Báo cáo khoa học: Thermal unfolding and aggregation of actin Stabilization and destabilization of actin filaments doc

REVIEW ARTICLE
Thermal unfolding and aggregation of actin
Stabilization and destabilization of actin filaments
Dmitrii I. Levitsky
1,2
, Anastasiya V. Pivovarova
1,3
, Valeria V. Mikhailova
1
and Olga P. Nikolaeva
2
1 A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia
2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia
3 School of Bioengineering and Bioinformatics, Moscow State University, Russia
Actin is one of the most abundant and highly con-
served proteins found in all eukaryotic cells. It is
involved in many different cellular processes that are
essential for growth, differentiation and motility.
Moreover, bacterial homologs of eukaryotic actin,
ParM and MreB, have recently been identified. It is
now clear that prokaryotic cells also possess actin and
that a dynamic, actin-like cytoskeleton is involved in a
variety of essential cellular processes in bacteria [1].
The 43 kDa actin monomer (globular actin or
G-actin) spontaneously assembles in vitro to form long
polar filaments (filamentous actin, or F-actin) upon
the addition of neutral salts (usually 50–100 mm KCl,
2–4 mm MgCl
2
, or both). Actin filaments have a
crucial role in biological motility as the main partners

of the myosin-based motor systems and as the major
constituent of the cytoskeleton. The polymerization of
G-actin into F-actin is accompanied by the hydrolysis
Keywords
actin; actin filaments; cofilin; differential
scanning calorimetry; heat-induced
aggregation; inorganic phosphate analogs;
phalloidin; small heat shock proteins;
thermal stability; thermal unfolding
Correspondence
D. I. Levitsky, A.N. Bach Institute of
Biochemistry, Russian Academy of
Sciences, Leninsky Prosp. 33, 119071
Moscow, Russia
Fax: +7 495 954 2732
Tel: +7 495 952 1384
E-mail:
(Received 14 April 2008, revised 31 May
2008, accepted 24 June 2008)
doi:10.1111/j.1742-4658.2008.06569.x
Actin is one of the most abundant proteins in nature. It is found in all
eukaryotes and plays a fundamental role in many diverse and dynamic
cellular processes. Also, actin is one of the most ubiquitous proteins because
actin-like proteins have recently been identified in bacteria. Actin filament
(F-actin) is a highly dynamic structure that can exist in different conforma-
tional states, and transitions between these states may be important in cyto-
skeletal dynamics and cell motility. These transitions can be modulated by
various factors causing the stabilization or destabilization of actin filaments.
In this review, we look at actin stabilization and destabilization as expressed
by changes in the thermal stability of actin; specifically, we summarize and

analyze the existing data on the thermal unfolding of actin as measured by
differential scanning calorimetry. We also analyze in vitro data on the heat-
induced aggregation of actin, the process that normally accompanies actin
thermal denaturation. In this respect, we focus on the effects of small heat
shock proteins, which can prevent the aggregation of thermally denatured
actin with no effect on actin thermal unfolding. As a result, we have pro-
posed a mechanism describing the thermal denaturation and aggregation of
F-actin. This mechanism explains some of the special features of the thermal
unfolding of actin filaments, including the effects of their stabilization and
destabilization; it can also explain how small heat shock proteins protect
the actin cytoskeleton from damage caused by the accumulation of large
insoluble aggregates under heat shock conditions.
Abbreviations
AlF
4
)
, anions of aluminum fluoride; BeF
x
, anions of beryllium fluoride; DSC, differential scanning calorimetry; sHSP, small heat shock
proteins; T
m
, midpoint of thermally induced unfolding transition.
4280 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
of bound ATP followed by a slower release of P
i
;asa
result, each F-actin protomer contains either tightly
bound ADP or ADP-P
i
. In vivo, actin polymerization

is a highly regulated process controlled not only by
nucleotide binding and hydrolysis, but also by the
action of a number of actin-binding proteins that can
nucleate, cleave, cross-link, bundle, stabilize or desta-
bilize the filaments [2,3].
Monomeric G-actin is a globular protein consisting of
a single polypeptide chain of 375 residues. It contains
one tightly bound nucleotide, ATP or ADP, and a
single, high-affinity divalent cation, Ca
2+
or Mg
2+
.In
order to be crystallized, actin must be rendered
non-polymerizable and, thus, almost all actin crystal
structures available to date either represent complexes
of G-actin with actin-binding proteins (e.g. DNase I [4],
gelsolin [5] or profilin [6]) and organic toxins [7], or were
obtained from G-actin that had been changed in differ-
ent ways (e.g. by chemical modifications [8–10], proteol-
ysis [11] or mutation [12]). The first atomic resolution
(2.8 A
˚
) structure of actin co-crystallized with DNase I
was published in 1990 by Kabsch et al. [4]. This publica-
tion, as well as other 3D structures of actin, revealed
that it consists of two easily distinguishable domains
separated by a deep cleft, each domain being subdivided
into two subdomains (Fig. 1A). The nucleotide is bound
in the cleft between the two domains. Subdomains 1 and

2 comprise the so-called ‘small’ domain and are linked
to subdomains 3 and 4 (the so-called ‘large’ domain) by
a ‘hinge’ or connecting piece between subdomains 1 and
3. In fact, the ‘small’ domain is not significantly smaller
than the ‘large’ domain [13]. Numerous solution studies
suggest that the nucleotide-binding cleft between the
two domains can exist in two main states, closed and
open, and nucleotide-induced conformational changes
in G-actin are associated with a transition between these
two states of the cleft. However, among the numerous
crystal structures that have been published, significant
opening of the nucleotide-binding cleft has been
observed only in profilin-bound actin crystals [14]. It is
possible that crystal packing interactions favor a closed
state for G-actin even though the state of the cleft in
solution may be shifted by the bound nucleotide
[11,12,15].
Actin normally exists in the monomeric G-form only
in solutions with a very low ionic strength, and it poly-
merizes rapidly upon the addition of 100 mm KCl with
the formation of long polar filaments of F-actin, which
are double-stranded spiral polymers of actin molecules
(Fig. 1B). Polymerization of actin fully prevents its
crystallization which needs high protein and salt
concentrations. As a result, the atomic resolution
structure for F-actin remains unknown, and only
models are available. The most important model,
generated by Holmes et al. [16], used a rotational
and translational search to place the G-actin crystal
D

D
D
D
T
D
D
D
D
D
D
D
DD
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
T
T

T
D
D
D
D
DD
D D
D
D
D
D
D
T
D
DT
D D
D
D
D
D
T
T
T
T
T
T
D
D
D
D

T
T
T
T
T
T
D
ATP (T)
P
i
D
D
(+)
(–)
ADP (D)
A
B
Fig. 1. (A) Three-dimensional atomic structure of monomeric
G-actin shown in approximately the same orientation as originally
illustrated by Kabsch et al. [4] (Protein Data Bank ID code 1ATN).
This actin structure was obtained from co-crystals of actin–DNase I
from which the DNase I component has been removed [4]. The four
subdomains are indicated by the numbers encircled. D-loop, DNase-
I-binding loop. A molecule of nucleotide bound in the nucleotide-
binding cleft is indicated as ATP, and the metal cation (Ca
2+
or
Mg
2+
) is indicated as a sphere. (B) Schematic representation of

actin polymerization and treadmilling of actin filaments. Actin mole-
cules are shown by circles. Monomeric G-actin contains bound ATP
(T inside the circles). Actin polymerization includes three steps,
namely monomer activation, nucleation and filament elongation, the
latter being accompanied by ATP hydrolysis. Actin filaments initially
grow with terminal subunits containing ATP; at later stages, after
ATP hydrolysis, subunits containing ADP-P
i
transiently accumulate
(not shown here); at steady states, the filament is made of subunits
containing tightly bound ADP (denoted as D inside the circles)
except for terminal subunits at the barbed end of the filament
(denoted as ‘+’) that contain ATP or ADP-P
i
. Polar actin filaments
depolymerize from their pointed ends (denoted as ‘)’). After disso-
ciation from the filament and replacement of bound ADP by ATP,
actin monomers can again bind to the barbed ends of the filaments.
D. I. Levitsky et al. Actin unfolding and aggregation
FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS 4281
structure into a helical filament so as to best match the
observed X-ray actin fiber diffraction pattern from an
oriented F-actin gel. The original ‘Holmes model’ [16]
and its variations [17] contain the explicit assumption
that no large-scale conformational change is needed
between a G-actin monomer and an F-actin protomer.
The only change in actin structure that was introduced
involved the movement of a hydrophobic loop (resi-
dues 264–273 in subdomain 4) from the body of the
subunit to form a contact with subunits on the oppo-

site strand of a double-stranded spiral polymer [15].
According to the Holmes model of F-actin [16], sub-
domains 3 and 4 of each actin protomer are located
close to the filament axis, whereas subdomains 1 and 2
are at high radius near the surface of the filament.
Because of the helical symmetry of F-actin, each pro-
tomer in the filament may be in contact with four
adjacent protomers [13]. Residues involved in the
actin–actin contacts are located in all four subdomains
of the protomer. For example, there are contacts
between several sites in subdomain 3 and sites on
subdomains 4 and 2, as well as contact between sub-
domains 1 and 4 [13], and the DNase I-binding loop
(D-loop) at the top of subdomain 2 is proposed to
interact with the C-terminal region in subdomain 1 of
the adjacent protomer [18,19] (Fig. 1A).
A growing body of data suggests that the actin fila-
ment does not exist in a single state, but can be quite
dynamic [15]. One of the most remarkable properties
of F-actin is that although subunits have a very fixed
axial rise in the filament (of  27 A
˚
), the rotation
between adjacent subunits can be quite variable [20].
An actin-binding protein, cofilin, has been shown to
change the average twist of subunits in F-actin by
 5
o
per subunit (from  167 to  162
o

) [21]. How-
ever, detailed analysis of pure actin filaments has sug-
gested that cofilin stabilizes an already existing twisted
state of F-actin that can be formed spontaneously in
the absence of other proteins [22]. Another dynamic
mode within F-actin involves the ability of subunits to
undergo a substantial tilt (up to  30
o
) [15]. This tilt
was first observed within actin filaments decorated by
cofilin [22], but was subsequently observed within
F-actin in the absence of other proteins [23].
Thus, according to recent studies, F-actin can exist
in different structural states, and transitions between
these states may play an important role in cytoskeletal
dynamics and in the contractile cycle of actomyosin
[15]. Many factors (such as the binding of nucleotides,
drugs and actin-binding proteins) may modulate such
transitions, i.e. isomerizations of F-actin from one
structural state to another, thus stabilizing or destabi-
lizing the actin filaments.
In this review, we look at actin stabilization or
destabilization as expressed by changes in its thermal
stability; specifically, we describe and analyze existing
data on the thermal unfolding of actin as measured
using differential scanning calorimetry (DSC), which is
the most effective and commonly used method to study
the thermal unfolding of proteins [24,25]. In the fol-
lowing sections, we summarize and discuss the effects
of stabilization or destabilization for both G-actin and

F-actin that are expressed by an increase or decrease
in actin thermal stability. We also summarize the
in vitro data on the heat-induced aggregation of actin,
a process that normally accompanies actin thermal
unfolding. In this respect, we focus mainly on the
effects of small heat shock proteins (sHSPs), which can
effectively prevent the aggregation of thermally dena-
tured actin with no effect on its thermal unfolding.
Thermal unfolding of monomeric
G-actin
In 1984 Tatunashvili and Privalov [26] used DSC to
investigate the thermal denaturation of monomeric
G-actin and suggested the presence of at least two inter-
acting domains in the molecule. The existence of two
domains in the G-actin molecule was also proposed by
Bertazzon et al. [27] after computer deconvolution of
the G-actin heat sorption curve into two individual ther-
mal transitions. It should be noted that the thermal
denaturation of actin is irreversible, and the use of such
approaches to analyze irreversible thermal transitions is
rather controversial [28]. However, the existence of a
domain structure in the G-actin molecule, initially pro-
posed from the DSC data [26,27], was confirmed by the
3D atomic structure of the G-actin published in 1990
[4], and showed the presence of two easily distinguish-
able domains separated by a deep cleft (Fig. 1A).
The D-loop at the top of subdomain 2 (Fig. 1A)
seems to play a very important role in the thermal
stability of G-actin. Binding of DNase I to G-actin
strongly increased the thermal stability of both

proteins. Separate proteins had thermal transitions
with single maxima of  61 °C for G-actin and
 56 °C for DNase I. In the complex, both proteins
were mutually stabilized and were denatured as a
unit, resulting in a new sharp thermal transition with
maximum at 70 °C (data not shown).
A very similar effect was observed in DSC studies
on the binding of cofilin to G-actin [29,30]. In this
case, both interacting proteins also formed a complex
in which they stabilized each other and denatured
together resulting in a new, highly cooperative thermal
transition with maximum at 66.4 °C [29] or  68 °C
Actin unfolding and aggregation D. I. Levitsky et al.
4282 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
[30]. Thus, as in the case with DNase I, the binding of
cofilin to G-actin significantly increased the thermal
stability of both proteins.
According to recent views, cofilin binds preferentially
to G-actin either in the cleft between subdomains 1 and
3 [31] or in the cleft between subdomains 1 and 2 [32],
or it may bind in both these sites [31]. The D-loop in
subdomain 2 is also proposed to be involved in a weak
binding of cofilin to G-actin [31,33]. Cofilin binding
induces closure of the nucleotide-binding cleft of
G-actin [32,34], and this closure, together with changes
in the D-loop, may be one of the main reasons for the
increased thermal stability of G-actin. In favor of this
assumption are the DSC data on the thermal unfolding
of G-actin complexed with thymosin b
4

[35]. It has been
proposed that this small protein, a member of a family
of actin-sequestering proteins, binds to subdomains 1
and 3 on actin, as well as to the D-loop in subdomain 2,
and this binding causes changes in the spatial orienta-
tion of G-actin subdomains, closing the nucleotide-bind-
ing cleft [35,36]. It has been shown using DSC that the
binding of thymosin b
4
results in the thermal stabiliza-
tion of G-actin, shifting its thermal transition by 1.8 °C
towards a higher temperature and making the transition
more cooperative [35]. However, the opposite effect was
observed by DSC with G-actin specifically cleaved by a
bacterial ECP32 protease which has been shown to
cleave actin at a single site between Gly42 and Val43
within the D-loop [37]. It has been shown that cleavage
of G-actin with ECP32 dramatically decreases its
thermal stability, lowering the thermal transition (T
m
)
by 7–8 °C (A. V. Pivovarova, S. Yu. Khaitlina &
D. I. Levitsky, unpublished results). These DSC results
are in good agreement with data showing a more open
conformation for the nucleotide-binding cleft between
two domains in ECP32-cleaved actin, as evidenced by
the increased nucleotide exchange rate and its higher
susceptibility to limited proteolysis [38,39].
It thus seems possible that the thermal stability of
G-actin is mainly determined by the conformational

state of the nucleotide-binding cleft between the two
domains in the actin molecule. The thermal stability of
G-actin increases when the cleft is closed (e.g. due to
the binding of various proteins to the D-loop), whereas
opening of the cleft leads to significant destabilization
of G-actin.
Thermal unfolding of F-actin filaments
Polymerization of G-actin to F-actin
The use of DSC allows very clear probing of the
changes caused by actin polymerization, i.e. the trans-
formation of monomeric G-actin into F-actin filaments
(Fig. 1B). These changes, observed by many authors
[27,30,40–42], are expressed as a significant increase
in the denaturation temperature and a sharp change
in the shape of the peak (the peak becomes much
narrower, indicating a significant increase in the coo-
perativity of thermal denaturation) (Fig. 2). It is evi-
dent that the changes in thermal unfolding that
accompany actin polymerization are due to numerous
contacts established between adjacent actin protomers
in the filament.
Effects of nucleotides on the thermal unfolding
of F-actin
Although bound nucleotide is not required for actin
polymerization [43], it is very important for the stabil-
ization of actin. It is well known that actin lacking
bound nucleotide is very unstable and denatures easily
[44] unless protected by high sucrose concentrations,
which permit actin to retain its stability and the ability
to form filaments of F-actin [43,45]. Under normal

conditions, ADP is tightly bound in F-actin subunits
and is unable to exchange with free nucleotides.
Surprisingly, our DSC experiments have shown that
40 50 60 70 80 90 100
0
50
100
150
ΔC
p
(kJ·mol
–1
·K
–1
)
Temperature (°C)
G-actin
F-actin
F-actin + phalloidin
F-actin + AlF
4
–
F-actin + phalloidin + AlF
4
–
Fig. 2. Temperature dependences of the excess heat capacity
(DC
p
) of G-actin, F-actin in the presence of 1 mM ADP and F-actin
stabilized by phalloidin or AlF

4
)
, or simultaneously by both these
stabilizers. The actin concentration was 1 mgÆmL
)1
. Other condi-
tions: for G-actin, G-buffer (2 m
M Tris ⁄ HCl, pH 8.0, 0.2 mM ATP,
0.2 m
M CaCl
2
, 0.5 mM b-mercaptoethanol and 1 mM NaN
3
); for
F-actin, 30 m
M Hepes, pH 7.3, containing 100 mM KCl, 1 mM
MgCl
2
and 1 mM ADP. Concentrations of stabilizers: 33 lM phalloi-
din and 0.5 m
M AlF
4
)
(5 mM NaF and 0.5 mM AlCl
3
). Heating rate
1KÆmin
)1
. Adapted from Levitsky et al. [41] and Levitsky [54] with
some changes and additions.

D. I. Levitsky et al. Actin unfolding and aggregation
FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS 4283
the thermal stability of F-actin depends strongly on
the concentration of ADP added. The transition tem-
perature (T
m
) of F-actin increased by 5–6 °C with
increasing ADP concentrations up to 1 mm, and
reached a plateau at higher ADP concentrations, a
half-maximum increase in T
m
being observed in the
presence of 0.1 mm ADP [46]. The stabilizing effect of
ADP was highly specific, being observed only with
ADP, and not with other nucleoside diphosphates
(IDP, UDP, GDP, CDP). A similar effect of the
F-actin thermal stabilization was also seen in the
presence of ATP; however, this effect was much less
specific, and was observed, although to a lesser extent,
for other nucleoside triphosphates (ITP, UTP and
GTP). Another difference between the effects of ATP
and ADP was that an increase in ATP concentration
from 1 to 5 mm led to further significant increase in
the thermal stability of F-actin (T
m
increased by
>3°C), whereas a similar increase in ADP concentra-
tion had no influence on the thermal unfolding of
F-actin [46]. These findings suggest that the stabilizing
effect of ATP [46,47] differs significantly in its mecha-

nism from the effect caused by ADP [46].
It seems very likely that the stabilizing effect of ATP
and other nucleoside triphosphates is caused not only
by their binding to high-affinity sites, but also by their
interaction with some additional (‘second’) nucleotide-
binding site on F-actin. The presence of a second,
low-specificity and low-affinity nucleotide-interacting
site on actin has been postulated by some studies [47–
50]. Nucleotide binding in this site occurred at milli-
molar concentrations, ITP and CTP being even more
effective than ATP [50].
It is important to note that the most striking
changes in the thermal stability of F-actin were
observed at low concentrations of added nucleotide
(0.1–0.2 mm), and under these conditions the effects of
ADP and ATP were very similar [46]. It is difficult to
explain these effects by some interaction of ADP
or ATP with the second nucleotide-binding site on
F-actin, which needs much higher concentrations of
nucleotide. It seems most likely that these effects,
which are observed at low ADP or ATP concentra-
tions, are caused by their binding with highly specific,
high-affinity sites in the nucleotide-binding cleft of
actin subunits, whereas the additional stabilization of
actin filaments, which occurs at high ATP concentra-
tions, is caused by ATP binding to low-affinity sites
that are able to bind ATP and other nucleoside
triphosphates, but not ADP.
It seems to us that the most likely explanation for
the stabilizing effect of nucleotides on the thermal

unfolding of F-actin is as follows. We have proposed
that irreversible thermal unfolding of F-actin is pre-
ceded by a reversible stage involving nucleotide (ADP)
dissociation from specific nucleotide-binding sites in
actin subunits [46]. Reversible dissociation of ADP
from actin subunits probably occurs only upon heat-
ing, just before the irreversible denaturation of the
protein. In the absence of free nucleotides, actin-bound
ADP dissociates easily and, as a result, actin denatures
easily and rapidly. However, the presence of free nucle-
otide (ADP or ATP) in solution prevents the dissocia-
tion of actin-bound ADP, and this may explain the
nucleotide-induced increase in the thermal stability of
F-actin.
Stabilization of F-actin by phalloidin
It is well known that F-actin interacts specifically with
a cyclic heptapeptide, phalloidin – one of the principal
toxins of the mushroom Amanita phalloides. Phalloidin
binds to F-actin with very high affinity and causes sub-
stantial stabilization of the actin filaments, preventing
the depolymerization of F-actin and protecting it from
proteolytic cleavage. Therefore, actin filaments stabi-
lized by phalloidin are often used in experiments, e.g.
in vitro motility assays. Le Bihan and Gicquaud [51]
showed that the binding of phalloidin to F-actin signif-
icantly increases the temperature of F-actin thermal
denaturation, raising the temperature at which thermal
transition occurs by 14 °C. This effect of phalloidin
(Fig. 2), also observed by us [41,52] and other authors
[53], is very useful for DSC studies on F-actin com-

plexes with other proteins, and we often use phalloi-
din-stabilized F-actin to obtain a better separation of
the thermal transitions of F-actin and actin-bound
proteins (e.g. myosin head, tropomyosin) on DSC
curves [54].
Phalloidin binds to F-actin at the interface of three
adjacent actin protomers [17,55] and stabilizes lateral
interactions between the two filament strands. These
physical contacts appear to be the most important
factors for the thermal stabilization of F-actin by
phalloidin. It has been shown using electron micros-
copy that phalloidin can stabilize F-actin with a high
cooperativity, at a 1 : 20 molar ratio with actin [56].
The cooperativity of the phalloidin-induced stabil-
ization of F-actin has also been observed in DSC
experiments [57].
Stabilization of F-actin by P
i
analogs
During the ATP hydrolysis that normally accompanies
actin polymerization, intermediate states are formed in
actin subunits, which are actin complexes with ATP or
Actin unfolding and aggregation D. I. Levitsky et al.
4284 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
ADP-P
i
. These intermediate states can be studied using
stable complexes of F-actin with beryllium fluoride
(BeF
x

) or aluminum fluoride (AlF
4
)
) anions, which have
been found to be good structural analogs of P
i
. Com-
beau and Carlier [58] found that AlF
4
)
and BeF
x
(BeF
x
stands for the BeF
3
)
and BeF
2
(OH)
)
complexes) bind
strongly to F-actin with an affinity three orders of mag-
nitude higher than P
i
and compete with P
i
for binding
to the nucleotide-binding cleft of ADP–F-actin protom-
ers in the place of the c-phosphate of ATP. Both these

P
i
analogs strongly stabilize F-actin by decreasing the
rate of protomer dissociation from filaments by 150-fold
[58]. In the case of BeF
x
, this stabilization was shown to
be associated with structural changes in subdomain 2,
as indicated by the strong inhibition of its proteolytic
cleavage [59] and by electron microscopy [60].
Stabilization of actin filaments by BeF
x
or AlF
4
)
can be clearly seen in DSC studies on the thermal
unfolding of F-actin (Fig. 2). It has been shown that
both these P
i
analogs increase the thermal stability of
F-actin drastically, increasing the thermal transition
temperature by more than 16 °C [41,61]; the effects of
BeF
x
and AlF
4
)
are very similar [61].
Additive effect of F-actin stabilization by
phalloidin and P

i
analogs
It is interesting to note that phalloidin and AlF
4
)
(Fig. 2) (or BeF
x
) [41] have similar effects on the thermal
denaturation of F-actin, and these effects are expressed
as a significant increase of the transition temperature.
However, when we simultaneously added both phalloi-
din and BeF
x
(or AlF
4
)
) to F-actin we observed an addi-
tional stabilization of F-actin expressed as an increase in
the thermal transition temperature of more than 25 °C
[41] (Fig. 2). This means that phalloidin and BeF
x
(or
AlF
4
)
) stabilize F-actin in different ways independent of
each other and most likely affect different sites on the
actin molecule. This is consistent with the literature data
that phalloidin and BeF
x

bind to different sites on actin.
Indeed, bound phalloidin is located at the contact region
of three actin subunits thus stabilizing their interactions
[55], whereas BeF
x
(or AlF
4
)
) binds to the nucleotide-
binding cleft, affects the structure of subdomain 2 and
its interaction with the adjacent longitudinal protomer
in ADP–F-actin [59,60], and probably favors a closed
state for the cleft.
Stabilization and destabilization of F-actin by
cofilin
Very little is known how the actin-binding proteins
affect the thermal unfolding of F-actin. One of the first
studies in this direction was our attempt to apply the
DSC method to investigate the thermal unfolding of
F-actin in complexes with cofilin, a small actin-binding
protein belonging to the actin-depolymerizing factor
(ADF) ⁄ cofilin family of proteins.
ADF ⁄ cofilin proteins have attracted much attention
because of their important role in regulating actin
dynamics in cells [2,3,62,63]. These proteins can depo-
lymerize [64] and sever [65,66] actin filaments by weak-
ening longitudinal [22,67] and lateral [68,69]
interprotomer contacts in F-actin. It has been shown
that the binding of cofilin induces a transition in sub-
domain 2, which is accompanied by disordering of the

D-loop [70], and these cofilin-induced conformational
changes in F-actin expose subdomain 2 to proteolysis
[71]. Also, cofilin binding was shown to change the
twist of F-actin by decreasing the rotation of one
protomer with respect to its neighbors [21]. All these
conformational changes in the actin filament are
predicted to lead to filament destabilization.
It was shown for the first time in our DSC experi-
ments [29] and then confirmed by Bobkov et al. [30]
that cofilin has a dual effect on the thermal unfolding
of F-actin, depending on the molar ratio of cofilin to
actin. At saturating concentrations, cofilin strongly
increases the thermal stability of F-actin increasing the
temperature at which thermal transition occurs by
7 °C and increasing the cooperativity of the transition
(Fig. 3) [29]. The stabilizing effect of cofilin on F-actin
was very similar to that observed with G-actin in the
presence of cofilin [29,30]. This suggests that cofilin
binding to F-actin subunits induces conformational
changes similar to those seen in monomeric G-actin
(presumably the closure of the nucleotide-binding cleft)
[32], and these changes result in a significant increase
in the thermal stability of F-actin. According to mod-
els of cofilin–F-actin complexes derived from electron
microscopy studies [21–23,70], cofilin binds to subdo-
main 2 of a lower protomer and subdomain 1 of an
upper protomer in F-actin and overlaps the inter-
protomer interface between these subdomains. This
interprotomer interaction may also contribute to the
cofilin-induced thermal stabilization of F-actin.

Interestingly, the stabilization of F-actin by cofilin
at saturating concentrations fully abolished the effects
of the other F-actin stabilizers, phalloidin or AlF
4
)
[29]. In the case of AlF
4
)
, this is consistent with a
recent observation that cofilin can dissociate BeF
x
(another P
i
analog, which is very similar to AlF
4
)
in
its effects on F-actin) [29,58] from the actin filament,
whereas BeF
x
does not bind to F-actin saturated with
cofilin, presumably because of the cofilin-induced
changes in the nucleotide-binding cleft of F-actin
D. I. Levitsky et al. Actin unfolding and aggregation
FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS 4285
subunits [72]. Phalloidin, unlike BeF
x
and AlF
4
)

, can
bind to F-actin that is fully saturated with cofilin and
compete with cofilin for F-actin [72]. However, cofilin-
induced structural changes in the actin filament (such
as the changes in twist [21] and the weakening of
lateral contacts in the filament [68]) may alter the
contacts between actin protomers in the filament and
prevent the simultaneous binding of phalloidin with
three adjacent protomers, thereby abolishing its stabi-
lizing effect on F-actin.
Contrary to the stabilizing effect of cofilin on F-actin
thermal unfolding at saturating concentrations of cofi-
lin, a strong decrease in the thermal stability of F-actin
was observed at sub-saturating concentrations of cofilin
[29,30]. Under these conditions, the temperature at
which the thermal transition of F-actin occurred was
lowered by 1–6 °C, depending on the cofilin ⁄ actin
monomer molar ratios. The most pronounced effect was
observed at cofilin to actin molar ratios between 1 : 6
and 1 : 1.5. Under these conditions, two peaks were
simultaneously observed: a cofilin-stabilized F-actin
peak at 65–67 °C and a cofilin-destabilized peak at
56–58 °C (Fig. 3) [29]. On decreasing the cofilin ⁄ actin
monomer molar ratio, the cofilin-stabilized peak decrea-
sed, whereas the cofilin-destabilized peak increased.
At low cofilin ⁄ actin monomer molar ratios (< 1 : 12)
only the destabilized peak was observed (Fig. 3) and its
maximum shifted to higher temperatures as the cofilin ⁄
actin molar ratio decreased [29]. The destabilizing effect
of cofilin was highly cooperative as it was observed

even at cofilin ⁄ actin molar ratios as low as 1 cofilin per
100–200 actin protomers [29,30]. It has been suggested
from these DSC results that cofilin, when bound to
F-actin, stabilizes only those actin subunits to which it
binds directly, whereas it destabilizes with a very high
cooperativity neighboring regions of the actin filament
that are free of cofilin [29]. This is consistent with
electron microscopy observations showing that actin
subunits within cofilin-free regions of cofilin-decorated
actin filaments differ in their orientation from those in
undecorated F-actin [22]. The DSC data also suggest
that cofilin-induced changes in the conformation of
F-actin can be propagated over a long distance along
the filament from subunits stabilized by cofilin to sub-
units free of cofilin. Obviously, these changes become
weaker at low cofilin concentrations, i.e. when the
average separation between cofilin molecules bound to
F-actin increases.
Cofilin-induced destabilization of F-actin was fully
prevented by the addition of phalloidin or AlF
4
)
[29],
thus indicating that actin subunits destabilized by
cofilin retained their ability to bind other stabilizers of
F-actin.
Interestingly, the cofilin-induced destabilization of
F-actin was observed by DSC even with F-actin, in
which Gln41 in the D-loop on subdomain 2 was cross-
linked to Cys374 near the C-terminus on subdomain 1

of the adjacent protomer within the same strand of the
long-pitch helix [30]. This may suggest that cofilin,
when it binds to F-actin at low molar ratios, weakens
the lateral contacts between actin subunits rather than
the longitudinal contacts.
The destabilizing effect of cofilin demonstrated by
DSC studies may play an important role in actin
dynamics in living cells. In particular, it may be impor-
tant to provide a possible molecular mechanism
for the actin-severing and depolymerizing activities of
cofilin.
Destabilization of F-actin by formins
Members of the formin family of proteins are known
to be key regulators of actin polymerization that nucle-
ate actin filaments and play essential roles in the regu-
lation of the actin cytoskeleton. It has been shown
using DSC that the binding of formins to F-actin
decreases the F-actin thermal stability slightly, lower-
ing the T
m
by 1.5 °C [73]. This effect was observed at
a low formin ⁄ actin molar ratio (1 : 20). The authors
assigned this decrease in the thermal stability of
F-actin to an increase in the flexibility of the filament.
40 50 60 70
0
10
20
30
40

50
F-actin (25
µ
M)
F-actin + cofilin (1
µ
M)
F-actin + cofilin (8
µ
M)
F-actin + cofilin (32
µ
M)
Tem
p
erature (°C)
Apparent heat capacity (
µ
W)
Fig. 3. DSC curves of F-actin alone and in complexes with cofilin
obtained at various cofilin ⁄ actin monomer molar ratios. The F-actin
concentration (25 l
M) was held constant, and the cofilin concentra-
tions were as indicated for each curve. Other conditions:
30 m
M Hepes, pH 7.3, 2 m M MgCl
2
and 0.2 mM ADP. Heating rate
1KÆmin
)1

. Adapted from Dedova et al. [29] .
Actin unfolding and aggregation D. I. Levitsky et al.
4286 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
It has been proposed that formins can regulate actin
filament flexibility via long-range allosteric interactions
in the filament.
Other interactions affecting F-actin thermal
stability
Actin filaments can interact with many other proteins.
First, interaction of F-actin with myosin heads and
muscle regulatory proteins (tropomyosin and troponin
in striated muscle or caldesmon and calponin in
smooth muscle) plays a key role in the molecular
mechanism and regulation of muscle contraction. We
used the DSC method to investigate how these
proteins affect the thermal stability of F-actin.
Although the binding of myosin heads to F-actin
significantly increased the thermal stability of myosin,
it had no appreciable influence on the thermal unfold-
ing of F-actin either in the absence [74] or presence
[41,75] of phalloidin, presumably because the thermal
denaturation of actin-bound myosin heads occurred
at a much lower temperature than that of F-actin.
Also, we did not observe any changes in the thermal
unfolding of F-actin in complex with smooth muscle
tropomyosin [52], caldesmon and calponin, as well as
in complex with skeletal a-tropomyosin [76,77]. In the
case of tropomyosin, this is easily explained by the
fact that it dissociates from F-actin and denatures
before the thermal denaturation of F-actin [52,76,77].

However, we observed a pronounced change in the
thermal denaturation of F-actin upon addition of
troponin I, one of the components of the troponin
complex. These changes were expressed as a significant
decrease in the enthalpy and cooperativity of the melting
of F-actin [76]. The data indicated that there is a direct
interaction between troponin I and F-actin. The effect
was much weaker if troponin I was added to F-actin
stabilized by AlF
4
)
[76]. Apparently, stabilization of
F-actin by AlF
4
)
prevents the effects of troponin I on
the actin filament structure.
A very interesting effect was observed by Gicquaud
[78] who used the DSC method for studies on the direct
interaction of F-actin with membrane lipids. It has been
shown that due to the interaction with liposomes, actin
undergoes to a major conformational change resulting
in the complete disappearance of its thermal transition
on the DSC profile.
Proposed mechanism for the thermal
denaturation of F-actin
It is obvious that the thermal denaturation of such
a complicated system as the actin filament cannot be
explained by simple models that describe the thermal
unfolding of proteins. We attempted to describe a

mechanism for F-actin thermal denaturation using
special DSC approaches.
It is known that, upon irreversible thermal denatur-
ation of many oligomeric proteins and enzymes, the
maximum thermal transition temperature increases
remarkably with the increase in protein concentration.
Recent views, such as the dependence of T
m
on the
protein concentration, suggest the presence of a revers-
ible dissociation stage for the subunits of the oligomer
prior to their irreversible denaturation [79,80]. We
applied this approach to F-actin and have shown that
the T
m
value depends strongly on the protein concen-
tration. The T
m
of the F-actin thermal transition
increased by more than 3 °C when the concentration
of F-actin was increased from 0.5 to 2.5 mgÆmL
)1
[46].
A similar dependence of the T
m
value on the protein
concentration was demonstrated for F-actin stabilized
by phalloidin: in this case, the T
m
increased from 81 to

84 °C as the actin concentration increased from 0.5 to
2.0 mgÆmL
)1
. However, such dependence was much
less pronounced in the presence of AlF
4
)
; in this case,
the T
m
value increased by only 1.1 °C. As expected,
for monomeric G-actin, the T
m
value for its thermal
transition was independent of the protein concentra-
tion [46]. These results are consistent with a dissocia-
tive mechanism proposed for the irreversible thermal
denaturation of other oligomeric proteins [79,80].
Combining these DSC results with the above-
described effects of ADP on the thermal unfolding of
F-actin, we proposed the following dissociative mecha-
nism for the thermal denaturation of F-actin [46]. We
propose that at least two reversible stages precede the
irreversible thermal unfolding of F-actin. One is the
dissociation of nucleotide (ADP) from the nucleotide-
binding sites of actin subunits, and the other is the
fragmentation of actin filaments or the dissociation of
relatively short oligomers from the filament. The
proposed mechanism for the thermal denaturation of
actin filaments is described in Fig. 4.

During heating, destabilization of actin filaments
occurs, leading to increased mobility of the filament
and weakening of the bonds between subunits. As a
result, short oligomers dissociate from one end of
the polar actin filament [presumably from the
pointed (‘)’) end where the dissociation rate is much
higher than on growing barbed (‘+’) end of the fila-
ment]. These oligomers either lose bound ADP and
then immediately denature and aggregate, or the
ADP-containing oligomers may bind again to the
actin filament, but they most likely bind to another
filament and to the other end (the ‘+’-end). This
D. I. Levitsky et al. Actin unfolding and aggregation
FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS 4287
mechanism (Fig. 4) explains why the thermal unfold-
ing of F-actin depends on both ADP and protein
concentration. The presence of excess free ADP in
solution impedes the thermally induced reversible dis-
sociation of tightly bound ADP from actin subunits,
whereas the increase in the protein concentration
increases the number of actin filaments and, corre-
spondingly, the number of ‘+’-ends to which short
actin oligomers can bind.
It is important to note that two F-actin stabilizers,
phalloidin and AlF
4
)
, differ from one another in the
extent to which they influence the dependence of T
m

on the protein concentration [46]. Both stabilizers
increase the thermal stability of F-actin substantially
(Fig. 2), although independently of each other. Phalloi-
din increases the thermal stability of F-actin by
strengthening the bonds between adjacent subunits in
the actin filament, whereas AlF
4
)
demonstrates a simi-
lar effect by trapping ADP in the nucleotide-binding
site of actin. Thus, the complex ADP–AlF
4
)
–actin,
which mimics the ADP–P
i
–actin intermediate state of
actin polymerization, should prevent the dissociation
of ADP from actin subunits and, by contrast, stimu-
late the binding of actin monomers or short oligomers
to the barbed (‘+’) end of the filament. According to
the mechanism proposed above, this means that AlF
4
)
may influence both reversible stages preceding the irre-
versible thermal denaturation of F-actin, thus making
the dependence of T
m
on the F-actin concentration less
pronounced and more similar to those characteristic of

monomeric proteins.
We are aware that the proposed mechanism for the
thermal denaturation of F-actin [46] (Fig. 4) is based
mainly on indirect data (i.e. the dependence of T
m
on the actin and ADP concentrations). However,
Gicquaud and Heppel directly observed two reversible
steps preceding the irreversible thermal unfolding of
F-actin [81]. These steps were revealed by measuring
the temperature dependences of the fluorescence of
F-actin labeled with pyrene at Cys374 and e-ATP
bound to F-actin at the nucleotide-binding site [81].
Although the authors did not interpret these results, it
is possible that the changes in fluorescence observed
before the irreversible thermal denaturation of F-actin
may correspond to dissociation of nucleotide from the
actin subunits and the dissociation of subunits from
the filament.
Let us briefly consider the main statements of the
proposed mechanism (Fig. 4). One is that the actin
filament denatures, not as a whole, but as separate
short oligomers which dissociate from the filament
during heating. In favor of this assumption are the
results of recent studies on the interaction of F-actin
with sHSPs [82,83]. It has been found that these pro-
teins effectively prevent the aggregation of thermally
denatured actin by forming small soluble complexes
(Fig. 4), the size of these complexes being much less
than that of intact F-actin [82,83] (see below).
The other important statement of the proposed

dissociative mechanism of the F-actin thermal dena-
turation is that the ADP-containing short oligomers
dissociated from the ‘)’-end of the actin filament may
bind again to the ‘+’-ends of the filaments (Fig. 4). In
favor of this assumption is a well-established pheno-
menon of end-to-end annealing of actin filaments, i.e.
the formation of long polar actin filaments from very
short filaments due to an interaction between the ‘+’-
and ‘)’-ends of the filaments [84–86]. The annealing
process proceeds spontaneously, and, unlike actin
polymerization, the presence of ATP-containing
G-actin monomers and ATP hydrolysis is not required
[84]. Although the annealing is a rather slow process,
its rate increases linearly with actin concentration [87],
D
D
D
D
D
D
D
D
D
DD
D
D
D
D
D
D

D
D
D
D
D
D
D
D
D
DD
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
DD
D
D

D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
DD
D D
D
D
D
D
D
D
D
D
D
D
D

D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D

DD
D
D
D
(–))+(
(+)
(–)
(+)
(–)
D
D
D
F-actin
Denaturation
Aggregation
sHSP
Fig. 4. The proposed mechanism for the thermal denaturation and
aggregation of F-actin filaments, as well as the protective effect of
sHSPs on the aggregation of thermally denatured actin. ADP mole-
cules, both free in solution and bound to actin monomers, are
marked as D. The hatched short oligomers of actin are those that
dissociate upon heating from the pointed (‘)’) end of actin filament
and can bind to the barbed (‘+’) end of the same or other filament.
See the text for more details.
Actin unfolding and aggregation D. I. Levitsky et al.
4288 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
in good agreement with the above proposed dissocia-
tive mechanism for F-actin thermal denaturation. Fur-
thermore, it has been shown that the annealing rate
near a hydrophobic surface in the presence of crowd-

ing agents is substantially higher (by a factor 20) than
in solution, and this rate is also much higher than the
rate of actin polymerization [87]. These results support
our viewpoint that short oligomers which have dissoci-
ated from the actin filament in the initial stages of
F-actin thermal denaturation may again bind to the
filaments (Fig. 4).
Aggregation of thermally denatured
actin
The irreversible thermal denaturation of actin is nor-
mally accompanied by aggregation of the protein.
However, the aggregation process is quite different for
G-actin and F-actin.
‘Inactivated’ G-actin
It has been found that thermal denaturation of mono-
meric G-actin (after 30 min incubation at 60 or 70 °C)
leads to the formation of so-called ‘inactivated actin’,
which is represented by stable homogeneous aggregates
consisting of a limited number of unfolded protein
molecules [88–91]. Inactivated actin can be obtained by
both heat treatment and at moderate concentrations of
urea (4 m) or guanidinium chloride (1.5 m) [90,91].
According to recent studies, inactivated actin is the
off-pathway misfolded state stabilized by aggregation
of partially folded actin molecules [92]. It has been
shown that inactivated actin represents a specific stable
aggregate (named ‘monodisperse associate’ by the
authors), which is characterized by a sedimentation
constant of  20S (compared with  3.3S for intact
G-actin) [88,90]. The hydrodynamic radius of ther-

mally denatured G-actin, as measured by dynamic
light scattering, was  12 nm (V. V. Mikhailova,
unpublished results). A Stokes radius of  8 nm (com-
pared with 2.8 nm for intact G-actin) and an apparent
molecular mass of  700 kDa have been estimated for
inactivated actin using size-exclusion chromatography
[90,91]. These data suggest that the inactivated actin
associate contains up to 15–17 monomers of denatured
actin [91].
It is important to note that inactivated actin can
exist in the form of stable monodisperse associates
only under very specific conditions, at very low ionic
strength. Addition of salts induces the aggregation of
inactivated actin, which is accompanied by a signifi-
cant increase in light scattering [93].
Heat-induced aggregation of F-actin and effects
of sHSPs
F-actin aggregates easily when denatured, and its ther-
mally induced aggregation is accompanied by a signifi-
cant increase in light scattering. Very good correlation
was found between thermal denaturation and the
aggregation of F-actin [46,82] (Fig. 5). Upon thermal
stabilization of F-actin (e.g. in the presence of ADP
[46] or phalloidin), both the thermal transition
measured by DSC and the aggregation curve measured
by light scattering shifted to a higher temperature.
F-actin denatures and aggregates at a rather high
temperature, > 50 °C (Fig. 5), much higher than the
40 50 60 70
0

100
200
300
400
0
5
10
15
20
25
F-actin
F-actin + Hsp27-3D
Aggregation (Light scattering)
Light scattering (rel. u.)
Tem
p
erature (°C)
F-actin
F-actin + Hsp27-3D
Apparent heat capacity (
µ
W)
Thermal unfolding (DSC)
A
B
Fig. 5. Thermal unfolding measured by DSC (A) and heat-induced
aggregation measured as an increase in light scattering at 350 nm
(B) of F-actin (1.0 mgÆmL
)1
) in the absence and presence of small

heat shock protein Hsp27 with mutations mimicking its phosphory-
lation, denoted as Hsp27-3D (1.0 mgÆmL
)1
). The DSC and light-scat-
tering measurements were performed under the same conditions
(30 m
M Hepes, pH 7.3, 100 mM KCl and 1 mM MgCl
2
) and at the
same heating rate of 1 KÆmin
)1
. Adapted from Pivovarova et al.
[82,83].
D. I. Levitsky et al. Actin unfolding and aggregation
FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS 4289
temperature in the cell. The question arises as to
whether the thermal denaturation and aggregation of
actin filaments can occur at lower temperatures. To
answer this, we performed special experiments and
found that F-actin denatures and aggregates, although
rather slowly, upon prolonged incubation at heat-
shock temperature (43 °C) in the absence of added nu-
cleotides (A. V. Pivovarova & D. I. Levitsky, unpub-
lished results). This means that different types of
stress, for example heat shock, can induce actin
unfolding, leading to the disruption of actin filaments
and the aggregation of fully or partially denatured
actin. The accumulation of aggregated proteins is dan-
gerous for the cell, and this is especially important in
the case of abundant proteins, such as actin. There are

different mechanisms for preventing formation of
insoluble aggregates, and the sHSPs play an important
role in this process.
sHSPs comprise a large and diverse family of pro-
teins with molecular masses from 12 to 43 kDa.
Almost all sHSPs assemble into large oligomeric
complexes that vary in structure and the number of
monomers [94]. The dynamic organization of sHSP
oligomers seems to be important for regulation of their
activity. In vitro, sHSPs act as molecular chaperones in
preventing partially or fully unfolded proteins from
irreversible aggregation and insolubilization [94–96].
Different protein kinases phosphorylate sHSPs, and by
this means might affect their oligomeric structure and
chaperone activity [94,97].
The expression of some sHSPs (Hsp27, aB-crystal-
lin) is increased in response to different kinds of injury,
such as heat shock and ischemia, and their content is
particularly high in muscle tissues, where the expres-
sion of actin is also very high. It seems likely that one
of the main functions of sHSPs in muscles is their
interaction with actin under unfavorable conditions.
Multiple publications indicate that different stress con-
ditions might induce the translocation of sHSPs from
the cytosol to the cytoskeleton and this translocation
can result in the stabilization of actin filaments [98–
101]. Very recently, it has been shown that, under
heat-shock conditions (upon incubation at 43 °C),
aB-crystallin, a member of the sHSP family, interacts
directly with actin in immunoprecipitation experiments

and associates with actin filaments in living cells, and
that this in vivo interaction of aB-crystallin prevents
the heat-induced disorganization of actin filaments
[102]. However, no effects of aB-crystallin were
observed in unstressed cells. These facts agree with the
data that in vitro sHSPs do not interact with intact
actin filaments [82,103], but prevent heat-induced
aggregation of actin [82,93,103,104]. Thus, it seems
probable that sHSPs interact with actin filaments only
under stress conditions, such as heat shock, but the
exact molecular mechanism by which this interaction
occurs is not clearly understood.
We have recently shown that, in solution, some
recombinant sHSPs (chicken Hsp24, human Hsp27
and their 3D mutants mimicking phosphorylation)
have no influence on the thermal unfolding of F-actin
as measured by DSC, but they effectively prevent the
aggregation of thermally denatured actin [82,83]. Fur-
thermore, we used co-sedimentation experiments to
analyze the interaction between denatured actin and
the S15D ⁄ S78D ⁄ S82D mutant construct of Hsp27
(denoted as Hsp27-3D), which has been proposed to
mimic the properties of phosphorylated Hsp27 in vitro
[105]. It has been shown that, after heating F-actin in
the presence of Hsp27-3D, denatured actin does not
precipitate upon high-speed centrifugation and is
found in the supernatant together with Hsp27-3D,
whereas both intact F-actin and F-actin heated in the
absence of Hsp27-3D precipitate fully under the same
conditions [82]. From these data, we propose that

Hsp27-3D and other sHSPs can form relatively small,
stable and highly soluble complexes with denatured
actin, and this is the mechanism by which sHSPs
prevent the aggregation of F-actin.
In further studies, we applied dynamic light scatter-
ing, analytical ultracentrifugation and size-exclusion
chromatography to examine the properties of the com-
plexes of Hsp27-3D with denatured actin. Hsp27-3D
was particularly useful for such experiments, due to its
very small size, it being much smaller than wild-type
human Hsp27 and many other sHSPs, which usually
form large oligomers [106–108]. Our results clearly dem-
onstrated that, upon heating, the thermal unfolding of
F-actin is accompanied by the formation of stable, solu-
ble complexes of Hsp27-3D with denatured actin [83].
All the methods showed that the size of actin–Hsp27-3D
complexes decreases as the Hsp27-3D concentration in
the incubation mixture increases and that saturation
occurs at approximately equimolar concentrations of
Hsp27-3D and actin. Under these conditions, the com-
plexes exhibit a hydrodynamic radius of  16 nm, a
sedimentation coefficient of 17–20S and a molecular
mass of  2 MDa [83]. We suppose that Hsp27-3D (and
probably other sHSPs) binds to denatured actin mono-
mers or short oligomers dissociated from the actin fila-
ment upon heating and protects them from subsequent
aggregation by forming relatively small and highly
soluble complexes, whose size is much less than that of
intact F-actin or the aggregates of denatured actin. This
mechanism (Fig. 4) might explain how sHSPs prevent

the aggregation of denatured actin and so protect the
Actin unfolding and aggregation D. I. Levitsky et al.
4290 FEBS Journal 275 (2008) 4280–4295 ª 2008 The Authors Journal compilation ª 2008 FEBS
cytoskeleton and the whole cell from damage caused by
the accumulation of large insoluble aggregates upon
heat shock conditions.
Concluding remarks
There is little doubt that the actin filament is a highly
dynamic structure. Recent years have produced a
growing body of data suggesting that F-actin can exist
in different structural states, and that transitions
between these states may play an important role in the
cytoskeleton dynamics and in the contractile cycle of
actomyosin. The stabilization or destabilization of
actin filaments by different factors can be explained by
transitions of F-actin from one structural state to
another, and these transitions can be clearly demon-
strated in DSC studies as the changes in the F-actin
thermal unfolding. Thus, DSC in combination with
other methods provides a promising approach for
studying highly cooperative structural changes in actin
filaments.
In this review, we have summarized the DSC data
that exist on the thermal unfolding of actin, paying
special attention to the effects of stabilization and
destabilization of F-actin by different factors,
expressed as the changes in the thermal stability of
F-actin. In general, all the DSC data summarized here
are in good agreement with the data obtained by other
methods. Furthermore, in some cases, the DSC results

can provide insight into the mechanism of structural
changes occurring in the actin filament (e.g. in the case
of highly cooperative destabilization of F-actin by
cofilin).
We have also summarized and discussed the existing
data on the aggregation of actin induced by its thermal
denaturation. It has been shown for the first time using
DSC that sHSPs have no influence on the thermal
unfolding of F-actin, although they effectively prevent
its subsequent aggregation by forming relatively small
soluble complexes with denatured actin. These results
provide new insight into the mechanism by which
sHSPs prevent the aggregation of F-actin induced by
its thermal denaturation.
Finally, combining the DSC results with data
obtained from other methods, we have proposed a
model that can describe the mechanism of the thermal
unfolding of F-actin and its subsequent aggregation,
including the effects of sHSPs (Fig. 4). This mecha-
nism may explain some specific features of F-actin
thermal unfolding and, furthermore, how sHSPs
protect the cytoskeleton against damage caused by
accumulation of large, insoluble aggregates under heat
shock conditions.
Acknowledgements
This study was supported by the Russian Foundation
for Basic Research (grant 06-04-48343 to DI Levitsky)
and the program ‘Molecular and Cell Biology’ of the
Russian Academy of Sciences.
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