43 °Cresultsinsome
changes in actin structure, but this treatment does not
Fig. 5. Effect of heating on the kinetics of polymerization (A) and salt-
induced increase of the light scattering (B) of actin. Actin (15 l
M
)
containing 10% or pyrene-labeled actin in buffer G were incubated at 4
(1), 43 (2), or 60 °C (3) for 1 h. After cooling and diluting with buffer
G, so that the concentration of actin becomes equal to 10 l
M
,the
reaction was started by the addition of KCl and MgCl
2
up to the final
concentrations 50 and 2 m
M
, respectively. Polymerization was fol-
lowed by an increase in fluorescence at 407 nm excited at 366 nm.
Light scattering (I/I
o
) was followed at 560 nm.
896 O. O. Panasenko et al.(Eur. J. Biochem. 270) Ó FEBS 2003
completely prevent actin polymerization. In addition, this
heating regime resembles that occurring in vivo under
certain physiological conditions. In preliminary experiments

we have shown that the increase of the time of heating at
43 °C is accompanied by exponential decrease of parameter
A(I
320
/I
365
), that tends to a limit equal to 1.8 (data not
shown). This value is significantly higher than the corres-
ponding parameter obtained after heating at temperatures
higher than 55 °C, which is equal to 1.3 (Fig. 1). Actin
(containing 10% of pyrene-labeled protein) was heated at
43 °C for different periods of time, and samples of actin
having different parameter A (2.2–2.7) were analyzed for
their ability to polymerize. In these particular experiments
we measured the initial rate of polymerization that was
initiated by simultaneous addition of actin nuclei and salts.
In good agreement with earlier presented results, we found
that the wild type HSP25 has almost no effect on the initial
rate of polymerization of intact actin (A > 2.55), whereas
the 3D mutant of HSP25 slightly decreased the initial rate of
polymerization (15–25%, Fig. 7). The effect of HSP25
became negligible when parameter A of actin was close to
2.4. Increase of the time of heating at 43 °C leading to
decrease of parameter A up to 2.2–2.3 was accompanied by
further decrease of the initial rate of polymerization. When
parameter A was in the range of 2.20–2.35 the wild type
HSP25 had no effect on the initial rate of polymeri-
zation, whereas its 3D mutant activated the initial rate of
polymerization by 25–35% (Fig. 7). This means that
depending on the state of actin the 3D mutant of HSP25

can either increase (if A < 2.4) or decrease (if A > 2.4) the
initial rate of polymerization.
We also analyzed the effect of HSP25 and its mutants
on the extent of heated actin polymerization. In this case,
actin (containing 10% of pyrene-labeled protein) was
heated at 43 °C until parameter A reached 2.2. Polymeri-
zation was initiated by salt addition and was followed for
1 h. Under the conditions used, wild type HSP25 did not
affect actin polymerization, whereas the 3D mutant of
HSP25 increased the rate of polymerization without
affecting the maximal extent of polymerization (data not
presented).
We suppose that this effect of the 3D mutant of HSP25
on the initial rate of actin polymerization can be explained
by the prevention of nonspecific aggregation of partially
denatured actin that can trap intact actin. The 3D mutant
prevents aggregation and by this means increases the
quantity of available actin monomers and therefore increa-
ses the initial rate of polymerization.
Fig. 6. Effect of recombinant wild type HSP25 and its mutants
mimicking phosphorylation on polymerization of intact actin. (A) Effect
of HSP25 and its 3D mutant on the kinetics of actin polymerization.
Samples containing 1–4 l
M
of actin (10% of pyrene-labeled protein)
were incubated for 5 min in the absence (1) or in the presence of 6 l
M
of HSP25 (2) or its 3D mutant (3). Polymerization was initiated by the
simultaneous addition of actin nuclei, KCl and MgCl
2

up to the final
concentrations 0.5 l
M
,50m
M
and 2 m
M
, respectively. The results
shown are representative of three experiments with three different
purified actin samples, and triplicate measurements of each experi-
mental point. If not shown the error bars are smaller than the size
of symbol. (B) Influence of HSP25 and its 3D mutant on the extent of
actin polymerization. Samples containing 4 l
M
of actin (10% of
pyrene-labeled protein) were preincubated for 5 min at 25 °Cinthe
absence (1) or in the presence of wild type HSP25 (2) or its 3D mutant
(3). Polymerization was initiated by addition of KCl and MgCl
2
up to
50 and 2 m
M
, respectively. The results shown are representative of
three independent experiments. (C) Effect of HSP25 and its mutants on
actin polymerization measured by ultracentrifugation. Samples con-
taining 0.12–0.48 nmol of unheated unmodified actin in 60 lLof
G-buffer were incubated for 5 min at 25 °C in the absence (1) or in the
presence of 10 l
M
of wild type HSP25 (2) or 10 l

M
ofits2D(3)or3D
(4) mutants. One fifth of the volume of buffer S was added and after
mixing the samples were immediately subjected to ultracentrifugation.
The quantity of actin in the pellet is plotted against the total quantity of
actin in the sample. The results are representative of five experiments
with four different preparations of actin.
Ó FEBS 2003 Actin and small heat shock protein (Eur. J. Biochem. 270) 897
Effect of HSP25 on the aggregation of partially
unfolded thermally inactivated actin
As already shown, heating of actin at a temperature higher
than 55 °C completely prevents its polymerization
(Fig. 5A). After this type of heating actin formed small
oligomers (Fig. 2), and an increase in ionic strength induced
further aggregation (Fig. 5B). We analyzed the ability of
HSP25 and its mutants to prevent salt-induced aggregation
of partially unfolded actin heated at 60 °C. In the first series
of experiments actin was heated at 60 °C for 1 h, cooled,
mixedwithdifferentspeciesofHSP25,andafterthe
addition of salt subjected to ultracentrifugation. Under
these conditions only aggregated actin was sedimented.
Therefore, by measuring the quantity of actin in the pellet
we were able to estimate the chaperone activity of HSP25
and its mutants. In the absence of HSP25 about 90% of
heated actin was found in the pellet after ultracentrifugation
(Fig. 8A). Addition of the wild type recombinant HSP25
reduced the quantity of sedimented actin up to 60%. Point
mutants mimicking phosphorylation of HSP25 by
MAPKAP-2 kinase were very effective in preventing salt-
induced aggregation of partially unfolded actin (Fig. 8A).

Only 10–20% of the total quantity of actin presented in the
sample was precipitated in the presence of the 1D, 2D or 3D
mutant of HSP25. The data presented indicate that HSP25,
and especially its mutants mimicking phosphorylation,
effectively prevent salt-induced aggregation of partially
folded actin.
To obtain more detailed information on the interaction of
HSP25 with partially unfolded actin, we analyzed the
effect of different quantities of HSP25 on the salt-induced
aggregation of actin. In this case actin was either kept on ice
or heated at 60 °C for 1 h. The samples of actin were
incubated with different quantities of HSP25 or its 3D
mutant. Polymerization (in the case of intact, unheated
Fig. 8. Effect of HSP25 on the salt-induced aggregation of partially
unfolded thermally inactivated actin. (A) The influence of different
HSP25 species on the salt-induced aggregation of thermally inactivated
actin measured by ultracentifugation. Actin (15 l
M
)inbufferGwas
heated for 1 h at 60 °C. After cooling and dilution to 2 l
M
,actinwas
mixed with different species of HSP25 (final concentration 4 l
M
)and
incubatedfor20minat25°C. Aggregation was initiated by the
addition of 1/5 of the sample volume of buffer S. Samples were incu-
bated for 1 h at 25 °C and subjected to ultracentrifugation (1 h,
100 000 g). Actin in the pellet was determined by quantitative SDS gel
electrophoresis. C, control without HSP25; WT, wild type recombin-

ant HSP25; 1D, 2D and 3D, HSP25 mutants with replacement of one,
two or three S residues (S15, S77 and S81) by D. The results are
representative of four independent experiments with three different
preparations of actin and triplicate measurements of each experimental
point. (B) Concentration-dependent effect of the wild type HSP25 and
its 3D mutant on polymerization of intact actin (1,2) and salt-induced
aggregation of actin heated at 60 °C (3,4). 15 l
M
actininbufferGwas
kept at 4 °C (1,2) or at 60 °C (3,4) for 1 h. After cooling and dilution
to 2 l
M
, actin was mixed with different quantities of the wild type
recombinant HSP25 (1,3) or its 3D mutant (2,4) and incubated for
20 min. One fifth of the volume of buffer S was added and after
incubation for 2 h at 25 °C the samples were subjected to ultracentri-
fugation (1 h, 100 000 g). Actin in the pellet (percentage of the total
actin in the sample) was determined by quantitative SDS gel electro-
phoresis. The results are representative of six independent experiments
with three different actin samples.
Fig. 7. Dependence of the rate of polymerization upon parameter A of
actin. Actin (15 l
M
, containing 10% pyrene-labeled protein) in buffer
G was heated for 0–90 min at 43 °CandparameterA(I
320
/I
365
)was
recorded. The samples were cooled, diluted to a final actin concen-

tration of 4 l
M
and incubated in the absence (1) or in the presence of
6 l
M
of the wild type HSP25 (2) or its 3D mutant (3) for 5 min at
25 °C. Polymerization was initiated by the simultaneous addition of
actin nuclei, KCl and MgCl
2
at final concentrations of 0.2 l
M
,50m
M
and 2 m
M
, respectively. The initial rate of polymerization was deter-
mined during the first 2 min of the reaction by increase in fluorescence
at 407 nm excited at 366 nm. The results are representative of two
independent experiments with two different actin samples, with trip-
licate measurements of each experimental point.
898 O. O. Panasenko et al.(Eur. J. Biochem. 270) Ó FEBS 2003
actin) or aggregation (in the case of heated actin) was
initiated by salt addition and was allowed to proceed for 2 h
at 25 °C. After ultracentrifugation the quantity of actin in
the pellet was determined by quantitative SDS gel electro-
phoresis. Actin in the pellet (as a percentage of actin in the
total sample) was plotted against the HSP25 concentration
(Fig. 8B). As described earlier, neither the wild type HSP25
nor its 3D mutant affected the final extent of polymerization
of intact unheated actin (Fig. 8B, curves 1,2). Addition of

the wild type HSP25 reduced the quantity of aggregated
partially folded actin heated at 60 °C (Fig. 8B, curve 3).
However, even at a very high concentrations the wild type
HSP25 was not able to completely prevent aggregation of
partially unfolded actin. In contrast, even much lower
quantities of the 3D mutant of HSP25 completely prevented
aggregation of actin heated at 60 °C (Fig. 8B, curve 4).
We may conclude that HSP25 effectively prevents
aggregation of partially folded actin. Phosphorylation (or
mutations mimicking phosphorylation) increased the chap-
erone effect of HSP25 so that it becomes able to completely
prevent salt-induced aggregation of heated actin.
Discussion
Heating induces significant changes in the structure of
G-actin. Bertazzon et al. [4] suggested that upon heating
native (N) actin is irreversibly converted to denatured (D)
(or partially folded) actin. This first step of unfolding is
enthalpic and involves the denaturation of two independent
domains of approximately 11 and 31 kDa. Addition of a
high concentration of guanidine hydrochloride or urea can
reversibly convert D (or partially folded) actin to the
completely unfolded (U) state. This second step of unfolding
is reversible and purely enthropic. Thus, the mechanism of
unfolding of G-actin was described by a simple scheme:
N-actin ! D-actin Ð U-actin
However, the first transition from N-actin to D-actin was
not a one-step process. It has been shown [4] that the
calorimetric melting curve of actin was asymmetric, and that
the excess heat capacity curve of G-actin can be fitted into
two independent intermediate steps with T

m
of 52 and
57 °C, respectively. Therefore, the scheme of actin unfolding
is more complex and can be represented in the form:
N-actin Ð D
1
-actin ! D
2
-actin Ð U-actin
where D
1
-andD
2
-actins represent two states of denatured
actin and the reversibility or irreversibility of transitions
between N, D
1
and D
2
are unknown. Although D
1
and D
2
states of actin were postulated, their properties and even
their existence was not confirmed experimentally.
We propose that heating of actin at 40–45 °Cleadstoa
slow transition from N-actin to D
1
-actin. The D
1

state of
actin is different from both native actin and from the well-
characterized denatured (or D
2
) state of actin. Heating
under these mild physiologically relevant conditions results
in the accumulation of the protein with only a moderate
change in the W environment (Fig. 1), diminished ability to
interact with ANS (Fig. 3) and a small quantity of high
molecular mass aggregates (Fig. 2). In addition, actin in this
state was more resistant to proteolysis than intact protein
and in this respect was completely different from denatured
actin (Fig. 4). Moreover, even after prolonged heating at
43 °C actin retained its ability to polymerize (Figs 5 and 7).
This property was completely lost by denatured actin
(Fig. 5). We may suppose that heating at 43 °C induces
unfolding of a small domain proposed by Bertazzon et al.
[4]. At present it is difficult to locate this domain exactly in
the crystallographic structure of actin. However, it is known
that trypsin predominantly cleaves actin at residues 62 and
68, forming fragments with apparent molecular masses of
33 and 9 kDa [26]. Heating at 43 °C partially protects actin
from trypsinolysis (Fig. 4). This fact may indicate that
the above-mentioned small domain with molecular mass
9–11 kDa may include subdomains 1 and 2 of actin. After
heating at 43 °C, actin turns into a state that is different
from both the intact and denatured conformations. This
intermediate state may be of importance because under
physiological conditions the body temperature of warm-
blooded animals can rise up to 40–42 °C.

The denatured (D or D
2
) state of actin was analyzed in
detail [4–6]. This state is characterized by a very hydrophilic
environment of W residues, exposed hydrophobic sites
interacting with ANS and increased susceptibility to
proteolysis. Exposure of hydrophobic sites increases the
probability of self-aggregation and therefore partially folded
actin tends to aggregate. Self-aggregation may be the reason
for the irreversibility of transition from the native to the
partially folded state [5]. Transition of N-actin to the D (or
D
2
) state was observed after heating at temperatures higher
than 55 °C, after the removal of calcium or after the
addition of low concentrations of urea or guanidine
hydrochloride [5,6]. Accumulation of denatured actin can
be dangerous for the cell as it tends to form high molecular
mass aggregates.
Let us analyze the interaction of HSP25 with the N-, D
1
-
and D
2
-forms of actin. Miron et al. [11,12] claimed that
HSP25 effectively inhibits polymerization of N-actin by
increasing its critical concentration. Similar results were
obtained by Benndorf et al. [13], but only with the
monomeric unphosphorylated form of HSP25. These
results were obtained with HSP25 purified from avian or

human tissues. In both cases the starting steps of purifica-
tion of HSP25 were performed according to Feramisco and
Burridge [27] and an initial crude mixture contained a
number of different proteins with molecular mass in the
range of 20–80 kDa that were able to inhibit polymerization
of actin [28]. The ability of HSP25 to inhibit actin
polymerization was diminished or completely deteriorated
if the protein was purified on a hydroxyapatite column
under special conditions [12]; recombinant HSP25 was also
ineffective in the inhibition of actin polymerization [13,18].
All of these facts can be explained by the suggestion that
HSP25 purified from animal tissues contained trace
amounts of a highly effective inhibitor of actin polymeriza-
tion having a molecular mass of monomers close to that of
HSP25. Cofilin, which has an apparent molecular mass of
20–22 kDa, could be one of the candidates for this role. At
substoichiometric concentrations cofilin inhibits actin poly-
merization, induces depolymerization of actin and is able
to form oligomers with molecular mass in the range
22–100 kDa [29]. As heat shock is accompanied by the
simultaneous translocation of both cofilin and HSP25 to the
Ó FEBS 2003 Actin and small heat shock protein (Eur. J. Biochem. 270) 899
nucleus [30], we may suppose that these two proteins can
interact with each other.
Analyzing the interaction of native actin with HSP25
purified from avian tissues and with recombinant protein,
we found that the wild type HSP25 has little effect on the
rate or extent of actin polymerization. At the same time, the
3D mutant of HSP25 slightly decreased the initial rate of
actin polymerization (Fig. 6A) without affecting the maxi-

mal extent of polymerization. Mutations mimicking phos-
phorylation induce partial dissociation of high molecular
mass oligomers of HSP25 and accumulation of dimers and
tetramers [20]. Low molecular mass oligomers of HSP25
may interact with G-actin and in this way decrease the initial
rate of polymerization. However, this interaction seems to
be weak and therefore HSP25 (or its 3D mutant) does not
affect the final extent of polymerization.
Heating at 40–45 °C leads to transition of native actin to
the D
1
form and is accompanied by a decrease in the rate
and extent of actin polymerization (Figs 5 and 7). This
could be due to the fact that at this heating regime some
actin becomes aggregated (Fig. 2) and therefore is excluded
from polymerization. The wild type HSP25 has little effect
on the polymerization of D
1
-actin (Fig. 7), whereas the 3D
mutant of HSP25 increases the rate of polymerization
without affecting its maximal extent (Fig. 7). This effect of
the 3D mutant may be explained by preventing aggregation
of actin leading to an increase in the concentration of
G-actin available for polymerization.
Conversion of native actin to the D
2
-form completely
prevents polymerization (Fig. 5A). Denatured actin con-
tains exposed hydrophobic sites (Fig. 3) and tends to
aggregate upon addition of salt (Fig. 5B). HSP25 prevents

salt-induced aggregation of denatured actin (Fig. 8), and
HSP25 mutants mimicking phosphorylation possessed
higher chaperone activity than the wild type HSP25. The
chaperone activity of HSP25 strongly depends on the nature
of the target protein and on the state of HSP25 phosphory-
lation. For example, phosphorylation (or mutations
mimicking phosphorylation) decreases the chaperone
activity of human or murine HSP27 with citrate synthase,
insulin [16] and avian HSP25 with a-lactalbumin [20]. At the
same time, phoshorylation (or mutations mimicking phos-
phorylation) increases the chaperone activity of HSP25 with
alcohol dehydrogenase [20]. The same effect was observed in
the case of denatured actin. Different types of stress induce
phosphorylation of HSP25 [10], thus converting it to the
form that effectively prevents aggregation of actin, and in
this way protect the cell from accumulation of large
quantities of insoluble material.
Summing up we may conclude that depending on the
conditions HSP25 has multiple effects on polymerization
and aggregation of G-actin. Monomers or low molecular
mass oligomers of HSP25 weakly interact with G-actin and
thereby slightly inhibit the initial rate of polymerization of
intact actin. The HSP25 mutants, mimicking phosphoryla-
tion, stabilize partially denatured molecules of G-actin,
prevent formation of high molecular mass aggregates and in
this way increase the initial rate of polymerization of
partially denatured actin. Finally HSP25, and especially its
mutants, effectively prevent salt-induced aggregation of
denatured actin, thereby protecting the cell from the
accumulation of insoluble proteins.

Acknowledgements
The authors are grateful to Dr Alim S. Seit-Nebi (V.A. Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences) for the
cloning and expression of recombinant forms of HSP25 and its
mutants. This investigation was supported by Russian Foundation for
Basic Research and by the Wellcome Trust.
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