Tài liệu Báo cáo khoa học: Thermal unfolding of smooth muscle and nonmuscle tropomyosin a-homodimers with alternatively spliced exons docx

Thermal unfolding of smooth muscle and nonmuscle
tropomyosin a-homodimers with alternatively spliced
exons
Elena Kremneva
1
, Olga Nikolaeva
2
, Robin Maytum
3
*, Alexander M. Arutyunyan
2
,
Sergei Yu. Kleimenov
1
, Michael A. Geeves
3
and Dmitrii I. Levitsky
1,2
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 Department of Biosciences, University of Kent at Canterbury, UK
Tropomyosins (Tm) are a family of actin-binding,
a-helical coiled-coil proteins found in most eukaryotic
cells [1]. They bind to actin cooperatively along the
length of actin filaments and confer cooperativity
upon the interaction of actin with myosin heads [2].
The Tm molecules are parallel homo- or hetero-
dimers (encoded from the same or different genes) of
two a-helical chains of identical length, although the
length can vary according to isoform type. In mam-
malian cells, alternative splicing produces a variety

of muscle and nonmuscle isoforms from four differ-
ent genes [1]. Muscle cells express two major iso-
forms of Tm (a and b), each containing 284
residues. Smooth and skeletal muscles express differ-
ent isoforms resulting from alternative splicing of the
a and b genes.
There are two major classes of a-Tm: long (or high
relative molecular mass) Tm (284 residues) are
expressed in muscle and nonmuscle cells whereas short
(or low relative molecular mass) Tm (247 residues) are
Keywords
tropomyosin; actin; thermal unfolding;
differential scanning calorimetry; circular
dichroism
Correspondence
D.I. Levitsky, A.N. Bach Institute of
Biochemistry, Russian Academy of
Sciences, Leninsky prosp. 33,
119071 Moscow, Russia
Fax: +7095 9542732
E-mail:
*Present address
School of Biological Sciences, Queen Mary,
University of London, Mile End Road,
London E1 4NS, UK
(Received 14 September 2005, revised
2 December 2005, accepted 6 December
2005)
doi:10.1111/j.1742-4658.2005.05092.x
We used differential scanning calorimetry (DSC) and circular dichroism

(CD) to investigate thermal unfolding of recombinant fibroblast isoforms
of a-tropomyosin (Tm) in comparison with that of smooth muscle Tm.
These two nonmuscle Tm isoforms 5a and 5b differ internally only by
exons 6b ⁄ 6a, and they both differ from smooth muscle Tm by the
N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We
show that the presence of exon 1b dramatically decreases the measurable
calorimetric enthalpy of the thermal unfolding of Tm observed with DSC,
although it has no influence on the a-helix content of Tm or on the end-to-
end interaction between Tm dimers. The results suggest that a significant
part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds
noncooperatively, with the enthalpy no longer visible in the cooperative
thermal transitions measured. On the other hand, both DSC and CD stud-
ies show that replacement of muscle exons 1a and 2a by nonmuscle exon
1b not only increases the thermal stability of the N-terminal part of Tm,
but also significantly stabilizes Tm by shifting the major thermal transition
of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to
additional increase in the a-Tm thermal stability. Thus, our data show for
the first time a significant difference in the thermal unfolding between
muscle and nonmuscle a-Tm isoforms, and indicate that replacement of
alternatively spliced exons alters the stability of the entire Tm molecule.
Abbreviations
CD, circular dichroism; DSC, differential scanning calorimetry; Tm, tropomyosin; smTm, recombinant smooth muscle Tm; Tm5a and Tm5b,
recombinant fibroblast Tm isoforms with alternatively spliced exons 6b and 6a, respectively.
588 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS
found in nonmuscle cells [1,2]. In the short a-Tm iso-
forms, a single exon (exon 1b, encoding residues 1–43)
replaces the first two exons (exons 1a and 2 encoding
residues 1–80) in long a-Tm (see Fig. 1). The two other
alternatively spliced exons in a-Tm are exons 6 and 9.
Possible relationships between the alternatively spliced

exons and the functional properties of the a-Tm iso-
forms have been addressed in previous studies. The
actin binding properties are mainly determined by the
two terminal regions, encoded by exons 1 and 9 [3].
Amino acid replacements in the region encoded by
exon 2 of lobster muscle Tm altered end-to-end inter-
action between Tm molecules and actin affinity of Tm
[4]. The replacement of muscle-specific exon 6b by
nonmuscle exon 6a in recombinant rat smooth muscle
a-Tm was shown to increase the actin affinity of a-Tm
[5], and the same exon exchange has a similar effect
between fibroblast Tm5a and 5b isoforms [6]. This
replacement in rat fibroblast a-Tm has been shown to
increase the calcium sensitivity of the regulation of
acto-myosin interaction in the presence of troponin [7].
Our aim in the present study was to determine how
the alternatively spliced exons 1, 2, and 6 affect the
structural properties of the a-Tm molecule. For this
purpose, we have used the smooth muscle a-Tm
(smTm) and the two a-Tm fibroblast isoforms Tm5a
and Tm5b, as shown in Fig. 1. All of these a-Tm iso-
forms have a smooth muscle-like exon 9d. Both of the
fibroblast isoforms are shorter than smTm because
they lack exon 2 due to replacement of muscle exons
1a and 2a by nonmuscle exon 1b. The short fibroblast
isoforms Tm5a and Tm5b differ internally only by
exon 6. Confusingly, Tm5a has exon 6b, whereas
Tm5b possesses exon 6a (Fig. 1). Thus, comparison of
Tm5a with smTm allows study of the effects of
replacement of muscle exons 1a and 2a by nonmuscle

exon 1b, and comparison of Tm5a with Tm5b allows
the effects of exchange of exon 6 to be studied.
Studies of thermal unfolding of Tm may provide
valuable information on the structure of Tm both free
in solution and bound to actin. Thermal unfolding of
the Tm coiled-coil can be successfully studied by differ-
ent methods such as CD, fluorescence, and DSC.
Many authors have used DSC for detailed investiga-
tion of the thermal unfolding of Tm from skeletal and
smooth muscles [8–14]. Other authors successfully used
CD to study the thermal unfolding of homo- and het-
erodimers of skeletal [15,16] and smooth [17–19]
muscle Tm, their mutants [20–23], and numerous
coiled-coil model peptides corresponding to the N- and
C-terminal parts of the Tm molecule [24–29]. CD
measures whole the process of the unfolding of a-helical
coiled-coil of Tm, whereas DSC generally gives reliable
information only on the thermal unfolding of those
parts of Tm which melt cooperatively with significant
changes in enthalpy. On the other hand, DSC is capable
of monitoring the thermal unfolding of Tm when
bound to actin [13,14,30], whereas CD is of limited use
in the presence of actin as the signal from the six- or
sevenfold molar excess of actin dominates the signal.
Fluorescent labels can also be used in the presence of
actin but may report only the local unfolding of regions
close to the label. Thus, each method has strong and
weak sides, but combination of both the DSC and CD
provides a powerful approach for structural characteri-
zation of Tm and its interaction with actin.

In this work we have used DSC and CD to character-
ize the thermal unfolding of smTm, Tm5a, and Tm 5b.
We have shown that exon replacements alter the stabil-
ity of the entire Tm molecule. The replacement of mus-
cle exons 1a and 2a in smTm by nonmuscle exon 1b
in Tm5a (and Tm5b) dramatically decreases the total
measurable calorimetric enthalpy of the thermal unfold-
ing of Tm, although it has no influence on the a-helix
content of Tm and on end-to-end interaction between
Tm dimers. On the other hand, this replacement signifi-
cantly stabilizes Tm, increasing the temperature of the
cooperative thermal transitions of Tm. Replacement of
exon 6b in Tm5a by exon 6a in Tm5b leads to addi-
tional increase in the thermal stability of Tm.
Results
Thermal unfolding of recombinant Tm: DSC
studies
The excess heat capacity curves obtained for recombin-
ant smTm, Tm5a, and Tm5b are presented in Fig. 2.
Judging by the complete reproducibility of the calori-
metric traces after cooling the sample within the DSC
cell, the heat-induced unfolding of Tm was fully
reversible. For both fibroblast Tms the major trans-
ition takes place at a higher temperature than for
Fig. 1. Exon structure of smooth muscle a-Tm and the two fibro-
blast isoforms Tm5a and Tm5b with constitutive exons are shown
in white, smooth muscle exons in light grey, and nonmuscle exons
in dark grey.
E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin
FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 589

smTm (T
m
¼ 34.6 °C), by  5 °C for Tm5a (T
m
¼
39.3 °C) and by  8 °C for Tm5b (T
m
¼ 42.7 °C).
Surprisingly, the calorimetric enthalpy, DH
cal
, of the
thermal unfolding of both Tm5a and Tm5b was much
less than that for smTm, and it represented only
 60% of the DH
cal
value for smTm (Table 1).
Another difference between Tm5a, Tm5b and smTm
is that both fibroblast Tm isoforms possess, in addition
to the major sharp thermal transition and the shoulder
at 25–35 °C, a broad low-cooperative transition at 55–
65 °C (Fig. 2). It is important to note that it was diffi-
cult to reveal this broad high-temperature transition in
our apparatus as it was small and difficult to distin-
guish from the instrumental baseline. Therefore we
used a specially developed method to avoid artefacts
caused by subtraction of the instrumental baseline (see
Experimental procedures). The heat capacity curves
obtained in this way were subjected to deconvolution
analysis (Fig. 3), which shows that the profiles can be
decomposed into three separate thermal transitions

(calorimetric domains).
The first, the low-temperature transition at  33 °C
(Fig. 3) represents 15–20% of the total calorimetric
enthalpy (Table 1). The second transition, which repre-
sents more than 60% of the total enthalpy, is similar
in enthalpy but more stable in Tm5b (T
m
¼ 42.7 °C)
than in Tm5a (T
m
¼ 39.3 °C). The third transition
again represents  20% of the total enthalpy, and it is
very similar for Tm5a and Tm5b (T
m
 57.5 °C)
(Fig. 3, Table 1). Since the only major difference
between the two Tms is in transition 2 this is likely to
represent the unfolding of the region of Tm containing
the alternately spliced exon 6.
The heat capacity curve for smTm also reveals three
transitions. However, the first and third transitions are
broader and are not as clearly resolved from the second,
main transition. SmTm and Tm5a are identical in struc-
ture except for the alternative exons 1 and 2 therefore
differences in the unfolding thermogram should reflect
the role of these exons in thermal stability. As seen from
Table 1 all three transitions for smTm are at a lower
temperature than for Tm5a (by 2, 4.7, and 18.4 °C,
respectively) while the total enthalpy of smTm unfolding
is 1.6 times that of Tm5a. Note however, that the parti-

tion of the total enthalpy between the three transitions
remains at approximately 20, 60, and 20%, respectively.
The major difference between the thermal unfolding
of smTm and Tm5a is on the T
m
of the third trans-
ition which is destabilized by almost 20 °C. It is there-
fore most likely to reflect the N-terminal part of the
molecule dominated by the exchange of exon 1b for
exons 1a and 2a. However all three calorimetric
domains are destabilized by the exon change and the
total enthalpy is increased. This suggests that the
N-terminal part of the molecule is affecting the stabil-
ity of the entire molecule.
End-to-end interaction
One possible reason for the differences in the thermal
stability between smTm and Tm5a is that it is related
Fig. 2. Temperature dependence of the excess heat capacity (C
p
)
of smTm, Tm5a, and Tm5b. The protein concentration was
1.2 mgÆmL
)1
. Other conditions: 30 mM Hepes pH 7.3, 100 mM KCl,
and 1 m
M MgCl
2
. The heating rate was 1 KÆmin
)1
.

Table 1. Calorimetric parameters obtained from the DSC data for individual thermal transitions (calorimetric domains) of smTm, Tm5a, and
Tm5b. The parameters were extracted from Fig. 3. The error of the given values of transition temperature (T
m
) did not exceed ± 0.2 °C. The
relative error of the given values of calorimetric enthalpy, DH
cal
, did not exceed ± 8%.
Sample
Transition 1 Transition 2 Transition 3
Total DH
(kJÆmol
)1
)DH (kJÆmol
)1
) T
m
(°C) DH (kJÆmol
)1
) T
m
(°C) DH (kJÆmol
)1
) T
m
(°C)
smTm 150 30.9 515 34.6 125 39.0 790
Tm5a 100 32.9 310 39.3 85 57.4 495
Tm5b 75 33.5 300 42.7 110 57.9 485
Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al.
590 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS

to different end-to-end interactions of the Tm species.
Indeed, Tm5a not only differs from smTm by the
sequence encoded by the N-terminal exon 1 (Fig. 1),
but also because the recombinant smTm was expressed
with an N-terminal Ala-Ser extension to substitute for
the N-terminal acetylation of the native Tm [31]. The
fibroblast Tm5a (and Tm5b) has a natural five-amino
acid extension in exon 1b (Ala-Gly-Ser-Ser-Ser) in
comparison to exon 1a [6,7,32]. These differences in
the sequence might affect end-to-end interaction
between Tm dimers and could influence the observed
enthalpy of the thermal unfolding.
The strength of the end-to-end interaction between
Tm dimers can be estimated by viscometry, and the
interaction is known to be highly sensitive to ionic
strength [17,33]. We measured the viscosity and calori-
metric enthalpy (total D H
cal
) of smTm and Tm5a at dif-
ferent ionic strengths as shown in Fig. 4. The relative
viscosity of Tm5a was very similar to that of smTm at
all ionic strengths (Fig. 4A). At 500 mm KCl the visco-
sity was similar to that of water consistent with the
absence of any significant polymerization of Tm. Over
the range of ionic strengths studied, the DH
cal
value for
Tm5a was consistently smaller by 40–50% than that of
smTm (Fig. 4B). This means that end-to-end inter-
actions of Tm play little role in the difference in the

calorimetric enthalpy between smTm and fibroblast
Tm5a.
Thermal unfolding of recombinant Tm: CD
studies
The CD spectra of smTm, Tm5a, and Tm5b, which
are shown in Fig. 5, possessed the double minima at
208 and 222 nm characteristic of the correctly folded,
fully a-helical structure of Tm. The CD spectra of
Tm5a and Tm5b were virtually identical to each other
and to the spectrum of smTm.
Fig. 3. Deconvolution analysis of the heat sorption curves of smTm
(A), Tm5a (B), and Tm5b (C). Conditions were the same as in
Fig. 2. Solid lines represent the experimental curves after subtrac-
tion of instrumental and chemical baselines, and dotted lines repre-
sent the individual thermal transitions (calorimetric domains)
obtained from fitting the data to the nontwo-state model.
Fig. 4. Effects of ionic strength on the relative viscosity (A) and
calorimetric enthalpy DH
cal
(B) of smTm and Tm5a. The concen-
tration of Tm was 0.5 mgÆmL
)1
in viscosity experiments and
1.2 mgÆmL
)1
in DSC experiments.
E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin
FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 591
The thermal unfolding of the a-helix in smTm,
Tm5a, and Tm5b was measured by the temperature

dependence of the CD elipticity at 222 nm (Fig. 6).
These CD studies were performed under similar condi-
tions and at the same heating rate (1 °CÆmin
)1
) as the
DSC measurements except that lower protein concen-
tration (0.1 mgÆmL
)1
) and sodium phosphate buffer
was used instead of Hepes. However, the buffer
replacement had no significant influence on the ther-
mal unfolding of any Tm measured by DSC (data not
shown). Melting was fully reversible with a repeated
melting curve being identical to the initial ones. The
helix unfolding profile of recombinant smTm (Fig. 6A)
agrees with previous CD studies of the native aa-homo-
dimers of smooth muscle Tm [18,19]. The transition
midpoint for smTm ( 34 °C) is similar to the max-
imum temperature ( T
m
¼ 34.6 °C) of the heat capacity
curve measured by DSC under similar conditions
(Fig. 2).
The CD melting curve of Tm5a and 5b are similar
and differ significantly from that for smTm, having
broader (lower cooperativity) changes in ellipticity
(Fig. 6A). The first derivative of the data, dh ⁄ dt, shows
three well distinguished peaks on the profile of Tm5a
(Fig. 6B), with the major peak at 39.6 °C and two
small peaks at  30 °C and  50 °C similar to those

observed in DSC. The CD melting profile of Tm5b
also shows, like Tm5a, significant changes in ellipticity
long before and well after the major peak at 41.2 °C
(Fig. 6B). In general, these CD data are in very good
agreement with DSC results presented above (Figs 2
and 3). Both DSC and CD show that the magnitude
of the main thermal transition is much higher for
smTm than for Tm5a and Tm5b. Thus, both methods
show a marked difference in the thermal unfolding
between smooth and nonmuscle Tms.
DSC studies of the thermal unfolding of Tm
in the presence of F-actin
Previous studies have shown that DSC can be success-
fully used for studies of the actin-induced changes in
the thermal unfolding of Tm [13,14,30]. Interaction of
smooth muscle Tm with F-actin caused a 2–6 °C shift
in the Tm thermal transition to higher temperature,
depending on the Tm : actin molar ratio [13]. In the
present work, we apply this approach to characterize
the thermal unfolding of smTm, Tm5a, and Tm5b
complexed with F-actin.
DSC experiments with Tm–F-actin complexes were
performed in the presence of excess of Tm, i.e. under
conditions where actin filaments should be fully satur-
ated with Tm molecules. The character of the thermal
Fig. 5. CD spectra in the far-UV region of smTm, Tm5a, and Tm5b.
The spectra were measured at 20 °C. Protein concentration was
0.1 mgÆmL
)1
in all cases. Other conditions: 50 mM sodium phos-

phate buffer pH 7.3 containing 100 m
M NaCl and 1 mM MgCl
2
.
Fig. 6. The thermal unfolding profiles of smTm, Tm5a, and Tm5b
as measured by CD. (A) The temperature dependence of a-helix
content measured as the ellipticity at 222 nm. The heating rate
was 1 KÆmin
)1
. The protein concentration was 0.1 mgÆmL
)1
in all
cases. The heating rate was 1 KÆmin
)1
. Other conditions: 50 mM
sodium phosphate buffer pH 7.3 containing 100 mM NaCl, 1 mM
MgCl
2
and 1 mM b-mercaptoethanol. (B) First derivative profiles for
the data shown in (A).
Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al.
592 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS
denaturation of Tm was noticeably changed when
bound to F-actin. This is reflected in the appearance
of a new highly cooperative thermal transition at
higher temperature (Fig. 7). The interaction of Tm
with actin had no effect on the thermal denaturation
of F-actin stabilized by phalloidin, which denatures
irreversibly at much higher temperature (80 °C), as
was previously shown for smooth and skeletal muscle

Tms [13,14]. Thus, after heating of the Tm–F-actin
complex to 90 °C (i.e. after complete irreversible dena-
turation of actin) and subsequent cooling, only the
peaks corresponding to the thermal denaturation of
free Tm were observed during a second heating
(dashed line curves on Fig. 7). Thus, we conclude that
the appearance, in the presence of F-actin, of new
peak at higher temperature reflects the actin-induced
changes in the thermal unfolding of Tm. This peak on
Fig. 7 is named as peak 2, whereas the peak named as
peak 1 corresponds to the thermal unfolding of non-
actin-bound Tm in the presence of F-actin, and peak 3
corresponds to the thermal unfolding of free Tm dur-
ing re-heating after complete irreversible denaturation
of actin. It should be noted that in this case we did
not analyse the small thermal transitions of Tm5a and
Tm5b at  57.5 °C (Figs 2 and 3), because irreversible
thermal denaturation of F-actin began in this temper-
ature range.
In the case of smTm, the actin-induced shift in the
thermal transition (i.e. the difference in T
m
between
peak 2 and peak 3) was equal to 3.8 °C, while this
effect was less pronounced for Tm5a (T
2
) T
3
¼ 3 °C)
and Tm5b (T

2
) T
3
¼ 1.4 ° C) (Table 2). However, it is
clearly seen on Fig. 7 that the actin-induced increase in
the enthalpy of thermal unfolding of Tm5a and Tm5b
is more pronounced than for smTm. Indeed the
enthalpy of the actin-induced peak 2 (DH
2
) for Tm5a
is now almost exactly six-sevenths of the value for
smTm consistent with similar enthalpy of unfolding
per unit length. To calculate the actin-induced increase
in enthalpy we measured the enthalpy of the actin-
induced peak 2 (DH
2
) and determined the difference
between DH
2
and the enthalpy of free Tm (DH
3
)
with the enthalpy of peak 1 subtracted (DH
3
) DH
1
)
(Table 2). (It is noteworthy that in the absence of actin
the enthalpy of reversible unfolding of Tm did not sig-
nificantly change even after heating to 90 °C). As a

result, interaction with F-actin increased the enthalpy
of the thermal unfolding by 220–240 kJÆmol
)1
for
Fig. 7. Thermal denaturation of smTm (A), Tm5a (B), and Tm5b (C)
complexed with phalloidin-stabilized F-actin. A temperature region
above 55 °C corresponding to irreversible thermal denaturation of
F-actin stabilized by phalloidin is not shown. Accordingly, small ther-
mal transitions of Tm5a and Tm5b at  57.5 °C were not analysed
in this case. Curves shown by dashed lines (peak 3) were obtained
by reheating the same samples after the first heating to 90 °Cand
following cooling to 5 °C. The heating rate was 1 KÆmin
)1
. The
peaks 1, 2, and 3 are described in the text. In C peak 1 is highligh-
ted as a dotted line showing the curve fit. Concentration of Tm
was 10 l
M for smTm and 15 lM for Tm5a and Tm5b. Other condi-
tions: 46 l
M F-actin, 70 lM phalloidin, 30 mM Hepes, pH 7.3,
100 m
M KCl, 1 mM MgCl
2
,and1mM b-mercaptoethanol.
Table 2. Parameters of the thermal transitions observed by DSC
for smTm, Tm5a, and Tm5b in the presence of F-actin and of heat-
induced dissociation of the Tm–F-actin complexes. The calorimetric
parameters, T
m
and DH

cal
, were extracted from Fig. 7. The parame-
ters T
1
, T
2
, T
3
, DH
1
, DH
2
, and DH
3
correspond to peaks 1, 2, and 3
described in the text. Concentration of Tm was 10 l
M for smTm
and 15 l
M for Tm5a and Tm5b; concentration of phalloidin-stabil-
ized F-actin was 46 l
M. The error of the T
m
values did not
exceed ± 0.2 °C. The relative error of the DH
cal
values did not
exceed ± 10%. The values of T
diss
were calculated from light-scat-
tering data presented in Fig. 8. The error of the T

diss
values did not
exceed ± 0.2 °C.
Sample
T
diss
(°C)
T
m
(°C) DH
cal
(kJÆmol
)1
)
T
1
T
2
T
3
DH
1
DH
2
DH
3
DH
2
–(DH
3

–DH
1
)
smTm 38.5 33.8 38.3 34.5 120 685 680 125
Tm5a 43.2 38.7 43 40 65 575 400 240
Tm5b 43.9 40.3 44.4 43 60 530 370 220
E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin
FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 593
Tm5a and Tm5b, and by only 125 kJÆmol
)1
for smTm
(Table 2).
Thermally induced dissociation of Tm–F-actin
complexes
Previous studies have shown that Tm dissociates
from F-actin on heating, and this process can be
studied by light scattering measurements [13,14,34].
To examine the thermal dissociation of the Tm–
F-actin complexes, we measured the temperature
dependence of light scattering for the complexes of
phalloidin-stabilized F-actin with smTm, Tm5a, and
Tm5b. These measurements were performed under
conditions identical to those of the DSC experiments
presented in Fig. 7. When heated below the denatur-
ation temperature of actin under these conditions,
dissociation of the Tm–F-actin complexes was revers-
ible, as the light scattering intensity increased during
cooling after the first heating and decreased again
during the second heating. The fitted curves to the
normalized light scattering changes of dissociation of

the Tm–F-actin complexes are shown in Fig. 8. The
temperature of midpoint of dissociation (T
diss
), i.e.
the temperature at which a 50% decrease in light
scattering occurs, are presented in Table 2 and com-
pared with the maximum temperature of the actin-
induced transition 2 (T
2
) for the same samples
studied by DSC. This comparison shows a very good
correlation between the T
diss
and T
2
(Table 2). We
therefore conclude that actin-induced changes in the
thermal denaturation of Tm (i.e. the appearance of
the actin-induced peak 2) are associated with dissoci-
ation of Tm from F-actin. This confirms the DSC
data showing that both fibroblast Tms, Tm5a and
Tm5b, dissociate from F-actin at higher temperature
(T
diss
 43.5 °C) than smTm (T
diss
¼ 38.5 °C).
Discussion
In the present work we applied DSC combined with
CD to characterize the thermal unfolding of recombin-

ant fibroblast Tms, Tm5a and Tm5b, in comparison
with that for smooth muscle Tm. The thermal unfold-
ing of smooth muscle Tm isoforms has been investi-
gated in detail by DSC [11–13] and by CD [17–19] and
the results presented here agree with these earlier
works. Thus, smTm expressed with the addition of
Ala-Ser to the N-terminus is a good mimic of native
acetylated Tm.
The thermal unfolding of nonmuscle fibroblast Tms
has not been studied by DSC before. The two a-Tm
fibroblast isoforms Tm5a and 5b are identical in seven
of their eight exons and differ internally only by exon
6 (Fig. 1). The short 25-residue sequences encoded by
exons 6b and 6a do not differ in stabilizing or destabil-
izing clusters defined by Hodges and coworkers in the
hydrophobic core [35–37], both containing only one
stabilizing cluster of five residues. However, sequence
analysis of exon 6 using coiled-coil prediction software
[38] suggests that the coiled-coil propensity of exon 6b
(Tm5a) is lower than that of exon 6a (Tm5b) [7]. The
DSC data presented are consistent with this prediction,
showing that the replacement of exon 6b in Tm5a by
exon 6a in Tm5b increases the thermal stability of the
major thermal transition by 3.4 °C. In contrast the
exon swap has no appreciable influences on the a-helix
content at 20 °C (Fig. 5) and on the total calorimetric
enthalpy of the thermal unfolding (Table 1). Previous
CD studies also showed an increased thermal stability
of a recombinant smooth muscle a-Tm with exon 6b
replaced by exon 6a [5,39].

Previous studies of thermal unfolding of skeletal Tm
have allowed assignment of the thermal transitions to
specific regions of Tm for skeletal a-Tm [8,9,14]. Ther-
mal unfolding of smTm (Fig. 3A) is quite different
from that of skeletal Tm measured by DSC under the
same conditions, skeletal Tm has for example two
Fig. 8. Normalized temperature dependence of dissociation of the
F-actin complexes with smTm, Tm5a, and Tm5b. 100% corres-
ponds to the difference between light scattering of the Tm–F-actin
complexes measured at 25 °C and that of pure F-actin stabilized by
phalloidin which was temperature independent within the temper-
ature range used. A decrease in the light-scattering intensity
reflects dissociation of the Tm–F-actin complexes. Conditions were
the same as for DSC experiments presented in Fig. 7. The heating
rate was 1 KÆmin
)1
.
Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al.
594 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS
thermal unfolding transitions of similar enthalpy
compared to one major and two minor transitions
observed for smTm [14]. The less stable of the two
thermal transitions for skeletal Tm has been assigned
to the C-terminal part of the molecule but the C-ter-
minal region of the two Tms differ at exon 9 (and at
the N-terminal exons 1 and 2). Thus comparison
between skeletal and smTm isoforms does not allow
the assignment of the transitions of smTm.
Comparison between the three Tm molecules studied
here can allow assignment of the three thermal trans-

itions observed to specific regions of the Tm. The
observation that only the main thermal transition is
affected by exon 6 in Tm5a and 5b suggests that trans-
ition 2 represents the central part of the Tm including
exon 6. Note however, that the enthalpy of transition
2 is almost identical for Tm 5a and 5b. Transition 3 is
stabilized by almost 20 °C when the N terminal exons
1a and 2a of smTm are replaced by exon 1b in the
fibroblast Tm suggesting that transition 3 represents
unfolding of the N-terminal part of the Tm. In support
of this are the CD data on model peptides showing
that the peptide mimicking exon 1b is much more ther-
mostable than the peptide corresponding to exon 1a
[26,27,29]. Transition 1 is the least stable of the ther-
mal transitions and is similar in all three Tms. There-
fore this transition cannot be unambiguously assigned.
It might correspond to the C-terminal part of Tm. In
favour of this assumption are the CD data showing
that model peptides corresponding to the C-terminal
exon 9d are much less thermostable than the N-ter-
minal peptides [29]. In contrast, however, the data of
Paulucci et al. [40] show that the C terminus of Tm (in
particular the last 24 residues) is crucial for the stabil-
ity of Tm. The increased thermal stability of transition
3 in Tm5a and Tm5b in comparison with smTm can
be explained in part by recently proposed theory of
Hodges and coworkers [35–37] that the thermal stabil-
ity of a-helical coiled-coil depends on the presence of
stabilizing and destabilizing clusters in its hydrophobic
core. They defined these clusters as three or more con-

secutive core-forming a and d residues in a heptad
repeat motif (abcdefg)
n
of either stabilizing (Leu, Ile,
Val, Met, Phe, and Tyr) or destabilizing (the remaining
13 amino acids) residues, and postulated the presence
of very long destabilizing cluster (seven residues) in the
hydrophobic core of N-terminal part of muscle Tm
encoded by exons 1a and 2a [37]. The sequence of
fibroblast Tm5a [41], being analysed in the same way,
contained only a short destabilizing cluster (just three
residues) in the hydrophobic core of the N-terminal
region encoded by exon 1b. The shortening of the
destabilizing cluster from seven to three residues might
stabilize the transition 3 assigned to the N-terminal
part of the Tm.
SmTm is  15% larger than the fibroblast Tm (due
to inclusion of exon 2) and according to the CD spec-
tra the helical content of the two proteins appears sim-
ilar at 20 °C. Therefore the simple prediction is that
the two proteins should have a similar enthalpy of
unfolding (differing by 15% at most), yet the data in
Table 1 shows that the total energy of unfolding of
SmTm is 1.6-fold larger than that of Tm5a. The pro-
portion of the total enthalpy in the three domains of
each is very similar at 20 : 60 : 20, so this increase in
enthalpy is not simply due to the change in the stabil-
ity of the N-terminal part of the molecule, but a
change in the observed enthalpy of the whole mole-
cule.

There are two possible explanations for the
increased enthalpy in smTm compared to Tm5a, either
the addition of exon 2 influences enthalpy of the whole
molecule or there is some ‘unseen’ enthalpy in the
Tm5a.
Evidence that specific regions of Tm can have long-
range effects on the stability of the whole molecule
have been reported previously [40,42,43]. A recent
study by Singh and Hitchcock-DeGregori [22] has
shown that mutations in a region at the C-terminal
end of exon 2b in a-Tm2 (identical to smTm except
exon 2b vs. 2a) can cause a change in the melting pro-
file of several unfolding transitions. The mutations
which caused either an increase or decrease in mid-
point of the melting transition could both result in
an apparent major decrease in total enthalpy of un-
folding.
‘Unseen’ enthalpy may be the consequence of broad
noncooperative unfolding of parts of the molecule, giv-
ing a very slight slope to the heat capacity curve. This
proceeds over a broad temperature range and can be
difficult to precisely measure by DSC. Noncooperative
melting was earlier observed by DSC for some muscle
proteins (e.g. troponin T, troponin I [30], and calponin
[44]), which did not exhibit any detectable thermal
transitions upon heating up to 100 °C.
The assumption that noncooperative unfolding takes
place for some parts of nonmuscle Tm is corroborated
by our DSC studies on the complexes of Tm with
F-actin. Interaction with F-actin significantly increases

the thermal stability and unfolding enthalpy of Tm,
and reduces the difference in total enthalpy seen
between smTm and nonmuscle Tm isoforms 5a and 5b
(Fig. 7, Table 2). In particular the enthalpy of peak 2
for Tm 5a, corresponding to the actin-bound Tm, is
almost exactly six-sevenths of that of smTm as predic-
ted on a simple size comparison. This suggests that the
E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin
FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 595
stabilizing of Tm on actin results in almost all of the
unfolding occurring as a single cooperative process
simultaneous with dissociation from actin. It was con-
cluded from our previous studies that F-actin prevents
the actin-bound Tm from thermal denaturation, which
only occurs upon dissociation of Tm from F-actin
[14,45]. As a result, Tm unfolds at higher temperature
and with much higher cooperativity. This allows the
‘lost’ enthalpy of parts which unfold noncooperatively
at temperatures below T
diss
in the absence of actin, to
be ‘recovered’ in the highly cooperative dissociation
transition. It is possible that smTm also has some ‘lost’
enthalpy, but it is likely to be much less than in non-
muscle Tm isoforms, which demonstrate in the pres-
ence of F-actin much more pronounced ‘recovery’ of
enthalpy (by 70%) than smTm (by only 20%)
(Table 2).
The recent CD and DSC data of Dragan and Priv-
alov on the thermal unfolding of a leucine zipper

which, like Tm, is an a-helical, double-stranded
coiled-coil [46] support noncooperative transitions in
the unfolding pathway. These authors showed that
the initial almost linear change of leucine zipper ellip-
ticity prior to the sigmoidal change (that is very sim-
ilar to those of Tms in Fig. 6A) cannot be regarded
as a trivial optical effect but is associated with some
temperature-induced conformational changes of the
dimeric molecule from the very beginning of its heat-
ing. They concluded, that the enthalpy of cooperative
unfolding that is associated with dissociation of the
two strands and is observed as a cooperative thermal
transition by DSC, does not represent the full
enthalpy of unfolding of the molecule. The full
enthalpy also includes the enthalpy of all predissocia-
tion changes, which comprises almost 40% of the
total enthalpy. These temperature-induced changes in
protein structure, that occur before the cooperative
separation of strands, are believed to be associated
with some rearrangements in the coiled-coil, and they
are highly sensitive to modifications of the N termi-
nus [46]. It seems possible that somewhat similar
structural changes may also occur in nonmuscle Tm
isoforms due to replacement of the N-terminal muscle
exon 1a by nonmuscle exon 1b.
The noncooperative unfolding of a significant part of
the nonmuscle Tm molecule may suggest that this part
(or these parts) of the molecule becomes more flexible
due to replacement of the N-terminal muscle exons 1a
and 2a by nonmuscle exon 1b. Higher flexibility may

affect actin-binding (since Tm must match the actin
periodicity to bind effectively and flexibility may be
important in the ability of Tm to change position on the
actin surface [22,47,48]). This could explain in part why
the replacement of exon 1a by exon 1b strongly increases
the affinity of Tm for actin [3] despite having little effect
on Tm end-to-end interactions as shown by the Tm
polymerization measured by viscosity (Fig. 4A).
In conclusion, the data presented here comparing the
three tropomyosins is compatible with previous reports
that suggest there is no simple correlation between
specific ‘domains’ of Tm and the overall stability of the
molecule but there are long-range cooperative effects
on structure. Furthermore, in some cases local confor-
mation changes and unfolding occur as low enthalpy
noncooperative transitions that are not easily detected
by DSC. This missing enthalpy becomes apparent when
the Tm dissociates from actin and unfolds as a single
highly cooperative process. Understanding the nature
and location of the noncooperative unfolding regions
will be important to understand the way in which the
exons changes affect the stability of remote unfolding
domains. In the future, definitive identification of
specific regions of Tm with particular unfolding transi-
tions could be facilitated by the use of labels to report
local unfolding events.
Experimental procedures
DNA constructs
Clones of rat fibroblast tropomyosins 5a and 5b were
amplified from PET8c (gift from M. Gimona and D. Helf-

man) using PCR primers designed to introduce NdeI and
BamHI restriction sites for cloning into pJC20. The
sequences for the primers used were 5¢-GGAATTCCA
TATGGCGGGTAGCAGCTCGCTGGCG-3¢ (5¢-forward
primer) and 5¢-CGCGGATCCTCACATGTTGTTTAGCT
CCAGTAAAG-3¢ (3¢-reverse primer). Identical primers
were used for TPM5a and TPM5b as they differ only by an
internal alternatively spliced exon 6 (see Fig. 1). The
smooth muscle clone was amplified from a full-length clone
which also contained the 5¢ UTR in pGem4 (gift from
C. Smith, Cambridge), using PCR primers again designed to
produce NdeI and BamHI restriction sites. The 5¢ forward
primer also introduced bases coding for a three amino acid
Met-Ala-Ser N-terminal extension to substitute for the lack
of N-terminal acetylation. The sequence for the N-terminal
5¢ forward primer was 5¢-GGAATTCCATATGGCGAGC
ATGGACGCCATCAAGAAGAAGATGC-3¢. As smTm
uses the same C-terminal exon 9d as Tm5a and Tm5b,
the same 3¢ reverse primer was used. The ligated plasmids
were transformed into Escherichia coli XL-1 Blue for
plasmid replication. The entire coding regions of the
constructs were verified by automatic DNA sequencing on
Applied Biosystems 373A sequencer (Applied Biosystems,
Foster City, CA, USA) using a dye-based PCR sequencing
reaction.
Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al.
596 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS
Expression and purification of recombinant
tropomyosins
For expression, all the clones were transformed in the

BL-21 DE3 (pLys) cells and expressed and purified as pre-
viously described [7,49] with some modifications. In brief,
1-L cultures were grown to late-exponential phase and
induced overnight with 0.4 mm isopropyl-1-thio-b-d-gal-
actopyranoside. Cells were harvested, resuspended in
60 mL lysis buffer (20 mm Tris pH 7.0, 150 mm NaCl,
5mm EDTA, 5 mm MgCl
2
), and lysed by sonication (two
2-min pulses separated by 1-min rest phase). The majority
of E. coli proteins were precipitated by heating to 80 °C for
10 min, and the precipitated protein and cell debris were
removed by centrifugation. Then solution was incubated
with 5 mgÆL
)1
DNase and 10 mgÆL
)1
RNase for 1.5 h. The
soluble Tm was then isoelectrically precipitated at pH 4.5
using 0.3 m HCl. The precipitate was pelleted and resus-
pended in 10 mL running buffer (5 mm potassium phos-
phate pH 7.0, 100 mm NaCl). This was then further
purified using a 5 mL Hi-trap Q column (Amersham) and
eluted with a 200–500 mm NaCl gradient, with the Tm elut-
ing at 250–450 mm salt. Fractions were analysed by
SDS ⁄ PAGE [50], pooled, and concentrated by isoelectric
precipitation. Extinction coefficients for recombinant pro-
teins were calculated from the sequences using the software
antheprot (G. Deleage, IBCP-CNRS). Protein concen-
trations were estimated using extinction coefficients E

1%
at
280 nm of 1.41 cm
)1
for smTm and 1.61 cm
)1
for fibroblast
Tm 5a ⁄ 5b, and molecular masses of 32834.8, 28557.9, and
28697.2 Da for smTm, Tm5a, and Tm5b, respectively.
Protein molecular masses were determined by electro-
spray mass spectrometry to confirm that the expressed Tms
had the correct size. Small (50 lL) stock samples were dia-
lysed overnight against 30 mm Hepes pH 7.3 containing
100 mm KCl and 1 mm MgCl
2
, and applied to a Finnegan
Mat LCQ ion-trap mass spectrometer fitted with a nano-
spray device. Predicted molecular masses for proteins were
calculated using the AnTheProt with Delta Mass (ABRF)
used to determine mass differences among the Tm species.
Relative molecular masses determined by MS for smTm,
Tm5a, and Tm5b were in good correspondence with the
predicted masses.
Before experiments, all Tm samples were incubated with
20 mm b-mercaptoethanol at 60 °C for 60 min. Such treat-
ment results in Tm species in completely reduced state [14].
To maintain the reduced Tm species, 1 mm b-mercapto-
ethanol was added to the samples.
Preparation of actin
Rabbit actin was prepared by the method of Spudich and

Watt [51]. Its molar concentration was determined by its
absorbance at 290 nm using an E
1%
of 6.3 cm
)1
and a
molecular mass of 42 kDa. F-actin polymerized by the
addition of 4 mm MgCl
2
and 100 mm KCl was further sta-
bilized by the addition of a 1.5-fold molar excess of phalloi-
din (Sigma).
Viscosity measurements
Measurements were carried out at 18.5 °C using an Ostow-
ald type capillary viscosimeter (Institute for Biological
Instrumentation, Puschino, Russia), with a buffer outflow
time of 27.6 s. Before measurements, proteins (0.5 mgÆmL
)1
)
were dialysed against 30 mm Hepes pH 7.3 containing
100 mm KCl and 1 mm MgCl
2
.
Differential scanning calorimetry
DSC experiments were performed on a DASM-4 m differ-
ential scanning microcalorimeter (Institute for Biological
Instrumentation, Pushchino, Russia) as described earlier
[12–14,30]. All measurements were carried out at a scanning
rate of 1 KÆmin
)1

in either 30 mm Hepes, pH 7.3, or 50 mm
sodium phosphate, pH 7.3, both containing 100 mm KCl
and 1 mm MgCl
2
. The solution also contained 1 mm
b-mercaptoethanol to prevent disulfide cross-linking between
the chains in the Tm homodimers. In the case of Tm–
F-actin complexes, the final concentration of F-actin was
46 lm. F-actin was stabilized by the addition of a 1.5-fold
molar excess of phalloidin (Sigma) to obtain a better separ-
ation of the thermal transitions of actin-bound Tm and
F-actin [13,14]. The reversibility of the thermal transitions
was assessed by reheating of the sample immediately after
cooling from the previous scan. The calorimetric traces
were corrected for the instrumental background by sub-
tracting a scan with buffer in both cells. In some cases, to
reveal small and low-cooperative thermal transitions in
Tm5a and Tm5b, a special DSC approach was applied as
follows. DSC measurements were performed not only by
usual way, when the protein was placed into the sample cell
and the buffer was placed into the reference cell, but also
vice versa, with the same protein in the reference cell and
the buffer in the sample cell. As a result, in last case the
protein peak on the DSC curve turned over. This curve
with inverted protein peak was then subtracted from the
curve obtained by usual way. This procedure completely
eliminated the instrumental baseline and doubled the ampli-
tude of the protein signal. The resulting curve was then
divided by two. The point is that the instrumental baseline
is the own property of each calorimeter, which is independ-

ent of the procedures described above. The above DSC
approach allows us to subtract the instrumental baseline
without its direct measurement, and to avoid all possible
artefacts caused by the measurement of instrumental base-
line and by its following subtraction from the DSC profile
of the protein. This new approach makes it possible to per-
form DSC experiments with high precision and to reveal
rather small and low-cooperative thermal transitions, which
E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin
FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 597
often cannot be observed by usual DSC measurements. The
temperature dependence of the excess heat capacity was
further analysed and plotted using Origin software (Micro-
Cal Inc., Northampton, MA). The thermal stability of the
proteins was described by the temperature of the maximum
of thermal transition (T
m
), and calorimetric enthalpy
(DH
cal
) was calculated as the area under the excess heat
capacity function.
The DSC data, with instrumental baseline deducted, were
analysed by using the software package Origin 1.16 (Micro-
Cal). Deconvolution is based on the procedure described by
Freire and Biltonen [52]. The complex endotherm may be
resolved into several Gaussian distributions, each represent-
ing individual transitions, using a least-squares curve fitting
procedure. Thus, the software allows for determination of
the number of two-state transitions (calorimetric domains)

contributing to the complex endotherm. It was found that
no more than three independent domains are needed to
obtain adequate fits. The following parameters were consid-
ered for each domain: the DH
cal
which gives the size of the
transition, and T
m
which locates the mid-point of the ther-
mal transition of the domain.
CD measurements
Far-UV CD spectra (190–250 nm) were obtained using
Mark V dichrograph (Jobin Yvon) at 20 °C. The tempera-
ture dependence of mean residual ellipticity at 222 nm
(h
222
) was monitored from 10 °Cto60°C at heating rate
of 1 °CÆmin
)1
, i.e. at the same heating rate as for DSC
experiments. The samples contained 0.1 mgÆmL
)1
of Tm in
50 mm sodium phosphate buffer pH 7.3, 100 mm NaCl,
1mm MgCl
2
, and 1 mm b-mercaptoethanol.
Light scattering
Thermally induced dissociation of Tm–F-actin complexes
was detected by changes in light scattering at 90° as des-

cribed earlier [13,14]. All measurements were performed at
350 nm on a Cary Eclipse fluorescence spectrophotometer
(Varian Australia Pty Ltd, Mulgrave, Victoria, Australia)
equipped with temperature controller and thermoprobes.
Light scattering measurements were performed under the
same conditions and at the same heating rate as the DSC
experiments. Scattering of F-actin solutions containing the
same concentration of actin as in the Tm–F-actin samples
was measured before the experiments. This value increased
proportionally with the amount of Tm bound to F-actin.
When Tm dissociated from F-actin during heating, the
value of the light scattering intensity became equal to that
of F-actin, because the light scattering of free Tm mole-
cules was negligible [34]. Thus, a temperature-dependent
decrease in light-scattering intensity of the Tm–F-actin
complexes reflects dissociation of Tm from F-actin. The
dissociation curves, with temperature dependence of light
scattering for F-actin alone deducted, were analysed by
using the Origin software (MicroCal), according to a sig-
moidal decay function (Boltzman). The main parameter
extracted from this analysis is T
diss
, i.e. the temperature at
which a 50% decrease in light scattering occurs.
Acknowledgements
We thank Valeriya Mikhailova for her help in per-
forming DSC experiments and for the preparation of
actin, and Sam Lehrer (Boston Biomedical Research
Institute, Boston, MA, USA) for valuable comments
and advice. This work was supported by the Wellcome

Trust (grants 066115 to D.I.L and M.A.G and 055881
to M.A.G), the Russian Foundation for Basic
Research (grant 03-04-48237 to D.I.L), and by the
Program for the Support of Scientific Schools in Rus-
sia (grant NSH-813.2003.4 to D.I.L).
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