Báo cáo Y học: The inhibitory region of troponin-I alters the ability of F-actin to interact with different segments of myosin pot

The inhibitory region of troponin-I alters the ability of F-actin
to interact with different segments of myosin
Valerie B. Patchell
1
, Clare E. Gallon
2
, Matthew A. Hodgkin
3
, Abdellatif Fattoum
4
, S. Victor Perry
1
and Barry A. Levine
1,2
1
Department of Physiology, School of Medicine and
2
School of Biosciences, University of Birmingham, Birmingham, UK;
3
School
of Biological Sciences, University of Warwick, Warwick, UK;
4
CRBM, CNRS, INSERM U249, F-34090 Montpellier, France
Peptides corresponding to the N-terminus of skeletal
myosin light chain 1 (rsMLC1 1–37) and the short loop of
human cardiac b-myosin (hcM398–414) have been shown
to interact with skeletal F-actin by NMR and fluorescence
measurements. Skeletal tropomyosin strengthens the
binding of the myosin peptides to actin but does not
interact with the peptides. The binding of peptides cor-
responding to the inhibitory region of cardiac troponin I

(e.g. hcTnI128–153) to F-actin to form a 1 : 1 molar
complex is also strengthened in the presence of tropomyo-
sin. In the presence of inhibitory peptide at relatively
lower concentrations the myosin peptides and a troponin I
peptide C-terminal to the inhibitory region, rcTnI161–181,
all dissociate from F-actin. Structural and fluorescence
evidence indicate that the troponin I inhibitory region and
the myosin peptides do not bind in an identical manner to
F-actin. It is concluded that the binding of the inhibitory
region of troponin I to F-actin produces a conformational
change in the actin monomer with the result that inter-
action at different locations of F-actin is impeded. These
observations are interpreted to indicate that a major
conformational change occurs in actin on binding to
troponin I that is fundamental to the regulatory process in
muscle. The data are discussed in the context of tropo-
myosin’s ability to stabilize the actin filament and facilitate
the transmission of the conformational change to actin
monomers not in direct contact with troponin I.
Keywords: Cardiac troponin I, tropomyosin, myosin pep-
tides, actin, conformational change.
The property of troponin I (TnI) of being able to inhibit the
MgATPase of actomyosin in a manner that can be
neutralized by the calcium-binding protein troponin C in
the presence of calcium ions suggests that TnI occupies a
key position in the regulation of contraction in striated
muscle. In the absence of tropomyosin and the other
components of the troponin complex, TnI inhibits the
MgATPase of actomyosin maximally when there is
approximately one molecule of TnI per actin monomer

[1,2]. This implies that TnI prevents the interaction of actin
with the myosin head that leads to the activation of the
MgATPase. In the presence of tropomyosin, the inhibitory
influence of TnI is much increased and the maximum effect
is obtained when the stoichiometry approaches one mole-
cule of TnI to seven actin monomers [1–5]. When troponin
C and troponin T are absent this inhibition is calcium
insensitive [6] but otherwise corresponds to the ÔoffÕ state in
intact muscle.
The region of rabbit fast skeletal TnI represented by
residues 96–116, known as the inhibitory peptide (IP),
possesses properties that are very similar to the intact
molecule in that it binds to troponin C, and in the
presence of tropomyosin the inhibition of the MgATPase
of actomyosin by the peptide is markedly increased [7].
The inhibitory peptide in the presence of tropomyosin is
about 50% as effective as the intact TnI molecule when
assayed under similar conditions. Only about half of the
residues of IP, as originally isolated, appear to be essential
for this property because a synthetic duodecapeptide
comprising residues 104–115 (short IP) has equivalent
inhibitory activity [8]. Recent evidence suggests that
additional regions of TnI, C-terminal to the IP, may be
required to obtain inhibitory activity equal to the intact
molecule [9,10].
The mechanism of action of TnI on the regulation of
the contractile process is not as yet understood (see [11]
for a review). Despite the inhibitory properties of TnI the
current view is that tropomyosin regulates the actomyosin
ATPase in situ by a steric mechanism [12–14] and it has

been suggested that the role of TnI is to induce the
binding of tropomyosin to actin [3]. Nevertheless the
ability of TnI to bind to actin must be of special
significance, as by blocking the binding site it could
prevent the interaction with myosin that leads to the
activation of the MgATPase. Alternatively binding could
involve an allosteric mechanism whereby a conformational
change is induced in the actin monomer that results in
regions elsewhere on the molecule no longer being able to
interact with myosin to activate the MgATPase. Any
proposed mechanism must explain the ability of tropo-
myosin to extend the inhibitory activity of the troponin I
molecule from one to seven actin monomers.
Correspondence to S. V. Perry, Department of Physiology, School of
Medicine, University of Birmingham, Birmingham B15 2TT.
Fax: + 44 121414 6919, Tel.: + 44 121414 6930,
E-mail:
Abbreviations: IP, inhibitory peptide; TnI, troponin I; ATPase,
adenosinetriphosphatase; MLC1, myosin light chain 1; IAEDANS,
5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid;
SPR, surface plasmon resonance
(Received 14 June 2002, revised 19 August 2002,
accepted 5 September 2002)
Eur. J. Biochem. 269, 5088–5100 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03227.x
The nature of the interaction of the myosin head with
actin is still a matter for discussion but it is clearly complex
and may involve more than one actin monomer (for review
see [15]). The major contacts of myosin with actin appear to
involve several regions of the myosin heavy chain. It is
considered that there is a large primary contact site on the

surface of actin flanked on three sides by additional contacts
involving myosin surface loops [16]. One of these loops,
Pro402–Lys415, is modelled as interacting with actin near
residues 331–332 [16] at the junction of subdomains 1 and 3
of actin and appears to be important for normal muscle
activity.Deletionofthisloopregionresultedinthelossof
strong binding of myosin to actin [17] while a single amino
acid residue change, ArgfiGln, in this loop region of the
b-chain of human cardiac myosin is associated with familial
hypertrophic cardiomyopathy [18,19] and has been reported
to result in altered kinetic properties of the myosin
subfragment 1 ATPase [20].
Although there is no doubt that tropomyosin moves on
contraction it is difficult, in view of the somewhat limited
knowledge of the nature of the actin–myosin interaction, to
decide on the role of actin in the activation process. X-ray
analysis provides some evidence for movement of the actin
domains during contraction [21] and it is likely that in model
systems using mutant proteins the movement of tropo-
myosin observed in the presence of myosin and troponin is a
consequence of conformational changes in actin [22,23]. The
binding of ligands at discrete and specific binding sites on
actin during the contractile cycle would be expected to
induce conformational changes that influence its interaction
with myosin. Cross linking studies with the zero length
carbodiimidate reagent specific for lysine–carboxylate con-
tacts suggest that one such ligand, TnI, binds close to the
region represented by residues 1–12 of actin [24]. Proton
MR studies have indicated that IP interacts with residues
1–7 and 24–25 of the N-terminal region of actin [25]. These

observations and the fact that only about half of the
residues of the IP are required for inhibitory activity suggest
that the interaction of only a small region of TnI with the
N-terminal region of actin is the minimum requirement to
prevent activation of the myosin MgATPase.
To throw light on the role of TnI and its relation to that of
tropomyosin in the regulatory process we have studied how
the actin-binding properties of peptides representing regions
of the myosin molecule are affected by the interaction of
actin with peptides incorporating the inhibitory domain of
TnI. The myosin peptides are displaced from F-actin by the
IP but not by tropomyosin; indeed their binding is
strengthened in the presence of the latter protein. Evidence
is provided for the induction of conformational changes in
at least two regions of the actin molecule on binding the
inhibitory domain of TnI to a third independent site. These
observations have important significance for understanding
the role of TnI in the regulation of contraction in striated
muscle.
Some aspects of this work have been briefly described in
abstract form [26].
MATERIALS AND METHODS
Peptides
The N-terminal region of the myosin light chain (MLC1),
residues 1–37, was prepared as described by Henry et al.
[27]. The peptides encompassing the inhibitory region of
human cardiac TnI, hcTnI128–153, dansylated hcTnI128–
153 (T128 replaced by a dansyl group), hcTnI136–148 and
the other peptides used in this study (Table 1) were
synthesized by Alta Bioscience (Birmingham University)

using Fmoc polyamide chemistry and purified as described
previously [28]. The peptide comprising residues 398–414 of
human cardiac b-myosin was synthesized by Syntem
(Montpellier) and purified as reported previously [29]. The
composition and purity of all peptides was confirmed by
NMR and mass spectral analysis.
Muscle proteins
Freeze dried actin prepared by the method of Spudich and
Watt [30] was dissolved in 5 m
M
triethanolamine/HCl,
pH 8.0, 0.2 m
M
CaCl
2
,0.2 m
M
ATP, 0.2 m
M
dithiothreitol,
and dialysed for 3 h against the same buffer until fully
depolymerized. It was then centrifuged at 30 000 g for
30minandtheconcentrationoftheG-actininthe
supernatant determined by measuring absorbance at
290 nm using an absorption coefficient of 0.63 mgÆ
mL
)1
Æcm
)1
. The G-actin was polymerized by making the

solution 2 m
M
with respect to MgCl
2
and 50 m
M
with
respect to KCl. The F-actin was then dialysed overnight
against several changes of 5 m
M
sodium phosphate buffer,
pH 7.0, in H
2
Oor[
2
H]
2
O. F-actin–tropomyosin complex
was made by adding G-actin prepared as described above to
Table 1. Peptides used in this study. Unless otherwise indicated the N-terminus is acetylated and the C-terminus is in the amide form. The
N-terminus of the long MLC1 peptide is trimethylalanine. The trimethylalanine of wild type MLC 1–13 was replaced by N-acetyl alanine. The HA
peptide corresponds to the well characterized immunodominant epitope of influenza haemagglutinin, residues 306–318. TnI numbering based on
N-acyl terminus as occurs in vertebrate proteins and not methionine as occurs in recombinant TnI.
Name Sequence
hcTnI128–153 TQKIFDLRGKFKRPTLRRVRISADAM
hcTnI128–147 (IP) TQKIFDLRGKFKRPTLRRVR
hcTnI136–147 (short IP) GKFKRPTLRRVR
rcTnI161–181 (C-terminal to IP) AKETLDLRAHLKQVKKEDTEK
hcTnI161–181 (C-terminal to IP) AKESLDLRAHLKQVKKEDTEK
hcM398–414 (myosin loop) GLCHPRVKVGNEYVTKG

rsMLC1 1–37 (MLC1) APKKDVKKPAAAAAAPAPAPAPAPAPAKPKEEKIDL
rsMLC1 1–13 (MLC1) APKKDVKKPAAAA
HA306–318 (HA peptide) PKYVKQATLKLAT
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5089
a stock solution of 1 mgÆmL
)1
rabbit skeletal tropomyosin
in 50 m
M
Tris/HCl, pH 7.6, 100 m
M
KCl, to give a final
concentration of 2.5 mgÆmL
)1
actin, 0.5 mgÆmL
)1
tropo-
myosin, i.e. a molar ratio of actin : tropomyosin of
approximately 7 : 1. The complex was dialysed into several
changes of 5 m
M
phosphate buffer, pH 7.0, in H
2
Oor
[
2
H]
2
O. Complex formation and the absence of free protein
was confirmed by comparison of the electrophoretic

patterns of the free proteins by electrophoresis on 10%
nondenaturing polyacrylamide gels run in 10% (v/v)
glycerol, 25 m
M
Tris)80 m
M
glycine, pH 8.3.
Actin labelled at Cys374 with IAEDANS was prepared
according to the method of Miki et al. [31]. F-actin
(2 mgÆmL
)1
)in5m
M
triethanolamine/HCl, pH 8, 0.2 m
M
ATP, 0.2 m
M
CaCl
2
(Buffer A) to which 50 m
M
KCl and
2m
M
MgCl
2
had been added was incubated with 10-fold
molar excess of IAEDANS for 2 h at room temperature.
The reaction was terminated by the addition of dithiothreitol
to 2 m

M
. The actin was then centrifuged at 100 000 g for 1 h
and the pellet resuspended in Buffer A. This was dialysed
extensively against Buffer A to remove excess IAEDANS.
The concentration of the resulting G-actin was determined
using an absorption coefficient of 0.63 mgÆmL
)1
Æcm
)1
at
290 nm. A correction for the IAEDANS contribution at
290 nm was made using absorbance at 290 nm ¼
0.21 · absorbance at 336 nm, for bound IAEDANS.
The concentration of IAEDANS was determined using
the absorption coefficient of 6100
M
)1
Æcm
)1
at 336 nm. The
extent of labelling was normally 0.8–0.9 molÆmole
)1
G-actin. The labelled G-actin was polymerized by making
the solution 50 m
M
with respect to KCl, 2 m
M
with respect
to MgCl
2

, and stored frozen in aliquots.
Fluorescence measurements
All fluorescence emission spectra were obtained using a
Perkin-Elmer LS50B fluorescence spectrometer interfaced
to a computer. The excitation wavelength for tryptophan
was 280 nm and the IAEDANS probe was excited at
340 nm. The fluorescence emission spectra of the dansylated
TnI peptide was recorded between 400 and 550 nm after
excitation of the dansyl group at 340 nm. Emission
fluorescence intensity values were corrected for the corres-
ponding solvent emission fluorescence values and the
dilution effects (< 5%) resulting from the titration carried
out. The dissociation constants (K
d
) for the complexes
formed were calculated by using a nonlinear regression
procedure fitting the fluorescence data obtained in three
separate titrations in each case to a 1 : 1 binding curve
in combination with the use of a reciprocal linear plot
(F
o
)F
min
)/(F
o
)F) vs. (concentration of added peptide)
)1
.
For all calculations it was assumed that the fractional
change in fluorescence was directly proportional to the

fraction of the complexes formed. The accuracy of the K
d
values was gauged from curve fit obtained, the associated R
2
value (> 0.95) and the requirement that iterative fit of the
linear representation of the experimental data extrapolated
to an intercept value of 1.
Surface plasmon resonance
Direct binding of the TnI inhibitory peptide to actin was
investigated using surface plasmon resonance (SPR)
analysis to evaluate the association and dissociation rate
constants, K
a
and K
d
respectively, for the binding of the
peptide to immobilized F-actin using a BIAcore 3000
system. F-actin or BSA was covalently linked to carboxy-
methyldextran surfaces using standard amine coupling.
One surface was derivatized in the absence of protein.
Following immobilization the chip surfaces were capped
with ethanolamine and subject to surface equilibration
(BIApplications Handbook, 1993). Non-specific binding
was monitored using the control BSA and underivatized
flow cells. Sensorgrams were obtained using different
immobilization densities and the binding of the TnI
inhibitory peptide was assessed at various flow rates
(5–30 lLÆmin
)1
) and over a range of concentrations

(1–50 l
M
). Sensorgrams were analysed using
BIAEVALUA
TION
3 software taking account of the small amount of
nonspecific binding of the TnI peptide to the control
surfaces. The association and dissociation rate constants
were obtained from these sensorgrams by fitting the
experimental data to a model obeying 1 : 1 complex
formation and the Langmuir binding isotherm. The
apparent equilibrium constant (dissociation constant K
d
)
was calculated as K
d
/K
a
. There was no significant change
in the K
d
derived for the peptide concentrations in the
range 1–10 l
M
. Curve fitting of the dissociation phase for
each concentration was also separately carried out as for
an AB complex dissociation.
NMR studies
The NMR spectral assignment of peptide resonances was
carried out using standard TOCSY and NOESY proce-

dures. Spectra were obtained at 500 MHz on a Bruker
spectrometer at a sample temperature of 285K. Titration
of the peptides with F-actin was carried out by addition of
aliquots of F-actin (10 mgÆmL
)1
) or F-actin–tropomyosin
(5 mgÆmL
)1
F-actin). Titration of the inhibitory peptide
with F-actin or F-actin–tropomyosin was also carried out
by the addition of small aliquots (1–5 lL) of a stock
solution of the peptide to 0.5 mL of solution containing
F-actin at a concentration of 2.5–4.0 mgÆmL
)1
. The broad
signals of the spectrum of F-actin obtained at these
concentrations contributed relatively little to the spectra of
the peptides in the presence of actin. Two-pulse spin-echo
spectra (1024 transients) were obtained using a (180-t-90-t)
sequence with a delay time, t ¼ 60 ms, and an overall
interpulse delay of 3 s to enable complete magnetization
recovery. Signal amplitude in these experiments is modu-
lated by the corresponding coupling constant and relax-
ation time of each resonance and is a very sensitive
indicator of the effect of binding. As observed in previous
studies of actin binding [25,28] interaction results in
marked reduction of the bound peptide ligand resonances
consistent with the high molecular weight and slow
tumbling of the complex. Both direct signal linewidth
and the signal intensity in the two-pulse spin-echo spectra

were used therefore to evaluate the perturbation resulting
from interaction with actin. The spectral changes were
also visualized by difference spectra taken at each stage of
the titration. Quantification of the binding stoichiometry
and affinity of complex formation was confirmed by
equilibrium fluorescence measurements and by surface
plasmon resonance studies.
5090 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
The interaction of the different peptides with F-actin was
assayed using a variety of biophysical techniques to
characterize their interaction affinity, to determine the
nature of the residues involved and the extent of any
competition between the peptides on binding. A diagnostic
test for the binding of a peptide ligand to F-actin or
tropomyosin is the observation of significant broadening of
the ligand resonances detected in the NMR spectrum. The
flexibility in a peptide ligand is manifest in the narrow line
widths of the peptide NMR resonances since linewidth is a
monotonic function of the effective correlation time [32].
Upon complex formation the bound groups of the peptide
ligand would experience the longer rotational correlation
time of the macromolecular assembly and a decrease in
segmental mobility. Interaction is therefore apparent from
changes in the linewidth of the peptide resonances since
complex formation results in an increased relaxation rate
due to the longer correlation time of the protein and the
motional constraints imposed by binding of the peptide to
the protein surface. Spectral linewidth increases on addition
of F-actin to inhibitory regions of troponin I and caldesmon

have been reported elsewhere [25,33]. Resonance line width
changes can also originate, however, from any increase in
solution viscosity that significantly alters the rotational
diffusion of the ligand. As F-actin solutions have significant
viscosity we therefore first studied the effect of an increase in
solution viscosity on the linewidth characteristics of the IP.
Minimal spectral effects were observed for the hcTnI128–
153 peptide over a concentration range of 0–500 l
M
in 10%
(v/v) deuterated glycerol (MSD Isotopes). These observa-
tions indicated that viscosity effects on resonance and
linewidth in the peptide spectrum were not significant.
Evidence for the absence of viscosity effects on peptide
resonance and linewidth as a result the presence of actin
were obtained by comparing the spectrum of a control
peptide, the HA peptide (Table 1), in the presence and
absence of F-actin (Fig. 1). The absence of detectable
alterations in the spectrum of the peptide indicated that any
changes in viscosity due to the presence of F-actin have
negligible effects on the rotational diffusion in solution and
hence linewidth of the peptide resonances. These results
(Fig. 1) also served as control data indicating that there was
no nonspecific HA peptide interaction with F-actin.
Inspection of the spectrum of the HA peptide in the
presence of F-actin also indicates that although interaction
did not occur, there is a detectable contribution to signal
intensity deriving from F-actin at the relatively high
concentrations of the protein used in this control experiment
(Fig. 1). The broad signals of the spectrum of F-actin did

not, however, mask the resonances of other peptides used in
this study due to the lower protein concentrations required
to induce spectral broadening. Figure 1 shows that specific
resonance broadening occurred during titration of the
rcTnI161–181 peptide with F-actin indicating complex
formation characterized by fast exchange between the free
and actin-bound states of the peptide. Most markedly
altered by interaction with F-actin are the sidechain signals
of Arg168, His170, Gln173 and the composite methyl group
resonance while the sidechain resonance deriving from the
five lysine residues of the peptide remained relatively
unperturbed. Correlation with the peptide sequence
(Table 1) indicates that residues 165–174 represent that
part of the peptide rcTnI161–181 whose molecular motions
are most restricted by binding to the surface of F-actin.
The possibility of nonspecific binding of the TnI peptides
to protein was examined using BSA. As shown in Fig. 2 the
presence of an equimolar concentration of the protein did
not give rise to any significant changes in the spectrum of
peptide hcTnI128–147 indicating the absence of nonspecific
interaction with the protein. Clearly resolved in the spectra
is the composite signal of the five Arg dCH
2
groups of the
inhibitory peptide ( 3.2 p.p.m., Fig. 2) that, as in the case
of rcTnI161–181 peptide (Fig. 1), can be used to monitor
the effect of F-actin on the spectrum of the peptide. The
arginine residues of the IP are located in the central portion
of the region associated with inhibitory activity.
Interaction of the cardiac inhibitory peptide region

with F-actin
To investigate the interaction between actin and the
inhibitory region of TnI, we monitored the NMR spectral
Fig. 1. Proton magnetic resonance spectra demonstrating that the
presence of F-actin does not result in broadening of signals of peptide in
the absence of complex formation whilst interaction with F-actin results
in specific spectral changes. Spectra determined in 5 m
M
sodium
phosphate buffer, pH 7.4, T ¼ 285K. (A) HA306–318 peptide,
200 l
M
, (B) HA306–318 peptide, 200 l
M
, in the presence of F-actin,
200 l
M
. The spectral region between 1.2 and 1.4 p.p.m. under these
conditions is shown on an expanded scale as inset. The fine structure
for the HA306–318 peptide resonances is retained indicating lack of
interaction with F-actin and the absence of broadening due to non-
specific viscosity effects over the actin concentration range studied
(0–8 mgÆmL
)1
). Peak at 1.34 p.p.m. in inset B is due to actin. (C)
rcTnI161–181 peptide, 200 l
M
. (D) rcTnI161–181 peptide, 200 l
M
,in

the presence of F-actin, 35 l
M
. Specific resonance broadening occurs
during titration of the peptide rcTnI161–181 with increasing concen-
trations of F-actin indicating complex formation characterized by fast
exchange between the free and actin-bound states of the peptide
population. The residues whose signals are most markedly affected by
interaction (e.g. Arg168, His170 and Gln173) indicate the region of the
peptide whose molecular motions are most restricted by binding to the
surface of F-actin.
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5091
changes during titration with F-actin of two peptides,
hcTnI128–153 and hcTnI136–147, corresponding to over-
lapping regions of human cardiac TnI (Table 1). Peptide
hcTnI128–153 comprises the inhibitory region of TnI with
the additional six residue C-terminal sequence, ISADAM,
which is present in all of the mammalian isoforms of TnI
sequenced so far and may be of functional significance. The
smaller peptide, hcTnI136–147 represents the region cor-
responding to the minimal inhibitory sequence of TnI [8].
Addition of F-actin to the human cardiac inhibitory
peptide in molar excess produced a marked reduction in
resonance intensity of the side chain groups of the free
peptide, indicating that interaction had occurred (Fig. 3).
The progressive variation in linewidth and intensity for the
peptide signals occurred in the absence of any chemical shift
change as is characteristic of relatively rapid exchange on
the relaxation time scale [32,34]. The marked reduction of
the peptide ligand resonance intensity upon addition of
F-actin is consistent with the high molecular weight and

slow tumbling of the complex formed. Similar results were
reported earlier [25] for the binding to F-actin by the
inhibitory peptide from rabbit fast skeletal muscle TnI
(residues 96–116) that differs from the homologous human
cardiac peptide by four conservative replacements. Since
almost all the resonances of the peptide hcTnI128–153 were
affected in the presence of F-actin (Fig. 3) the extent of the
spectral changes suggests that the entire length of the
peptide is constrained by attachment to the actin filament.
The kinetics of the interaction of the TnI inhibitory
region with actin were characterized using surface plasmon
resonance to monitor binding to immobilized F-actin. The
sensorgrams obtained recorded the association and disso-
ciation phases of the interaction (Fig. 4A). Analysis of the
dissociation phase for hcTnI128–153 peptide concentrations
in the range 1–10 l
M
gave an off rate constant of 10
3
Æs
)1
consistent with the NMR observation of fast exchange on
the relaxation time scale. The equilibrium constant for the
interaction was obtained by fitting the sensorgram data to a
model employing 1 : 1 complex formation. The value of the
dissociation constant derived, 3 l
M
(Table 2) was consistent
with an analysis of the dependence of the equilibrium
plateau signal on the concentration of the TnI inhibitory

peptide.
Tropomyosin enhances the affinity of the TnI inhibitory
peptide for F-actin
Titrations of F-actin with peptide hcTnI128–153 were
carried out with the molar ratio peptide : actin varied over
the range of 1 : 1 to 6 : 1 in the presence and absence of
tropomyosin. As shown in Fig. 4B, a steady change was
observed in the signal corresponding to the side chains of
arginine as the molar ratio of peptide to actin was increased.
The broad resonance linewidth of the bound peptide
reduced to that of the free peptide as the ratio of the bound
to free peptide decreased during titration with the peptide.
In the presence of tropomyosin the continuous reversion of
the signal lineshape to that of the free peptide is found to
saturate at close to a 1 : 1 molar ratio. In the presence of
tropomyosin, saturation occurred at a lower peptide : actin
ratio than was the case in the absence of tropomyosin.
(Fig. 4B). The rate of the exchange process between actin-
bound and free peptide is therefore altered by the presence
Fig. 2. Proton magnetic resonance spectra demonstrating the absence of
non-specific interaction of the TnI inhibitory peptide with BSA. Spectra
determined in 5 m
M
sodium phosphate buffer, pH 7.4, T ¼ 285K. (A)
peptide hcTnI128–147, 120 l
M
. (B) BSA, 120 l
M
. (C) 120 l
M

peptide
hcTnI128–147, in the presence of 120 l
M
BSA. This spectrum is
indistinguishable from the algebraic sum of the individual spectra
(A + B) indicating lack of nonspecific interaction with BSA. Signals
deriving from the hcTnI128–147 are labelled.
Fig. 3. Interaction of the human cardiac TnI inhibitory peptide with
F-actin illustrated by proton magnetic resonance spectroscopy to show
the residues involved in complex formation. Spectra determined in 5 m
M
sodium phosphate buffer, pH 7.2, T ¼ 293K. (A) peptide hcTnI128–
153, 200 l
M
, (B) hcTnI128–153, 200 l
M
, in the presence of F-actin,
18 l
M
. (C) hcTnI128–153, 200 l
M
, in the presence of F-actin, 50 l
M
.
(D) difference spectrum, A–C, highlighting the residues whose side-
chain signals are perturbed by binding to F-actin. Signals of the
hcTnI128–153 are labelled. Complex formation characterized by rel-
atively fast exchange between the free and actin-bound states of the
peptide population is indicated by the resonance broadening that
occurs during titration with increasing concentrations of F-actin. Note

that the lack of spectral change for the signal originating from the
buffer (*) confirms the absence of nonspecific viscosity effects.
5092 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002
of tropomyosin. The observation that in the presence of
tropomyosin the signal linewidth returned to that of the free
peptide at a 1 : 1 ratio and altered more dramatically at low
peptide : actin ratios indicates a retarded exchange process
and tighter binding of hcTnI128–153 to actin–tropomyosin.
In this intermediate exchange range, the rate of the exchange
process also contributes to the relaxation process and
resonance linewidths are expected on this exchange time-
scale to revert to those of the free peptide in a manner
dependent upon the exchange off-rate constant [34–36]. The
NMR data are therefore consistent with a 1 : 1 complex
formation between the IP and actin–tropomyosin and an
increase in affinity for actin resulting from the presence of
tropomyosin.
To supplement the binding data obtained from the NMR
investigations fluorescence studies using dansylated
hcTnI128–153 were undertaken to evaluate binding stoi-
chiometry and affinity. The intrinsic fluorescence emission
of actin tryptophan residues was not significantly altered by
the presence of the IP whereas titration of the dansylated
peptide with F-actin or F-actin–tropomyosin resulted in
enhancement of the emission intensity of the dansyl group
(Fig. 5). The titration data gave excellent fits to a 1 : 1
binding curve and provided direct evidence of a significant
increase in affinity in the presence of tropomyosin (Fig. 5,
Table 2). Dansyl emission was unaltered in the presence of
tropomyosin alone while competition with unlabelled IP

reversed the enhancement seen in the presence of F-actin or
F-actin–tropomyosin in a manner consistent with the
derived affinity of the IP (Table 2). These data confirmed
that the IP formed a 1 : 1 complex with F-actin the affinity
of which is enhanced by tropomyosin.
Interaction of the myosin light chain N-terminal peptide
with actin and reversal by the TnI inhibitory peptide
The effect of the IP on the interaction with F-actin of the
myosin light chain peptides, MLC1 1–37 and MLC1 1–13
was studied in view of the evidence that this region, localized
to the head of the myosin molecule, can bind to the
C-terminal of actin [15,37,38]. The MLC1 peptides were
found to bind to F-actin both in the absence and presence of
tropomyosin with the interaction resulting in the reduction
of the NMR resonance intensity for the majority of the
peptide sidechain signals (Fig. 6). Tropomyosin alone did
not affect the MLC1 peptide spectra nor did it result in the
dissociation of the MLC1 peptides from F-actin. On the
contrary it increased their affinity. The progressive changes
observed with increasing concentrations of F-actin reflected
complex formation in fast exchange and indicated the
Fig. 4. Interaction of the TnI inhibitory peptide with F-actin determined by surface plasmon resonance (25 mm Hepes, pH 7.4, 150 m
M
NaCl) and by
proton magnetic resonance spectroscopy (5 mm sodium phosphate buffer pH 7.2). (A) Sensorgrams showing the kinetics of binding of the human
cardiac TnI128–153 inhibitory peptide to immobilized F-actin at the peptide concentrations indicated. The fit of these data to 1 : 1 complex
formation yielded a dissociation constant of 3 ± 2 l
M
for the F-actin complex (Table 2). (B) The cardiac TnI inhibitory peptide forms a complex
with F-actin whose affinity is enhanced by tropomyosin as shown by the influence of tropomyosin on the change in resonance line width of the

composite signal of the dCH
2
groups of the arginine residue side chains of the inhibitory peptide as a function of the peptide : actin molar ratio. j,
F-actin-tropomyosin (molar ratio 7 : 1), m, F-actin. The concentration of actin was in each case was 40 l
M
with < 5% dilution during titration
with the inhibitory peptide up to a concentration of 160 l
M
, pH 7.2. Saturation of the linewidth change at lower molar ratios of actin–tropomyosin
compared to actin alone is indicative of the enhanced affinity of F-actin for the peptide in the presence of tropomyosin. The dotted lines show that in
the presence of tropomyosin the return to the linewidth of the free peptide occurred at approximately a 1 : 1 ratio of peptide : F-actin–tropomyosin
indicative of 1 : 1 complex formation.
Table 2. Dissociation constants for the different peptide complexes with F-actin determined from fluorescence and surface plasmon resonance meas-
urements. The K
d
values quoted were derived from the nonlinear regression fit (GraphPad Prism) of the fluorescence data to a 1 : 1 binding curve
for peptide–actin complex formation. The corresponding standard errors are quoted. The K
d
obtained for the myosin loop peptide, hcM398–414,
upon interaction with F-actin is consistent with the value previously reported using a peptide labelled at Cys400 [29]. The K
d
quoted for the
unlabelled TnI inhibitory peptide was obtained by curve fitting of the dissociation phase of the SPR data to derive the off-rate constant and an
on-rate of 5 · 10
8
M
)1
Æs with curve fitting of the association phase.
F-actin (
M

)6
) F-actin–tropomyosin (
M
)6
)
Dansylated inhibitory peptide, hcTnI128-153 28 ± 5 13 ± 3
Inhibitory peptide, hcTnI128–153 3 ± 2
Myosin loop peptide, hcM398–414 32 ± 5 18 ± 5
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5093
involvement of the N-terminal residues of MLC1 in actin
binding both in the absence and presence of tropomyosin.
(Fig. 6i).
Addition of hcTnI128–153 at a much lower relative con-
centration than either MLC1 peptide brought about disso-
ciation of the latter from F-actin and F-actin–tropomyosin.
Fig. 5. The TnI inhibitory peptide forms a 1 : 1 complex with F-actin whose affinity is enhanced by tropomyosin as indicated by fluorescence emission
spectra. The experimental conditions were 5 m
M
phosphate buffer, pH 7.2, T ¼ 293K. The relative fluorescence intensity is shown in arbitrary
units. Excitation was at 340 nm and the spectra were recorded from 420 to 600 nm. (A) Fluorescence emission spectra of dansylated TnI inhibitory
peptide complexed with F-actin-tropomyosin. Titration of the dansylated TnI inhibitory peptide with F-actin or F-actin–tropomyosin (molar ratio
of actin : tropomyosin of  7 : 1) resulted in enhancement of the fluorescence emission intensity of the dansyl group. Shown are fluorescence
emission spectra of 10 l
M
dansylated hcTnI128–153 in the presence of increasing concentrations of F-actin–tropomyosin (2, 5, 20 and 60 l
M
actin,
in traces 2–5, respectively). Competition by 2 l
M
unlabelled hcTnI128–153 in the presence of 60 l

M
actin led to a reduction in emission
enhancement, trace 6, consistent with the dissociation constant (Table 2) derived from curve fitting of the data to 1 : 1 complex formation with
F-actin–tropomyosin. (B) Fluorescence changes observed upon titration of 10 l
M
dansylated hcTnI128–153 with F-actin (j), or F-actin–tropo-
myosin (m) (molar ratio of actin : tropomyosin of  7 : 1). The binding curves shown are the nonlinear regression fits obtained (R
2
>0.97)for
1 : 1 complex formation using data obtained in three separate titrations in each case.
Fig. 6. The interaction of the N-terminal region of MLC1 with F-actin is weakened by the binding of the TnI inhibitory region. Spectra determined in
5m
M
sodium phosphate buffer, pH 7.2, T ¼ 293K. (i) Proton magnetic resonance spectra of the MLC1 1–13 peptide during titration with F-actin
and upon subsequent addition of hcTnI136–147. (A) MLC1 1–13 peptide, 200 l
M
, (B) MLC1 1–13 peptide, 200 l
M
, in the presence of F-actin,
60 l
M
. (C) As for (B) but in the presence of 55 l
M
hcTnI136–147. (D) MLC1 1–13 peptide as in (A), but spectrum acquired by the use of a two-
pulse spin-echo sequence. (E) MLC1 1–13 peptide in the presence of F-actin as in (B) but spectrum acquired by the use of a two-pulse spin-echo
sequence. Spectral accumulation in this way is sensitive to even small changes in signal linewidth resulting in readily detectable changes in intensity.
The linebroadening of the MLC1 peptide signals resulting from interaction with F-actin is markedly diminished by the presence of hcTnI136–147.
(ii) Spectra of MLC1 1–37 during titration with F-actin and upon subsequent addition of hcTnI128–153. (A) 200 l
M
peptide MLC1 1–37, in the

presence of 25 l
M
F-actin. (B) As for A and upon addition of 10 l
M
cardiac inhibitory peptide, hcTnI128–153
.
(C) Difference spectrum, B-A,
showing the sidechain groups of MLC1 1–37 whose resonances displayed actin-dependent broadening that is reversed by the presence of the
inhibitory peptide. The increase in signal intensity of the proton NMR spectra of the MLC1 peptide indicates that its interaction with F-actin is
abolished in the presence of the inhibitory peptide.
5094 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002
This was clearly indicated by the reversal of the actin-
associated spectral changes for resonances unique to the
MLC1 peptide, e.g. the trimethylalanine signal (Fig. 6ii).
Taken together these results suggested that, while tropo-
myosin on its own did not hinder the binding of MLC1 to
actin, the dissociation of the MLC1 1–37 by the IP binding
to F-actin or F-actin–tropomyosin may have resulted from
a conformational change in subdomain 1 of actin rather
than as a consequence of competition for binding at
identical or overlapping sites. The possibility that the IP
produced its effect by inducing a conformational change in
actin was explored further by studying its influence on the
binding of the loop peptide hcM398–414. This region of the
myosin molecule is believed to dock at the junction between
subdomain 1 and 3 of actin [16,39] whereas the LC1 peptide
binds close to the C terminus of actin.
Interaction of the myosin loop peptide, residues
398–414, with F-actin occurs at a region
that does not overlap with the binding site

for the TnI inhibitory peptide
Characterization of the interaction of cardiac b-myosin
residues 398–414 with F-actin was carried out so as to
explore any influence of tropomyosin on the inhibitory
region of TnI. The binding affinity of the myosin loop
peptide hcM398–414 to F-actin and F-actin–tropomyosin
was initially determined from the changes in intrinsic
tryptophan fluorescence of F-actin observed upon titration
with the peptide. The dissociation constant for the F-actin
complex formed was calculated using a nonlinear regression
procedure in each case to fit the data to a 1 : 1 binding curve
(Fig. 7A). The K
d
value obtained in the presence of
tropomyosin was 18 ± 4 l
M
. The affinity of the complex
with the peptide was higher than that found for F-actin
alone (Table 2) indicating that tropomyosin enhanced the
binding of hcM398-414 to F-actin.
Since the loop region of the myosin head, comprising
residues 398–414, is believed to interact near the C-terminus
of actin, F-actin labelled with 1,5 IAEDANS at Cys374 was
titrated with increasing concentrations of the hcM398-414
peptide. This was undertaken in order to determine an
alternative value for the binding affinity using as a readout
the spectral properties of a probe located on subdomain 1 of
actin in the vicinity of the presumed binding site. Addition
of hcM398–414 led to quenching of IAEDANS emission
with an overall intensity reduction of some 3% at saturation

(Fig. 7B). Comparable quenching effects were observed in
the presence of tropomyosin (1 : 7, tropomyosin : actin)
while the titration data were consistent with 1 : 1 complex
formation as judged by the goodness of fit of the data to a
1 : 1 binding curve. The derived K
d
values were similar to
those obtained by monitoring the actin–tryptophan fluor-
escence changes on the addition of the hcM398–414 peptide
(Table 2).
Titration of IAEDANS-labelled F-actin with the inhi-
bitory peptide, hcTnI128–153 was also carried out in the
absence and presence of tropomyosin. Under both condi-
tions the inhibitory peptide led to enhancement of the
IAEDANS emission ( 16% enhancement at saturation,
Fig. 7B) with a shift of the fluorescence emission maximum
from 475–470 nm. These titration data were consistent with
1 : 1 complex formation and yielded K
d
values similar to
those obtained using unlabelled F-actin (Table 2). The
observations that binding of the TnI inhibitory region led to
fluorescence enhancement and a blue-shift of the emission
maximum are consistent with the IAEDANS label on
Cys374 experiencing a less polar environment upon complex
formation. This contrasts with the change in environment of
the label upon interaction of actin with the hcM398–414
myosin loop peptide. The markedly different response of the
IAEDANS label to the binding of the two peptides provides
direct experimental evidence that the myosin loop and the

TnI inhibitory peptides bind on different sites on F-actin.
Resolution ofthe nature of the residuesof the hcM398–414
myosin loop peptide involved in interaction with F-actin was
achieved by monitoring the NMR spectral changes resulting
from complex formation in the presence and absence of
tropomyosin. As was the case with the other peptides used in
this study that bound to F-actin and F-actin–tropomyosin,
peptide hcM398–414 did not interact with tropomyosin
alone under the conditions described. Titration of peptide
hcM398–414 with F-actin resulted in marked spectral
broadening of the readily identifiable sidechain resonances
Fig. 7. The binding of hcM398–414 and hcTnI128–153 to F-actin as monitored by intrinsic (A) and extrinsic (B) fluorescence emission changes.
Experimental conditions were 5 m
M
sodium phosphate buffer, pH 7.4, T ¼ 293K. (A) Intrinsic tryptophan fluorescence emission spectra of the
F-actin–tropomyosin complex during titration with hcM398–414. The inset shows the decrease in fluorescence emission observed as a function of
increasing hcM398–414 concentration (0–50 · 10
)6
M
). The curve shown is the fit of the data to 1 : 1 complex formation at an F-actin concen-
tration of 5 · 10
)6
M
. (B) Variation of the IAEDANS emission upon titration of Cys374-labelled F-actin with hcM398–414 (m) or hcTnI128–153
(j), at a concentration of F-actin equal to 5 · 10
)6
M
. The curves shown are the nonlinear regression fits to 1 : 1 complex formation. The derived
dissociation constants are reported in Table 2.
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5095

of His401, Arg403, Asn408, Tyr410 and Thr412 that
indicated complex formation with F-actin (Fig. 8). Less
notably perturbed is the sidechain signal of Val411. The
nature of the residues affected was unchanged upon interac-
tion with F-actin–tropomyosin while the increased broaden-
ing effects observed at low peptide : actin in the presence of
tropomyosin are consistent with an enhanced affinity result-
ing from a decrease in peptide dissociation kinetics.
The binding of the TnI inhibitory region simultaneously
displaces peptides bound at nonoverlapping sites
on actin
Competition experiments were carried out to monitor the
ability of different peptides to simultaneously bind to
F-actin. The peptide derived from TnI, rcTnI161–181,
bound to F-actin in the presence or absence of tropomyosin
without displacing the myosin loop peptide (Fig. 9i). This
TnI peptide represents a region C-terminal to the IP of TnI
and has been proposed as an additional actin-binding site
[9,10]. Binding of the rcTnI161–181 peptide was judged
from the spectral broadening of its clearly distinguishable
sidechain signals (e.g. His170, c.f. Fig. 1) that occurred
without any concurrent changes in the resonances unique to
the hcM398–414 myosin loop peptide (His401 and Tyr410,
c.f. Fig. 8). Competition from the myosin loop peptide with
peptide rcTnI161–181 for interaction with actin would have
resulted in its displacement and the consequent appearance
of signals broadened as a consequence of interaction with
F-actin. These results indicated that the myosin loop peptide
and rcTnI161–181 are bound simultaneously at different
sites on actin as might be expected from the differences in

the composition of the two peptides.
Titration of the hcTnI128–153 inhibitory peptide into this
system up to a concentration equimolar to that of F-actin
resulted in the dissociation of both hcM398–414 and
rcTnI161–181 from actin whether in the absence or presence
of tropomyosin (Fig. 9i). This dissociation was also induced
by the shorter peptides encompassing the TnI inhibitory
region, hcTnI128–147 and hcTnI136–147 although
consistent with its lower actin affinity (Table 2), higher
hcTnI136–147 peptide : actin ratios were required to
achieve dissociation of hcM398–414 and rcTnI161–181.
Their simultaneous displacement was readily seen from the
reappearance of the signals unique to these peptides that
had been broadened by their interaction with F-actin in the
absence of the inhibitory peptide (Fig. 9i). At the same time
signals unique to the IP broadened in the manner described
above (Figs 3 and 4). Since the TnI inhibitory peptide forms
a 1 : 1 complex with F-actin these observations indicate that
the association of the TnI inhibitory region with F-actin
antagonized the binding of the myosin loop peptide and
rcTnI161–181 to their individual binding sites on actin.
These effects cannot be ascribed to site competition and
simple steric displacement.
Since the binding of the TnI inhibitory peptide appeared
to induce a conformational change in the actin molecule
that altered the ability of F-actin to interact with different
segments of myosin we went on to monitor the binding of
hcM398–414 at an actin : Tm : TnI peptide ratio of
7 : 1 : 1 over a range of myosin peptide concentrations.
Figure 9ii presents data obtained using a myosin peptide

concentration of 50 l
M
. The presence of the TnI inhibitory
peptide (0.55 l
M
) led to a decrease in the amount of myosin
peptide bound to F-actin–tropomyosin (with hcM398–414
at  100-fold excess over TnI peptide). This is seen from the
Fig. 8. Proton MR spectral changes upon titration of hcM398–414 with F-actin identifying the residues involved in complex formation. Spectra
determined in 5 m
M
sodium phosphate buffer, pH 7.2, T ¼ 293K. (i) (A) Peptide hcM398–414 (200 l
M
). (B) In the presence F-actin, 28 l
M
.(C)
Difference spectrum, A-B, highlighting the residues whose sidechain signals are perturbed by binding to F-actin. (ii) as in (i) but spectra acquired by
the use of a two-pulse spin-echo sequence. Spectral accumulation in this way distinguishes signals on the basis of their J-coupling patterns and
highlights even small changes in signal linewidth resulting in readily detectable changes in intensity. Signals of hcM398–414 are labelled. Complex
formation characterized by relatively fast exchange between the free and actin-bound states of the peptide population is indicated by the resonance
broadening that occurs during titration with increasing concentrations of F-actin. The unique sidechain resonances of His401, Arg403, Tyr410,
Val411 and Thr412 display marked perturbation upon complex formation.
5096 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002
change in the myosin peptide signals, for example, Arg403,
Val411 and Thr412 that, as highlighted by difference
spectroscopy (Fig. 9ii), revert towards those of the free
peptide in the presence of hcTnI128–147 at an
actin : Tm : TnI peptide ratio of 7 : 1 : 1. These observa-
tions reinforce the suggestion that conformational changes
which occur when one molecule of troponin I interacts with

the actin monomer are transmitted to other actin monomers
in the filament not associated with TnI.
DISCUSSION
The TnI inhibitory region is an early example of a growing
family of short peptide sequences capable of emulating the
ability of the parent proteins to interact with their physio-
logical targets. The biological activity characteristic of the
whole molecule is held to derive from the retention of
specific protein–protein recognition by such isolated pep-
tides and their resulting ability to inhibit receptor/effector
interactions. Examples of such intervention range from the
inhibition of the replication of simian virus 40 DNA by the
Proliferating Cell Nuclear Antigen-binding peptide of
p21
WAF1
[40] to the myosin loop peptide, hcM398–414,
used in this study. In keeping with its apparent contribution
to the actomyosin interface [16,17,20] the latter peptide
inhibited actin-activated MgATPase activity [29] while the
short TnI inhibitory peptide, some 6% of the parent
molecule, preserves both the inhibitory and the tropomyosin
accentuation effects characteristic of troponin-I.
The NMR data clearly indicate that peptides corres-
ponding to the N-terminus of myosin LC1 interact speci-
fically with F-actin in the absence of inhibitory peptide
derived from TnI. This conclusion is consistent with the
results of earlier NMR investigations [38,39] and cross
linking studies [37,41] that the N-terminal region of skeletal
LC1 is one of the sites involved in the interaction of myosin
with actin. From these studies and electron microscopy of

C-terminally labelled actin [42] it can be concluded that the
N-terminus of MLC1 binds close to the C-terminus of actin.
The N-terminal region APKK (residues 1–4) of MLC1
appears to be particularly important since modification of
these residues by recombinant DNA technology results in
changes in the kinetics of the actomyosin MgATPase [41].
Other residues at the N-terminus of MLC1 are involved in
binding and have indeed been shown to be important for the
activity of cardiac myosin. A peptide corresponding to
residues 5–14 of human ventricular MLC1 increased the
contractility of intact and skinned human heart fibres [43]
and a similar peptide added to rat cardiac myofibrils
induced a supramaximal increase in the MgATPase activity
at submaximal calcium levels [44].
The NMR and fluorescence studies both indicate inter-
action of F-actin with another region of myosin, the loop
peptide, hcM398–414. The interaction appears to occur at a
region on F-actin that is different from that involved in
binding the TnI inhibitory peptide as shown by the
distinctive response of the IAEDANS probe to each
peptide. This is consistent with the earlier observations that
Fig. 9. (i) The TnI inhibitory region displaces both peptide hcM398–414 and peptide rcTnI161–181 that interact concurrently with F-actin at distinct
binding locations. The aromatic sidechain NMR resonances are shown since these provide unique reporter signals for each of the peptides. Spectra
determined in 5 m
M
phosphate buffer, pH 7.2, T ¼ 293K. (A) Myosin loop peptide (hcM398–414), 108 l
M
. (B) hcM398–414, 108 l
M
in the

presence of 74 l
M
F-actin. The signals of His401 and Tyr410 of the myosin loop peptide are markedly broadened by complex formation. (C) As for
(B), but upon the addition of 186 l
M
rcTnI161–181. Binding of this TnI peptide is indicated by broadening of its His171 resonances. No
displacement of the myosin loop peptide has occurred since its signals remain broad. The two peptides are therefore concurrently bound to F-actin.
(D) As for (C) but upon titration with inhibitory peptide hcTnI128–153 (here 102 l
M
). The reappearance of the myosin loop peptide signals and
those of rcTnI161–181 indicates their simultaneous displacement by the inhibitory peptide. The signals unique to the inhibitory peptide, Phe132 and
138, are broad indicating that the peptide is bound to F-actin. (ii) The binding of hcM398–414 to F-actin–tropomyosin is altered by the presence of
hcTnI128–153 at an actin : Tm : nI inhibitory peptide ratio of 7 : 1 : 1. This is detected by the increased spectral contribution of free hcM398–414
signals clearly identified by difference spectroscopy. The two-pulse spin-echo spectra shown (c.f. Fig. 8ii) were obtained in 5 m
M
phosphate buffer,
pH 7.2, T ¼ 293K. (A) Myosin loop peptide (hcM398–414), 50 l
M
, (B) as for (A) but in the presence of F-actin–tropomyosin (3.7 l
M
F-actin). (C)
As for (A) but in the presence of an actin : Tm : TnI inhibitory peptide ratio of 7 : 1 : 1 (0.55 l
M
hcTnI128–153). (D) Difference spectrum (C-B)
showing the reappearance of the myosin loop peptide signals.
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5097
the inhibitory peptide interacts at the actin N-terminus and
with modelling studies that have placed the myosin loop
residues hcM398–415 close to actin residues 332–334
[16,39].

Under the conditions of low ionic strength at which these
studies were carried out there was no evidence that
tropomyosin inhibited the binding of either myosin peptide,
indeed the evidence was that their affinity for actin was
increased. This implies that the binding site(s) occupied by
these peptides are different from those involved in binding
tropomyosin.
The results of the competition experiments and the
observations that the affinities of the TnI inhibitory peptide
and the myosin peptides for actin are all very similar suggest
that displacement does not explain our results. A more
likely explanation is that all the peptides have specific
binding sites and that the binding of the IP in a 1 : 1
complex induces a conformational change that is wide-
spread in the actin molecule leading to the dissociation of
the MLC1–37, hcM398–414 and rcTnI161–181 peptides
(Fig. 10). On the other hand any conformational change
induced in actin by binding of the myosin loop peptide or
rcTnI161–181 is more restricted since any allosteric effect
does not extend from the regions of actin where these
peptides are bound.
Both NMR and fluorescence studies indicate that tropo-
myosin enhances the binding of the inhibitory peptide
region to actin presumably by enabling slower dissociation
of the peptide. From the results of early ultracentrifugation
studies [3,45] in which tropomyosin–actin binding was
assayed by co-centrifugation it has been assumed that these
two proteins did not interact at low Mg concentration and
low ionic strength. The fact that the K
d

sfortheactin–
peptide complexes reported in this communication had all
decreased in the presence of tropomyosin implies that
interaction had taken place between actin and tropomyosin
at low ionic strength and in the absence of added
magnesium. Earlier reports of the stimulatory effect of
tropomyosin on the inhibitory action of TnI on the
actomyosin MgATPase were also carried out at low ionic
strength [2]. The results obtained with IPs in this study
likewise indicate that interaction occurs under these condi-
tions. It would appear that the interaction of tropomyosin
with actin is much more subtle than has been supposed and
that the standard procedure for evaluating actin–myosin
interaction by co-centrifugation has its limitations. Further,
under the conditions in which our results have shown that
tropomyosin interacts with F-actin, the peptides represent-
ing presumptive binding regions on myosin and TnI can still
bind to actin. This suggests that in the presence of
tropomyosin regions of the actin monomer are available
for interaction with intact TnI and myosin.
It would appear that the ligand responsible for modula-
ting the interaction with myosin is the inhibitory region of
TnI which on binding to actin renders the molecule unable
to bind to the N-terminus of MLC1 and the myosin loop
peptide and possibly other sites of interaction with myosin
essential for activation of the MgATPase. The results
reported here strongly suggest that the binding of IP or TnI
to an actin monomer produces conformational changes at
least two other sites on the actin molecule. Although other
regions were not probed in this study it is possible that

widespread conformational changes in the actin monomer
occur on interaction with TnI. Inspection of the actin
structure indeed reveals intramonomer contacts within actin
subdomain 1 that may underlie the ability of the inhibitory
region of TnI to influence the surface activity of actin
towards other actin binding proteins. The site of binding of
the inhibitory region is the N-terminal of actin that is
structurally linked to the C-terminal and residues 99–101.
Displacement of myosin contacts would therefore be
facilitated by small conformational changes distributed
through the residue network making intramolecular con-
tacts between the N- and C-terminal regions on subdomain
1ofactin.
To maintain the integrity of the F-actin filament
structure after interaction with TnI with a monomer it
would be expected that the conformational changes would
be transmitted to neighbouring actin monomers. These
could take place through the contact points between actin
monomers in the F-actin filament, of which it has been
postulated that there are at least four per actin monomer
[46]. Biochemical and physiological data suggest that the
response of the actin protein assembly involves longitu-
dinal cooperativity along the thin filament. The regulatory
interactions would be expected to be the same as the
nearest neighbour interactions that govern the actin
monomer contacts upon which thin filament assembly is
based. Several studies have suggested that structural
changes in the actin monomer result from polymerization
to form F-actin [47,48]. These changes may be fundamen-
tal for the longitudinal cooperativity observed in func-

tioning F-actin filaments.
The results obtained in this study suggest that the role of
tropomyosin in the myofibrillar system may not be to block
sites on actin that interact with myosin to activate the
MgATPase but rather to stabilize the F-actin filament
and facilitate the transmission of conformational changes
Fig. 10. Schematic representation of the interaction sites on rabbit fast
skeletal actin. Outline of actin monomer taken from Kabsch et al.1990
[52]. Domains are labelled and the C and N termini of actin are
identified by bold letters. Full arrows indicate regions of interaction
whereas dotted arrows represent suggested allosteric effects on the
actin molecule. Although the position of the second TnI binding site on
actin is placed in domain 1 in the scheme the actual location of this site
is not known.
5098 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002
between the actin monomers that are induced by interaction
with TnI and myosin. While care should be exercised in
extrapolating the results obtained with peptides to those
associated with the intact proteins in the functioning
myofibril, nevertheless there is now widespread evidence
that certain properties of intact proteins are preserved in
peptide fragments derived from them [49]. Further, a
number of observations have been reported that are
explained by conformational changes occurring in actin
[2,21,22,50–52] while it has been reported [28] that the
affinity of the myosin loop peptide, hcM398–414, for
F-actin was enhanced by the presence of myosin subfrag-
ment-1 at a concentration substoichiometric with respect to
F-actin. These data also suggest that a conformational
change is propagated along the actin filament. The studies

described here provide direct of evidence of sites of
interaction and of conformational changes occurring in
actin that are an important aspect of the regulatory process
(Fig. 10). They also imply that the role of tropomyosin in
filament function may be to stabilize the actin filament and
facilitate its cooperative function rather than directly
blocking the interaction of actin with myosin as postulated
by the steric hypothesis.
ACKNOWLEDGEMENTS
The work described has been supported by grants from the British
Heart Foundation and the Wellcome Trust. The work is part of the
Bioinformatics Initiative at the University of Birmingham supported by
the Medical Research Council.
REFERENCES
1. Schaub, M.C. & Perry, S.V. (1971) The regulatory proteins of the
myofibril. Characterisation and properties of the inhibitory factor
(Troponin B). Biochem. J. 123, 367–377.
2. Perry, S.V., Cole, H.A., Head, J.F. & Wilson, F.J. (1972) Loca-
lisation and mode of action of the inhibitory component of the
troponin complex. Cold Spring Harbor Symp Quant Biol. 37, 251–
262.
3. Eaton, B.L., Kominz, D.R. & Eisenberg, E. (1975) Correlation
between the inhibition of the acto-heavy meromyosin ATPase and
the binding of tropomyosin to F-actin: effects of Mg
2+
,KCl,
troponin I and troponin C. Biochem. 14, 2718–2725.
4. Lehrer, S.S., Chai, M. & Geeves, M.A. (1997) Effect of troponin I
(TnI) on actin S1 ATPase and S1 binding in the absence and
presence of rabbit skeletal tropomyosin. Biophys. J. 72,MP198.

5. Geeves, M.A., Chai, M. & Lehrer, S.S. (2000) Inhibition of actin-
myosin ATPase activity by troponin I and IC: relationship to the
thin filament states of muscle. Biochemistry 39, 9345–9350.
6. Hartshorne, D.J., Schaub, M.C. & Perry, S.V. (1967) A protein
factor inhibiting the magnesium activated adenosine tripho-
sphatase activity of desensitised actomyosin. Biochem. J. 104, 907–
913.
7. Syska, H., Wilkinson, J.M. & Perry, S.V. (1976) The relationship
between biological activity and primary structure of troponin I of
white skeletal muscle of the rabbit. Biochem. J. 153, 375–387.
8. Talbot, J.A. & Hodges, R.S. (1981) Synthetic studies on the
inhibitory region of rabbit skeletal troponin I. J. Biol. Chem. 256,
2798–2802.
9. Tripet, B., Van Eyk, J.E. & Hodges, R.S. (1997) Mapping of a
second actin tropomyosin and a second troponin C binding site
within the C terminus of troponin I, and their importance in the
Ca
2+
-dependent regulation of muscle contraction. J. Mol. Biol.
271, 728–750.
10. Rarick, H.M., Tu, X H., Solaro, R.J. & Martin, A. (1997) The C
terminus of cardiac troponin I is essential for full inhibitory
activity and Ca
2+
sensitivity of rat myofibrils. J. Biol. Chem. 272,
26887–26892.
11. Perry, S.V. (1999) Troponin I: Inhibitor or facilitator. Mol. Cell
Biochem. 190, 9–32.
12. Haselgrove, J.C. (1972) X-ray evidence for a conformational
change in the actin-containing filaments of vertebrate striated

muscle. Cold Spring Harbor Symp Quant Biol. 37, 341–352.
13. Huxley, H.E. (1972) Structural changes in the actin- and myosin-
containing filaments during contraction. Cold Spring Harbor
Symp Quant Biol. 37, 361–378.
14. Parry, D.A.D. & Squire, J.M. (1973) Structural role of tropo-
myosin in muscle regulation: analysis of the x-ray diffraction
patterns from relaxed and contracting muscles. J. Mol. Biol. 75,
35–55.
15. Milligan, R.A. (1996) Protein–protein interactions in the rigor
actomyosin complex. Proc.NatlAcad.Sci.USA93, 21–26.
16. Rayment,I.,Holden,H.M.,Whittaker,M.,Yohn,C.B.,Lorenz,
M., Holmes, K.C. & Milligan, R.A. (1993) Structure of the actin–
myosin complex and its implications for muscle contraction.
Science 261, 58–65.
17. Sasaki, N., Asukagawa, H., Yasuda, R., Hiratsuka, T. & Sutoh,
K. (1999) Deletion of the myopathy loop of Dictyostelium myosin
II and its impact on motor functions. J. Biol. Chem. 274, 37840–
37844.
18. Cuda, G., Fananapazir, L., Zhu, W.S., Sellers, J.R. & Epstein,
N.D. (1993) Skeletal-muscle expression and abnormal function of
beta-myosin in hypertrophic cardiomyopathy. J. Clin. Invest. 91,
2861–2865.
19.Geisterfer-Lowrance,A.A.T.,Kass,S.,Tanigawa,G.,Vosberg,
H.P.,McKenna,W.,Seidman,C.E.&Seidman,J.G.(1990)A
molecular basis for familial hypertrophic cardiomyopathy – a
beta-cardiac myosin heavy-chain gene missense mutation. Cell 62,
999–1006.
20. Cuda, G., Fananapazir, L., Epstein, N.D. & Sellers, J.R. (1997)
The in vitro motility activity of beta-cardiac myosin depends on
the nature of the beta-myosin heavy chain gene mutation in

hypertrophic cardiomyopathy. J. Muscle Res. Cell Motil. 18,
275–283.
21. Squire, J.M. & Morris, E.P. (1998) A new look at thin filament
regulation in vertebrate striated muscle. FASEB J. 12, 761–771.
22. Rosol, M., Lehman, W., Craig, R., Landis, C., Butters, C. &
Tobacman, L.S. (2000) Three-dimensional reconstruction of thin
filaments containing mutant tropomyosin. Biophys. J. 78, 908–
917.
23. Lehman, W., Hatch, V., Korman, V., Rosol, M., Thomas, L.,
Maytum, R., Geeves, M.A., Van Eyk, J.E., Tobacman, L.S. &
Craig, R. (2000) Tropomyosin and actin isoforms modulate the
localisation of tropomyosin strands on actin filaments. J. Mol.
Biol. 302, 593–606.
24. Grabarek, Z. & Gergely, J. (1987) Troponin-I binds to the
N-terminal 12-residue segment of actin. Biophys. J. 51, A331.
25. Levine, B.A., Moir, A.J.G. & Perry, S.V. (1988) The interaction of
troponin-I with the N-terminal region of actin. Eur. J. Biochem.
172, 389–397.
26. Patchell, V.B., Gallon, C., Hodgkin, M.A., Fattoum, A., Perry,
S.V. & Levine, B.A. (2001) The inhibitory region of troponin I is
able to block binding at different locations on F-actin. J. Muscle
Res. Cell Motil. 22, 594–595.
27. Henry, G., Trayer, I.P., Brewer, S. & Levine, B.A. (1985) The
widespread distribution of alpha-N-trimethylalanine as the
N-terminal amino-acid of light chains from vertebrate striated
myosins. Eur. J. Biochem. 148, 75–82.
28. Levine, B.A., Moir, A.J.G., Patchell, V.B. & Perry, S.V. (1990)
The interaction of actin with the N-terminal region of dystrophin.
FEBS Lett. 263, 159–162.
Ó FEBS 2002 F-actin interactions inhibited by Troponin-I (Eur. J. Biochem. 269) 5099

29. Bartegi, A., Roustan, C., Chavanieu, A., Kassab, R. & Fattoum,
A. (1997) Interaction of F-actin with synthetic peptides spanning
the loop region of human cardiac beta-myosin heavy chain con-
taining Arg403. Eur. J. Biochem. 250, 484–491.
30. Spudich, J.A. & Watt, S. (1971) The regulation of rabbit skeletal
muscle contraction. Biochemical studies of the interaction of the
tropomyosin-troponin complex with actin and the proteolytic
fragments of myosin. J. Biol. Chem. 246, 4866–4487.
31. Miki, M., Dos Remedios, C.G. & Barden, J.A. (1987) Spatial
relationship between the nucleotide-binding site, Lys-61 and Cys-
374 in actin and a conformational change induced by myosin
subfragment-1 binding. Eur. J. Biochem. 168, 339–345.
32. Jardetzky, O. & Roberts, G.C.K. (1981) NMR in Molecular
Biology p.59. Academic Press, New York/London.
33. Huber, P.A.J., Gao, Y., Fraser, I.D.C., Copeland, O., El-Mez-
gueldi,M.,Slatter,D.A.,Keane.N.E.,Marston,S.B.&Levine,
B.A. (1998) Structure-activity studies of the regulatory interaction
of the 10 kilodalton C-terminal fragment of caldesmon with
actin and the effect of mutation of caldesmon residues 691–696.
Biochemistry 37, 2314–2326.
34. Lian, L Y. & Roberts, G.C.K. (1993) Effects of chemical
exchange on NMR spectra. In NMR of Macromolecules (Roberts,
G.C.K., ed.), pp 151–182. IRL Press, Oxford.
35. Jardetzky, O. & Jardetzky, C.D. (1962) Introduction to magnetic
resonance spectroscopy methods and biological applications.
Methods Biochem. Anal. 9, 225.
36. McConnell, H.M. (1958) Reaction rates by nuclear magnetic
resonance. J. Chem. Phys. 28, 430–431.
37. Sutoh, K. (1982) Identification of myosin-binding sites on the
actin sequence. Biochemistry 21, 3654–3661.

38. Trayer, I.P., Trayer, H.R. & Levine, B.A. (1987) Evidence that the
N-terminal region of A1-light chain of myosin interacts directly
with the C-terminal region of actin – a proton magnetic resonance
study. Eur. J. Biochem. 64, 259–266.
39. Timson, D.J., Trayer, H.R. & Trayer, I.P. (1998) The N-terminus
of A1-type myosin essential light chains binds actin and modulates
myosin motor function. Eur. J. Biochem. 255, 654–662.
40. Warbrick, E., Lane, D.P., Glover, D.M. & Cox, L.S. (1995) A small
peptide inhibitor of DNA replication defines the site of interaction
between the cyclin-dependent kinase inhibitor p21
WAF1
and
proliferating cell nuclear antigen. Current Biol. 5, 275–282.
41. Timson,D.J.,Trayer,H.R.,Smith,J.S.&Trayer,I.P.(1999)Size
and charge requirements for kinetic modulation and actin binding
by alkali 1-type myosin essential light chains. J. Biol. Chem. 274,
18271–18277.
42. Milligan, R.A., Whittaker, M. & Safer, D. (1990) Molecular
structure of F-actin and location of surface binding sites. Nature
348, 217–221.
43. Morano,I.,Ritter,O.,Bonz,A.,Timek,T.,Vahl,C.F.&Michel,
G. (1995) Myosin light–chain actin interaction regulates cardiac
contractility. Circ. Res. 76, 720–725.
44. Rarick, H.M., Opgenorth, T.J., von Geldern, T.W., Wu-Wong,
J.S.R. & Solaro, R.J. (1996) An essential myosin light chain
peptide induces supramaximal stimulation of cardiac myofibrillar
ATPase activity. J. Biol. Chem. 271, 27039–27043.
45. Martonosi, A. (1962) Studies on actin VII. Ultracentrifugal ana-
lysis of partially polymerised actin solutions. J. Biol. Chem. 237,
2795–2803.

46. Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990)
Atomic model of the actin filament. Nature 347, 44–49.
47. Steinmetz, M.O., Stoffler, D. & Hoenger, A. (1997) Actin: From
cell biology to atomic detail. J. Struct. Biol. 119, 295–320.
48. Moraczewska, J., Wawro, B., Seguro, K. & Strzelecka-
Golaszewska, H. (1999) Divalent cation, nucleotide and poly-
merization-dependent changes in the conformation of subdomain
2ofactin.Biophys. J. 77, 373–385.
49. Peptides as probes in Muscle Research. (1991) (J.C. Ruegg, ed.),
pp 1–174. Springer-Verlag, Berlin.
50. Bremel, R.D. & Murray, J.M. (1972) and. Weber. Manifestations
of cooperative behaviour in the regulated actin filament during
actin-activated ATP hydrolysis in the presence of calcium. Cold
Spring Harbor Symp Quant. Biol. 37, 267–275.
51. Butters, C.A., Willasden, K.A. & Tobacman, L.S. (1993)
Cooperative interactions between adjacent troponin-tropomyosin
complexes may be transmitted through the actin filament. J. Biol.
Chem. 268, 15565–15570.
52. Levine, B.A., Patchell, V.B. & Perry, S.V. (1999) Troponin I and
conformational changes in actin. J. Muscle Res. Cell Motil. 20,
828–829.
53.Kabsch,W.,Mannherz,H.G.,Suck,D.,Pai,E.F.&Holmes,
K.C. (1990) Atomic structure of the actin – DNase-I complex.
Nature 347, 37–44.
5100 V. B. Patchell et al.(Eur. J. Biochem. 269) Ó FEBS 2002