Eur. J. Biochem. 271, 589–600 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2003.03961.x
A specific C-terminal deletion in tropomyosin results in a stronger
head-to-tail interaction and increased polymerization
Adriana A. Paulucci, Angela M. Katsuyama, Aurea D. Sousa* and Chuck S. Farah
Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de São Paulo, São Paulo, SP, Brazil
Tropomyosin is a 284 residue dimeric coiled-coil protein that
interacts in a head-to-tail manner to form linear filaments at
low ionic strengths. Polymerization is related to tropomyosin’s ability to bind actin, and both properties depend on
intact N- and C-termini as well as a-amino acetylation of
the N-terminus of the muscle protein. Na-acetylation can be
mimicked by an N-terminal Ala-Ser fusion in recombinant
tropomyosin (ASTm) produced in Escherichia coli. Here we
show that a recombinant tropomyosin fragment, corresponding to the protein’s first 260 residues plus an Ala-Ser
fusion [ASTm(1–260)], polymerizes to a much greater extent
than the corresponding full-length recombinant protein,
despite the absence of the C-terminal 24 amino acids. This
polymerization is sensitive to ionic strength and is greatly
reduced by the removal of the N-terminal Ala-Ser fusion
[nfTm(1–260)]. CD studies show that nonpolymerizable
tropomyosin fragments, which terminate at position 260
[Tm(167–260) and Tm(143–260)], as well as Tm(220–284),
are able to interact with ASTm(1–142), a nonpolymerizable
N-terminal fragment, and that the head-to-tail interactions
observed for these fragment pairs are accompanied by a
significant degree of folding of the C-terminal tropomyosin
fragment. These results suggest that the new C-terminus,
created by the deletion, polymerizes in a manner similar to
the full-length protein. Head-to-tail binding for fragments
terminating at position 260 may be explained by the presence
of a greater concentration of negatively charged residues,
while, at the same time, maintaining a conserved pattern of
charged and hydrophobic residues found in polymerizable
tropomyosins from a variety of sources.
Skeletal muscle tropomyosin (Tm), a 284 residue, 410 Å
coiled-coil protein, is involved in the regulation of muscle
contraction through interactions with troponin and actin
[1–3]. The coiled-coil structure is the consequence of a
heptapeptide repetition (abcdefg) in the chemical nature of
the residues in the primary structure of the polypeptide
chain, where residues at positions a and d are often
hydrophobic and create a dimerization interface [4–6]. This
dimeric protein polymerizes at low ionic strengths, in a
head-to-tail manner, via interactions involving the first and
approximately the nine last residues of two independent Tm
molecules [5]. Tm polymerization in vitro is diminished
upon increasing the ionic strength [7–9] and polymerization
is important for its function in vivo, because the ability to
polymerize correlates with the binding to actin, cooperative
regulation of thin filaments by myosin, and binding to TnT
(the Tm-binding subunit of the troponin complex) [10–12].
Unlike native muscle Tm, the unacetylated protein
expressed in Escherichia coli cells is nonpolymerizable
[13–15]. The N-acetylated initiation methionine in the native
protein occupies an internal position in the coiled-coil
structure (position a). Repulsions between the positively
charged a-amino groups in the recombinant protein are
thought to destabilize the local coiled-coil, as well as the
head-to-tail, interactions [11,15,16]. The polymerization of
bacterially expressed nonacetylated recombinant skeletal
muscle Tm can be restored by the N-terminal fusion of two
(AS) or three (AAS) amino acids [15], and other studies
have provided evidence that specific N- and C-terminal
residues are important in the polymerization process
[10,13,17–19]. Furthermore, modifications within the central
portion of the Tm molecule can also abolish or diminish the
intensity of the head-to-tail overlap [20,21].
The structure of the complex between Tm N- and
C-termini (the head-to-tail complex) is not known, as
crystals of full-length tropomyosin only diffract to low
resolution (7 Å) [22,23]. However, high-resolution structures of isolated N- and C-terminal fragments have been
obtained by X-ray crystallography and by NMR. In the
2.7 Å crystal structure of a 31 residue C-terminal fragment
of Tm fused to a fragment of GCN4 leucine zipper, the
helices formed by the last 22 Tm residues (263–284) splay
apart and two symmetry-related molecules make intermolecular contacts in a tail-to-tail manner [24]. A recently
Correspondence to C. S. Farah, Departamento de Bioquı́mica,
Instituto de Quı́mica, Universidade de São Paulo CP 26.077,
CEP 05599-970 São Paulo, SP, Brazil.
Fax: + 55 11 3815 5579, Tel.: + 55 11 3091 3312,
E-mail: [email protected]
Abbreviations: ASTm, alanine-serine fusion in recombinant
tropomyosin; Tm, tropomyosin.
*Present address: Medical Biomolecular Research Building,
Room 5314, Department of Cell and Molecular Physiology,
University of North Carolina, 106 Mason Farm Road,
Chapel Hill, NC 27599-7545, USA.
Authors who contributed equally to this work.
(Received 4 November 2003, revised 3 December 2003,
accepted 9 December 2003)
Keywords: tropomyosin; head-to-tail interaction; protein
polymerization; protein folding; circular dichroism.
FEBS 2004
590 A. A. Paulucci et al. (Eur. J. Biochem. 271)
published solution structure [25] of a disulfide linked dimer
of peptide TM9a251–284, containing the last 34 C-terminal
residues of a-Tm, as well as a stabilizing mutation at
position 279, showed that residues 257–269 form a coiledcoil, residues 270–279 are in a parallel noncoiled-coil
arrangement and the last five residues (280, 281, 282, 283
and 284) are nonhelical.
Residues 258–284 of Tm are encoded by exon 9a, which
is specific for striated Tm in vertebrates [26]. The region
expressed by exon 9a is required for binding to TnT and for
troponin to promote the binding of tropomyosin to actin
[27–29]. Paulucci et al. [30] have shown that this region is
very important for the stability of the C-terminal region of
the molecule. Recently, Palm and co-workers [31] have
shown that a Tm C-terminal peptide, TM9a36ox (which
consists of the last 35 amino acids of rat striated muscle a-Tm
encoded by exon 9a cross-linked through an N-terminal
cysteine), was able to bind the N-terminal peptide AcTM1azip (which consists of the first 14 amino acids encoded by
rat a-Tm exon 1a followed by the 18 C-terminal residues of
the GCN4 leucine zipper). A mixture of these two peptides
showed an increased thermal stability when compared with
the sum of the individual components [31].
Here, we show that a truncated ASTm [ASTm(1–260)]
is able to polymerize, although it lacks the region encoded
by exon 9a. The polymerization of ASTm(1–260) is greatly
reduced upon removal of the Ala-Ser dipeptide fusion,
indicating that the polymerization is head-to-tail. Thermal
denaturation experiments with C-terminal fragments possessing Tm(220–284) and lacking the region encoded by
exon 9a [Tm(143–260) and Tm(167–260)] confirm the
importance of the Ala-Ser dipeptide and demonstrate that
the formation of the head-to-tail complex in the presence of
low ionic strength conditions is accompanied by a significant degree of a-helix formation, probably within the
C-terminal fragment.
Materials and methods
Construction of vectors for expression of Tm mutants
The plasmid vectors for the bacterial expression of Tm(167–
260), Tm(143–260) and Tm(220–284) fragments, and of the
nfTm269(5OHW) mutant, have been described previously
in Paulucci et al. [30] and in Sousa & Farah [9], respectively.
Plasmid vectors for the expression of ASTm(1–260),
ASTm(1–142), nfTm(1–260) and nfTm(1–142) were constructed by cloning NcoI/HindIII- or NdeI/HindIII-digested
PCR-amplified fragments into the NcoI/HindIII sites of
pET3d or the NdeI/HindIII sites of pET-3a [33]. PCR was
performed using the pET-MASTmy [for ASTm(1–260) and
ASTm(1–142)] or pET-Tmy [for nfTm(1–260) and nfTm(1–
142)] vector constructs [15] as templates. pET-MASTmy
contains the chicken skeletal a-tropomyosin cDNA cloned
in pET-3a and expresses Tm containing an N-terminal MetAla-Ser fusion (ASTm), whose N-terminal Met is removed
post-translationally. pET-Tmy expresses a nonfusion Tmy
(nfTm) [15]. The following oligonucleotides were used in
PCR reactions: *261Hind (5¢-CTTCAGTTTAAGCTT
TTAAAGCTCATCTTCTAG-3¢), *143Hind (5¢-CTGG
ATAAGCTTTTATTCCATCTTCTCTTCATCC-3¢) and
T7 (5¢-CACTATAGGGAGACCACAACGGTTTCC-3¢).
The pairs T7 and *261Hind were employed to produce
ASTm(1–260) and nfTm(1–260) fragments, while the
pairs T7 and *143Hind were employed to obtain the
ASTm(1–142) and nfTm(1–142) fragments.
Protein expression and purification
The fragments Tm(267–260), Tm(143–260) and Tm(220–
284), ASTm(1–142), nfTm(1–142), ASTm(1–260) and
nfTm(1–260) were expressed and purified according the
method described in Monteiro et al. [15]. nfTm269(5OHW),
which contains a 5-hydroxytryptophan at position 269 [9],
was expressed and purified as described previously [34].
Protein concentrations were determined as described previously [35].
Fluorescence experiments
The 5-hydroxytryptophan fluorescence of nfTm269(5OHW) (2 lM) [9] was monitored during its titration
with ASTm(1–142) or nfTm(1–142), in 25 mM Mops
(pH 7.0), 25 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol.
The spectra were obtained at 25 C, using an Aviv ATF105
spectrofluorimeter, with excitation at 312 nm and excitation
and emission bandwidths of 1 and 5 nm, respectively, with
5 min equilibration for each point. Emission intensity was
expressed as the total emission between 320 and 420 nm.
Viscosity experiments
Viscosity measurements were performed using a CannonManning semi-microviscosimeter (C30), with a buffer
outflow time of 242 s, at 21 ± 1 C. Proteins (8 lM) were
resuspended in buffer of 2 mM dithiothreitol, 10 mM
imidazol/HCl (pH 7.0), and equilibrated for 7 h before
each experiment.
CD spectroscopy
Proteins (10 lM) were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM EDTA and
1.0 mM dithiothreitol. Far-UV CD spectra (190–260 nm,
1 nm bandwidth) were collected, using a Jasco-720 spectropolarimeter, at 25 C, 100 nmÆmin)1 and a response time
of 0.25 s. Spectra shown are the sum of eight individual
scans. In experiments with mixtures of two proteins, the
experimental data were compared with the respective
theoretical curves obtained by summing the experimental
data obtained for each component in isolation. The a-helix
content was calculated based on the following algorithm,
developed by Holtzer et al. [36], for coiled-coil proteins:
1
Uh ¼ f½h ½hc þ ð2:55 I ½h1
h =nÞg=f½hh ½hc g;
where Fh ¼ fraction of residues in an a-helical conformation, h1
h ¼ mean residual ellipticity at 222 nm for a helix of
infinite length ()386Æcm)2Æmmol of peptide bonds)1),
hc ¼ mean residual ellipticity for a random coil at 222 nm
()10Æcm)2Æmmol of peptide bonds)1), I ¼ average number
of helical segments per chain (in this case equal to 1),
n ¼ number of peptide bonds and h ¼ mean residual
ellipticity at 222 nm.
FEBS 2004
Head-to-tail interaction of tropomyosin (Eur. J. Biochem. 271) 591
Thermal denaturation of Tm fragment mixtures
monitored by CD spectra
CD spectra were obtained using a Jasco J-810 spectropolarimeter, at the Laboratório Nacional de Luz Sincrotron
(Campinas, Brazil). Proteins (10 lM) were resuspended in
10 mM sodium phosphate (pH 7.0), 0.5 mM EDTA, 1 mM
dithiothreitol and measurements at 222 nm were collected
at 0.5 C intervals from 4 C to 80 C, and from 80 C to
4 C, at a velocity of 1 CÆmin)1 (1 nm bandwidth, 4 s
response time). Thermal denaturations were performed
individually on ASTm(1–142), nfTm(1–142), Tm(167–260),
Tm(143–260) and Tm(220–284), as well as on the following
combinations of Tm(167–260) + ASTm(1–142), Tm(167–
260) + nfTm(1–142),
Tm(143–260) + ASTm(1–142),
Tm(143–260) + nfTm(1–142), Tm(220–284) + ASTm(1–
142) and Tm(220–284) + nfTm(1–142). The denaturation
curves of the two-fragment mixtures were compared with
the sum of the curves obtained for each of the components.
Far-UV CD (190–260 nm) control spectra were also
obtained at 4 C, 80 C and 4 C (0.25 s response time,
100 nmÆmin)1, 1 nm of bandwidth; data not shown).
Results
We have recently characterized the stability and folding of a
number of C-terminal Tm fragments, where we demonstrated that a deletion of the last 24 residues severely reduced the
stability of the C-terminal half of the Tm molecule [30].
These deleted residues are encoded by exon 9a, which is
known to be expressed specifically in skeletal and cardiac
muscle [26] and is important for interactions with troponin
[28,37]. As previous studies have widely demonstrated, small
modifications in the N- and C-termini of Tm, such as
N-terminal deacetylation and C-terminal deletions (more
than three residues), abolish Tm polymerization and reduce
its affinity for actin in the absence of troponin
[10,11,13,14,17,38]. Therefore, when we decided to construct
two truncated tropomyosins – nfTm(1–260) and ASTm(1–
260) – the former lacking the N-terminal acetylation and the
latter with an N-terminal Ala-Ser dipeptide fusion, we had
reason to expect that both molecules would be polymerization deficient. We were therefore surprised to observe, in
viscosity experiments, that ASTm(1–260) appeared to
polymerize to a much greater extent than ASTm. Figure 1
shows the kinematic viscosity of solutions of Tm fragments
as a function of NaCl concentration. The viscosity of
ASTm(1–260) was much greater than that of full-length
ASTm at all ionic strengths tested. Similarly to the fulllength molecule, the polymerization of ASTm(1–260) is
strongly ionic-strength dependent. Furthermore, the corresponding fragment lacking the Ala-Ser N-terminal extension,
nfTm(1–260), has a greatly reduced viscosity when compared with ASTm(1–260). This parallels the effect of the
Ala-Ser dipeptide fusion in the full-length protein (Fig. 1)
[9,15] and suggests that ASTm(1–260) also polymerizes in a
linear head-to-tail manner. Note that at low NaCl concentrations, nfTm(1–260) produces solutions less viscous than
ASTm, but more viscous than nfTm (Fig. 1). Two smaller
fragments, terminating at position 260 but lacking the Tm
N terminus [Tm(167–260), Tm(143–260)], do not polymerize (Fig. 1), as expected by previously published analytical
Fig. 1. Viscosity assays of recombinant tropomyosin (Tm) and its
fragments as a function of ionic strength. Proteins (8 lM) in 10 mM
imidazol/HCl, pH 7.0, NaCl (concentration indicated) and 2 mM
dithiothreitol were equilibrated for 7 h before each experiment. Viscosities of the mutants ASTm, nfTm, ASTm(1–260) and nfTm(1–260)
are shown as a function of NaCl concentration. For clarity, the viscosity of ASTm(1–260) at 20 and 40 mM NaCl (7.5 and 4.5 centistokes)
is not shown. The viscosities of ASTm(1–142), nfTm(1–142), Tm(167–
260) and Tm(143–260) were also determined at 0 M NaCl. All show
essentially identical viscosities (1.0 centistokes) under these conditions
(black diamonds).
ultracentrifugation results [30]. Finally, two smaller
N-terminal fragments, which terminate at positions
142 [ASTm(1–142), nfTm(1–142)], do not polymerize, as
expected.
The unexpected results obtained for ASTm(1–260) led us
to investigate whether small N-terminal Tm fragments, with
or without the Ala-Ser dipeptide fusion, could be shown
to interact with small C-terminal Tm fragments, which
terminate at position 260 or 284. Instead of forming
polymers, these complexes, if formed, would be simple 1 : 1
dimers. The N-terminal fragments chosen were ASTm(1–
142) and nfTm(1–142), neither of which polymerize, as
shown above.
We first wished to demonstrate that ASTm(1–142) could
interact with the C-terminus of Tm in a manner similar to
that observed in the head-to-tail interaction of the fulllength protein. To achieve this we used a recombinant Tm
containing a 5-hydroxytryptophan probe at position 269, 15
residues from the C-terminus of the coiled-coil structure,
whose fluorescence intensity is sensitive to the polymerization state of the molecule [9]. While the fluorescence of
ASTm269(5OHW) is sensitive to the ionic strength of the
medium, the fluorescence of nfTM269(5OHW) is not, as
the absence of the Ala-Ser fusion results in a drastic
reduction in polymerization [9]. However, the C-terminus
of nfTm269(5OHW) would be expected to bind the
N-terminus of ASTm(1–142), resulting in a consequent
increase in fluorescence intensity. Figure 2 shows the
592 A. A. Paulucci et al. (Eur. J. Biochem. 271)
Fig. 2. Titration of nfTm269(5OHW) with ASTm(1–142) or nfTm(1–
142), as monitored by fluorescence. nfTm269(5OHW) (2 lM) was
titrated with ASTm(1–142) (gray circles) or with nfTm(1–142) (black
circles). Gray triangle: fluorescence signal of nfTm269(5OHW) titrated
with ASTm(1–142) after addition of 300 mM NaCl. Conditions:
25 mM Mops, pH 7.0, 5 mM MgCl2, 1 mM dithiothreitol at 25 C. All
samples were pre-equilibrated for 5 min at 25 C before determining
the fluorescence intensity at 339 nm.
titration of nfTm269(5OHW) with ASTm(1–142), as demonstrated by the fluorescence of the 5-hydroxytryptophan
probe. The results clearly reveal an interaction between
these two proteins. Therefore, a fragment of Tm without the
C-terminal region of the molecule does not impair its ability
to take part in a head-to-tail interaction, consistent with the
results of Palm and co-workers [31], who observed interactions between chimeric and covalently cross-linked Tm
peptides. Furthermore, titration of nfTm269(5OHW) with
nfTm(1–142) did not result in any significant increase in the
fluorescence intensity of the probe. Based on these results,
we conclude that the interactions between ASTm(1–142)
and nfTm269(5OHW) are specific and that ASTm(1–142)
can therefore be used to test head-to-tail interactions.
Figure 2 also shows that increasing the salt concentration to
300 mM NaCl after the titration of nfTm269(5OHW) with
ASTm(1–142), diminishes the fluorescence signal to 8%
below the initial value. This indicates that the head-to-tail
interaction formed between nfTm269(5OHW) and
ASTm(1–142) was abolished under conditions of high ionic
strength, as expected. The observation that the fluorescence
signal in 300 mM NaCl is less than the initial value is a result
of the fact that, although nfTm269(5OHW) is not acetylated, it has a basal level of polymerization, as seen for nfTm
in Fig. 1.
The results, described above, led us to perform a
combination of experiments to investigate the nature of
the Tm head-to-tail interaction involving the native
C-terminus, as well as the new C-terminus created by the
truncation at residue 260. In these experiments, we
combined the C-terminal fragments Tm(167–260),
Tm(143–260) and Tm(220–284), with the N-terminal fragments ASTm(1–142) and nfTm(1–142). We have previously
shown that the stability of the C-terminal Tm fragments is
strongly dependent on temperature and ionic strength [30].
While Tm(167–260), Tm(143–260) and Tm(220–284)
FEBS 2004
present significant helix content (> 60%) in buffer containing 100 mM KCl at 10 C [30], this secondary structure
is, in great part, lost in the absence of KCl and at 25 C
(Fig. 3 and Table 1). On the other hand, the two N-terminal
fragments remain relatively stable under these conditions
(Fig. 3 and Table 1). The observed difference in stabilities of
N- and C-terminal halves of the Tm molecule is consistent
with previous observations [39,40].
We therefore decided to perform CD experiments on
equimolar mixtures of N- and C-terminal fragments under
conditions where the C-terminal fragments on their own are
largely unfolded, while the N-terminal fragments are stable
dimers (10 lM of protein dimer in 10 mM phosphate buffer,
pH 7.0, 0.5 mM EDTA and 1 mM dithiothreitol at 25 C).
Figure 3 shows the CD spectra for all fragments on their
own and in equimolar mixtures (experimental curve, m).
The sum of the individual N- and C- terminal fragment
spectra is also shown for comparison (theoretical curve, d).
These results demonstrate that the experimental curves for
the mixtures of the three C-terminal fragments with
ASTm(1–142) all present a greater negative ellipticity than
that predicted by the theoretical curve. These differences
were either not observed or were observed to a much
lesser degree when nfTm(1–142) was used instead of
ASTm(1–142) (Fig. 3).
To characterize this phenomenon in greater detail, we
performed temperature denaturation experiments, monitored by CD, for each of the five fragments on their own and
for all six combinations between N- and C-terminal
fragments. Figure 4 shows that there was little or no
significant variation in the stability of the C-terminal
fragments combined with nfTm(1–142). We did however,
observe a considerable increase in the helical contents of the
mixtures containing ASTm(1–142). The results presented in
Figs 3 and 4 indicate that all three C-terminal Tm fragments
can interact with ASTm(1–142) and that this interaction is
accompanied by a significant degree of folding of one or
both of the polypeptide chains. As mentioned above,
N-terminal Tm fragments are more stable than C-terminal
fragments, with significantly greater melting temperatures
for fragments of the same size (Figs 3 and 4, and Table 1)
[39,40]. Therefore, it appears that the majority of the
conformational changes take place in the C-terminal fragments. We note that NMR and X-ray crystallography data
on N- and C-terminal Tm fragments have revealed helical, if
not coiled-coil, structures up to the last few residues. These
structures were, of course, determined under very different
conditions from those used here, the most significant
differences being greater ionic strength and greater peptide
concentrations [24,25]. High ionic strength is known to
stabilize the coiled-coil structure of Tm fragments and to
decrease Tm polymerization [7,8,30,40–44].
The insets in Fig. 4 show the fractional differences
between the experimental and the theoretical curves, as a
function of temperature, for the three mixtures containing
ASTm(1–142). These graphs shows that, for mixtures
containing ASTm(1–142), the differences between experimental and theoretical curves are most pronounced at 30 C
for Tm(143–260) and Tm(220–284) and at 25 C for
Tm(167–260). We note that mixtures of ASTm(1–142) with
Tm(167–260) or Tm(143–260) experience greater changes in
their negative ellipticities than mixtures of ASTm(1–142)
FEBS 2004
Head-to-tail interaction of tropomyosin (Eur. J. Biochem. 271) 593
Fig. 3. Far UV CD spectra of N- and C-terminal fragments of tropomyosin (Tm) and their mixtures. Tm fragments are indicated in the figure. White
circles: C-terminal fragments [Tm(220–284), Tm(167–260) or Tm(143–260)]. Gray diamonds: N-terminal fragments [ASTm(1–142) or nfTm(1–
142)]. Gray circles: sum of the individual spectra of the respective N- and C-terminal fragments (theoretical curve). Black triangles: mixture of
N- and C- terminal fragments (experimental curve). Conditions: 10 mM sodium phosphate (pH 7.0), 0.5 mM EDTA, 1 mM dithiothreitol, 25 C.
All protein (dimer) concentrations were 10 lM.
Table 1. The a-helix content of tropomyosin (Tm) fragments used in this study, as a function of temperature and ionic strength. The a-helix content was
calculated as described in the Materials and methods.
Tm C-terminal
fragment
% a-helix at 10 C
100 mM KCla
% a-helix at 25 C
100 mM KCla
% a-helix at 25 C
0 mM KClb
Tmc
(C)b
Tm(220–284)
Tm(167–260)
Tm(143–260)
ASTm(1–142)
nfTm(1–142)
68
64
82
97
99
48
28
69
91
92
32
7.7
54
90
81
22
16
23
40
40
a
25 mM sodium phosphate (pH 7.0), 1 mM dithiothreitol, 0.5 mM EDTA [30],
dithiothreitol, c temperature at midpoint of denaturation transition.
b
10 mM sodium phosphate, pH 7.0, 0.5 mM EDTA, 1 mM
594 A. A. Paulucci et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 4. Thermal denaturation curves of individual tropomyosin (Tm) fragments and of N- and C-terminal fragment mixtures. Graphs show the
ellipticities at 222 nm, as a function of temperature, of the Tm fragments indicated in the figure. Dark gray squares: C-terminal fragments [Tm(220–
284), Tm(167–260) or Tm(143–260)]. Black circles: N-terminal fragments [nfTm(1–142) or ASTm(1–142)]. Gray circles: sum of the individual
thermal unfolding curves of the N- and C-terminal fragments (theoretical curves). White triangles: thermal unfolding curves of mixtures of N- and
C-terminal fragments (experimental curves). The error bar represents the largest standard deviation observed for a set of two experiments. Inset:
percentage difference between experimental and theoretical curves, as a function of temperature. % ¼ [100*(hexperimental ) htheoretical)/
(htheoretical)]. Conditions: 10 mM sodium phosphate (pH 7.0), 0.5 mM EDTA, 1 mM dithiothreitol, 25 C. Protein concentration, 10 lM.
with Tm(220–284) (Figs 3 and 4). The temperature and
relative magnitude of the maximum difference between
experimental and theoretical curves is a function of both the
intrinsic stability of the C-terminal fragment at low
temperatures and of the stability of the head-to-tail complex
formed with ASTm(1–142). It is interesting to note that for
Tm(220–284) and for Tm(167–260), the differences at 5 C
are greater than that observed for Tm(143–260). This is
because the first two fragments are less stable than the latter
(see Table 1) [30].
The Tm primary structure has a sevenfold pseudo-repeat.
This so-called a/b repeat divides the Tm polypeptide chain
into seven regions, each of which comprises two alternative
pairs of negatively charged and positively charged zones.
These repeats are thought to correspond to the actinbinding sites of Tm [6,45] (L. M. F. Holthauzen and C. S.
Farah, unpublished observations). Integral repeats and a
continuous coiled-coil are required for binding of the
striated muscle tropomyosin to the regulated actin filament
[46], and four or more repeats are required for Tm to bind
FEBS 2004
Head-to-tail interaction of tropomyosin (Eur. J. Biochem. 271) 595
Fig. 5. Model for folding and interaction of tropomyosin (Tm) N- and C-terminal domains in the head-to-tail complex. (A) Division of the head-to-tail
interaction into two steps: folding of the C-terminal domain (transitions a fi b and d fi e with the associated free energy change DGFOLD) and the
interaction between the folded N- and C-terminal domains (transitions b fi c and e fi f with the associated free energy change DGINTER). We may
consider three possible scenarios. In the first scenario (i), at very low ionic strengths (low I), both Tm260 and Tm284 C-termini are essentially
unfolded in the absence of the Tm N-terminus (i.e. DGFOLD > 0), and the relative strengths of the head-to-tail interactions are determined by the
sum of the folding and interaction energies (DGFOLD + DGINTER). In the second scenario (ii), at relatively high ionic strengths (high I), both Tm260
and Tm284 C-termini may be significantly folded in the free state (i.e. DGFOLD < 0). In this case, the relative strengths of the head-to-tail interactions
are determined predominantly by the interaction energies (DGINTER), with very little contribution from the folding of the C-termini. In the third
scenario (iii), at intermediate ionic strengths (medium I), the Tm260 C-terminus is expected to be relatively unstable, while the Tm284 C-terminus is
relatively stable (i.e. DGFOLD260 > 0 and DGFOLD284 < 0). In all cases, the observed association energy is, in fact, a result of the relative differences
between the predominant (lowest energy) state of the polypeptides on their own (state a, b, d or e, indicated by asterisks) and the final head-to-tail
complex (c or f). Under specific conditions (i.e. the third scenario), the Tm260 C-terminus may be significantly unfolded, while the Tm284 C-terminus
may be significantly folded. Note that this scheme does not take into account possible conformational changes in the N-terminal domain. (B)
Illustration of the association between the Tm N-terminal fragment and non-native (left) or native (right) C-terminal fragments for the third scenario
described above. The greater concentration of negative charges at the non-native C-terminus (a, b) destabilizes the coiled-coil structure to a greater
extent than that in the native C-terminus (d, e). Thus the non-native C-terminus may be less stable than the native C-terminus in the absence of the
N-terminal fragment. Upon association, the non-native C-terminus (c) interacts more strongly with the Tm N-terminus than does the native
C-terminus (f). Here, the head-to-tail interaction for ASTm(1–260) is stronger than that of ASTm only if DGFOLD260 + DGINT260 < DGINT284.
to actin with reasonable affinity [47]. Although none of our
fragments contained an integral number of a/b repeats, and
all were less than four repeats in length, we did, nonetheless,
perform sedimentation experiments in order to investigate
whether the complexes formed between Tm(167–260),
Tm(143–260) or Tm(220–284) and ASTm1–142 could bind
596 A. A. Paulucci et al. (Eur. J. Biochem. 271)
stably to actin. We observed no significant Tm binding in
the presence of 10 mM, 25 mM or 50 mM NaCl in 10 mM
Mops (pH 7.0), 5 mM MgCl2, 0.5 mM EGTA and 1 mM
dithiothreitol (data not shown).
Discussion
The N- and C-terminal sequences of Tm are a determining
factor for the strength of the head-to-tail interaction, as well
as for the interaction of Tm with troponin [48–51]. The
N- and C-terminal sequences of Tm are expressed in a
tissue-dependent manner through the use of alternate gene
promoters and alternative splicing mechanisms [26].
Recently, Palm et al. [51] studied complex formation
between peptides derived from Tm sequences encoded by
exon 1a (N-terminus of muscle and some nonmuscle
isoforms), exon 1b (N-terminus of other nonmuscle isoforms), exon 9a (C-terminus of striated muscle Tm
isoforms) and exon 9d (expressed in smooth muscle and
nonmuscle Tm isoforms). They observed a head-to-tail
interaction between the four possible combinations of
N-terminal and C-terminal fragments and showed that
both C-terminal peptides formed a more stable binary
complex with the N-terminal encoded by exon 1b
(AGSSSLEAVRRKIRSL) than with the N-terminal coded
by exon 1a (MDAIKKKMQMLK). Note that the exon
1b-encoded N-terminus has a five-amino acid extension.
These studies agree with previous reports [50], in which a
recombinant fibroblast nonmuscle Tm (5aTm), whose
N- and C-terminal sequences are encoded by exons 1b
and 9d, presented a stronger head-to-tail interaction than
rat skeletal Tm, where the N- and C-terminal sequences are
encoded by exons 1a and 9a.
Some studies have demonstrated that smooth muscle
Tms polymerize to a greater extent than striated muscle
Tms. Sanders & Smillie [48] and Graceffa [49] showed that a
gizzard smooth muscle Tm presents a stronger head-to-tail
interaction than a rabbit skeletal muscle Tm. As mentioned
above, skeletal muscle isoforms and smooth muscle isoforms have identical N-termini, but different C-termini.
Similarly, the differences in the head-to-tail interactions that
we observed for ASTm and ASTm(1–260) are probably
caused by differences in the sequences present in the
C-termini of the polypeptides, as discussed below.
Although we have no direct data on how the N- and
C-terminal fragments are oriented in the complex, our
results are most easily interpreted in terms of a novel headto-tail interaction between the Tm N-terminus and a
truncated C-terminus. The C-terminus, which terminates
at position 260, possesses a greatly increased head-to-tail
affinity when compared with ASTm. In spite of this
difference in affinity, the interaction seems to be similar to
that observed for the wild-type C-terminus, as (a) polymerization is strongly ionic-strength dependent, confirming the
electrostatic nature of the interaction, (b) formation of both
types of complexes are accompanied with a significant
increase in helix content and (c) the interaction is greatly
diminished in the presence of the unacetylated nonfusion
N-terminal fragment, nfTm(1–142), confirming the head-totail nature of the interaction. We recognize that what we
have described here is the discovery of an altered C-terminus
that interacts with an altered N-terminus. However, the
FEBS 2004
Ala-Ser dipeptide fusion used here, in the recombinant Tms,
has been shown previously to faithfully mimic the
Na-acetylation of muscle Tm [15].
The strong electrostatic component of the head-to-tail
interaction is revealed by its ionic-strength dependence.
Several charged residues are present in both N- and
C-terminal overlap regions, and the N-terminus has a net
positive charge while the C-terminus has a net negative
charge [5]. Phosphorylation at serine 283 of the a-Tm
strengthens the head-to-tail interaction of Tm [52,53]. A
serine 283 fi glutamic acid mutation, which may mimic this
phosphorylation, also strengthens the interaction, whereas a
positive charge (lysine) at this position weakens it [54]. A
possible hypothesis to explain the increased binding presented by our fragments that terminate at position 260 is that the
new C-terminus (…LEKSIDDLEDEL) in some way mimics
the native C-terminus (…ELDNALNDITSL), in the sense
that important features for the interaction are conserved and
that the differences, in fact, stabilize the interaction instead of
destabilizing it. In this sense we may observe that the last 11
residues of the new C-terminus presents a much greater
concentration of negatively charged residues (14 per dimer,
including the C-terminal carboxylate) than that observed in
the native C-terminus (eight per dimer). To a crude first
approximation then, the new C-terminus may therefore
interact more strongly with the positively charged N-terminal
region (ASMDAIKKKMQMLK…).
We note that, while head-to-tail complexes involving
Tm(260) or Tm(284) C-termini are all destabilized under
increased ionic strengths, the head-to-tail complexes containing the Tm(260) C-terminus are apparently more stable
under all conditions (Fig. 1). Note also, that the extent of
folding of the Tm C-termini in the free (i.e. uncomplexed)
state are highly ionic-strength dependent and may be
significantly different for Tm(260) and Tm(284) C-termini
under specific conditions (Table 1). It may be expected that
the particularly greater concentration of negative charges at
the non-native C-terminus destabilize the coiled-coil structure to a greater extent than that in the native C-terminus.
This is consistent with the observation that Tm(167–260) is
less stable than the smaller fragment Tm(220–284) (Table 1)
[30]. Like-charge repulsions, under these conditions, would
not only favor a greater interhelical distance, as observed in
the crystal and solution structures of C-terminal Tm
fragments [24,25], but would also be expected to destabilize
the individual a-helices, favoring a more extended nonhelical structure for the polypeptide. Similarly, the presence
of a charged a-amino group at an internal a position in
nonacetylated nonfusion Tm disorders the coiled-coil
structure at the N-terminus [16,32] and reduces the headto-tail interactions [15]. The Ala-Ser N-terminal extension
recovers head-to-tail affinity presumably by dislocating the
a-amino group to an external f position, increasing the
distance and reducing repulsions between these groups in
the coiled-coil structure [15].
The above observations lead to an apparent paradox:
while destabilization of the N-terminal coiled-coil structure
destabilizes the head-to-tail interaction, conditions expected
or known to destabilize the helical and coiled-coil structure
of the C-terminus (increased negative charge density, low
ionic strength) in fact stabilize the head-to-tail interaction.
This paradox may be resolved by considering that the
FEBS 2004
Head-to-tail interaction of tropomyosin (Eur. J. Biochem. 271) 597
association energy is, in fact, caused by the relative
differences between the conformational energies of the
polypeptides on their own (i.e. in the free state before
interacting, see the asterisks in Fig. 5) and in the complex.
For illustrative purposes, it is useful to separate the head-totail interaction process into two steps: the folding of the
C-terminus (with free energy change DGFOLD) and the
subsequent interaction with the folded N-terminal domain
(with free energy change DGINTER). Here we assume that
the free N-terminal domain is well folded under all
conditions. We may then consider the expected ionicstrength dependence of each step and how they may be
rationalized in the light of the results presented in Figs 1 and
4 and Table 1. This leads to three possible scenarios,
presented in Fig. 5A. In the first scenario (i), at very low
ionic strengths, both Tm260 and Tm284 C-termini are
expected to be essentially unfolded in the absence of the Tm
N-terminus (i.e. DGFOLD > 0). Therefore, the relative
strengths of the head-to-tail interactions are determined by
the sum of the folding and interaction energies
(DGFOLD + DGINTER), and destabilization of the C-terminal structure in the free state may be compensated for by
a particularly strong interaction with the N-terminus in the
head-to-tail complex. In the second scenario (ii), at relatively
high ionic strengths, both Tm(260) and Tm(284) C-termini
may be partially folded in the free state (i.e. DGFOLD < 0).
In this case, the relative strengths of the head-to-tail
interactions are determined predominantly by the interaction energies (DGINTER), with relatively little contribution
from the folding of the C-termini (as they are already
significantly folded in the free state). In the third scenario
(iii), within a specific range of intermediate ionic strengths,
the Tm(260) C-terminus may be significantly unfolded,
while the Tm(284) C-terminus may be relatively more folded
(i.e. DGFOLD260 > 0 and DGFOLD284 < 0). Under these
conditions, the thermodynamics of the head-to-tail interactions, involving the Tm260 and Tm284 C-terminus, are
reflecting different processes: the former a process with a
significant folding component and the latter a process which
occurs beginning with a significantly folded free C-terminus
(as shown in Fig. 5B).
Our CD studies show that the head-to-tail interaction
between fragments which terminate at position 260 or 284
[Tm(167–260), Tm(143–260) and Tm(220–284)] with
ASTm(1–142) is accompanied with a significant degree of
folding (helix formation) of the C-terminal Tm fragment. As
all C-terminal fragments studied here, and especially
Tm(167–260), are not very stable under the low ionic
strength conditions of the assay (Table 1), we presume that
in the complex, the negative charges at the C-terminus are
shielded as a result of their proximity to the positively
charged N-terminus [ASTm(1–142)]. In this way, association with the N-terminus could stabilize a more compact
C-terminal structure in which intrahelical and interhelical
electrostatic repulsions between like charges are shielded.
These observations are consistent with the first scenario (i)
shown in Fig. 5A. At greater ionic strengths, head-to-tail
complexes, involving Tm(260) and Tm(284) C-termini, are
destabilized as shown in the second and third scenarios
(ii and iii) in Fig. 5A.
Differences in the net charges of the C-termini cannot,
of course, fully explain the differences in head-to-tail
interactions of ASTm and ASTm(1–260). Both must be
able to adopt structures whose complementary surfaces may
associate with significant favorable interaction energy. The
evidence presented here (ionic strength dependence, concomitant increase in helix content) suggests that the head-totail complex involving ASTm(1–260) is similar in nature
to that of ASTm. We do not, however, know the actual
structure of the Tm head-to-tail complex, so we can only
speculate as to necessary conformational features; for
example, a fully formed coiled-coil at the N-terminus
[16,32] and a helicoidal, but noncoiled-coil, structure at
the C-terminus (as seen in crystal and solution structures
of C-terminal fragments [24,25]). However, whether the Tm
C-terminal helices remain separated in the presence of the
positively charged Tm N-terminus is not clear.
Several lines of evidence point to a head-to-tail overlap
of approximately nine residues in native skeletal muscle
Tm [5]. This overlap should be two residues longer in
ASTm as a result of the Ala-Ser extension. We do not
know whether the head-to-tail complex involving the
truncated C-terminus in ASTm(1–260) involves an overlap
of a greater, equal or shorter length. In this sense, we note
that the C-terminal residues of ASTm(1–260) are not in
the same heptad position as are the C-terminal residues in
the native Tm molecule, i.e. the C-terminal Ile284 of the
native molecule occupies a d position in the heptad
repeat, while the C-terminal Leu260 of Tm(1–260) occupies an a position (Table 2). If the C-terminus in the
head-to-tail complex is a coiled-coil, this would argue for
an alignment between ASTm and ASTm(1–260) C-termini
that also aligns with respect to heptad repeat positions
[i.e. Ile284 of ASTm with Leu256 of ASTm(1–260), or
Met281 of ASTm with Leu260 of ASTm(1–260)]. However, if the last C-terminal residues of Tm do not adopt a
canonical coiled-coil structure, as proposed by Greenfield
et al. [25] and by Li et al. [24], the preference vanishes for
an alignment of the structures based on the heptad repeats
(we note, however, that the heptad repeat is maintained in
this region; Table 2). In this case, it is possible that the
C-terminal region of ASTm(1–260) adopts a structure
similar to that of the native C-terminus of Tm when in
complex with the N-terminus, in spite of terminating at
different heptad repeat positions. This common structural
feature would be expected to be reflected in a conserved
sequence in the two C-termini.
Table 2 shows an alignment of the 12 C-terminal
residues of ASTm(1–260) with the native C-terminal
portions of the a and b striated muscle and smooth muscle
Tm isoforms, which are known to bind to actin and to
polymerize in a head-to-tail manner [48–51]. While there is
a high degree of similarity between a and b isoforms of a
specific muscle type, there is also a lower, but significant,
degree of similarity between the striated and smooth
muscle isoforms, specifically at positions 274 (Leu), 275
(Asp), 278 (Leu), 280 (Asp/Glu), 281 (Met/Ile/Leu) and
284 (Ile/Leu/Met) (Table 2). The best sequence-based
alignment of the ASTm(1–260) C-terminus with the
C-termini of these four muscle isoforms is shown in
Table 2. ASTm(1–260) aligns particularly well with all but
the last (but see below) of these conserved positions: L249
of ASTm(1–260) with position 274 of the native C-terminus,
Glu250 with 275, Ile253 with 278, Asp255 with 280, and
598 A. A. Paulucci et al. (Eur. J. Biochem. 271)
FEBS 2004
Table 2. Alignment of the C-terminus of ASTm(1–260) with the C-termini of tropomyosin (Tm) muscle isoforms known to partake in head-to-tail
interactions. Alignment was performed using the CLUSTALW algorithm (http://www.ebi.ac.uk/clustalw/#). Symbols above and below the Tm(1–260)
sequence show the alignment between Tm(1–260) and striated and smooth muscle isoforms, respectively. * identity, :, conservation.
Leu256 with 281 (Table 2). Note that in this alignment, the
penultimate residue of ASTm(1–260) – Glu259 – is aligned
with the last residue (284) of the native protein terminus.
Therefore, the charged Glu259 side-chain of ASTm(1–260)
may, in fact, mimic the C-terminal carboxylate of the
native protein. Furthermore, at positions not well conserved between the native C-termini of striated and
smooth muscle isoforms, the ASTm(1–260) sequence
aligns with one of the isoforms (Table 2). For example,
residues Ser252, Glu257 and Asp258 align well with Thr277,
Asn282 and Asn283 of the smooth muscle proteins and
Asp254 aligns with Asn279 of the striated muscle proteins.
The above analysis suggests that the C-terminus of
ASTm(1–260) may, in fact, possess several of the features
necessary to adopt a structure similar to that of native
muscle Tms. While we have studied its interactions with
non-native N-terminal fragments (i.e. a nonacetylated AlaSer fusion or nonfusion forms), the N-terminus of ASTm
has been shown to behave in a manner very similar to
acetylated muscle Tm [15]. The strong reduction in stability
of the native head-to-tail complex in the presence of only
moderate amounts of monovalent ions strongly limits the
facility by which its structure may be studied by crystallography or NMR. The unusually strong interaction in headto-tail complexes involving Tm fragments terminating at
position 260 may prove useful substitutes for the native
C-terminus in such studies and its properties may provide
insights into the factors important for Tm function.
Acknowledgements
We thank the Laboratório Nacional de Luz Sincrotron, Campinas,
Brazil, for permission to use the Jasco J-810 spectropolarimeter. This
work was supported by grants from Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP). A. A. P. and A. D. S. received
graduate fellowships and A. M. K. received an undergraduate
fellowship from FAPESP.
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