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

(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

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.

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

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
.

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

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

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

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

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

–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

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].

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-

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

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

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

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

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-

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-

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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