(Received for publication, March 15, 1995; and in revised form, June 22, 1995)
From the
To investigate how Ca binding to troponin C
regulates muscle contraction, the Ca
-sensitive
properties of thin filament assembly were studied as the tropomyosin
binding, NH
-terminal region of troponin T was progressively
shortened. Troponin complexes were prepared that contained skeletal
muscle troponin C, troponin I, and either intact troponin T (TnT)
(residues 1-259) or fragment TnT-(70-259),
TnT-(151-259), or TnT-(159-259). In the absence of
Ca
their respective affinities for pyrene-labeled
tropomyosin were 2.3
10
M
, 1.2
10
M
, 1.9
10
M
, and 1.9
10
M
. Ca
had only a small
effect on these affinities: 1.1
10
M
for whole troponin, 2
10
M
for
troponin-(151-259), and 2.8
10
M
for troponin-(159-259). Forms of
troponin that bound weakly to tropomyosin in the absence of actin
increased the actin affinity of tropomyosin only 2-3-fold, even
in the absence of Ca
; weak binding of troponin to
tropomyosin correlated with weak effects on tropomyosin-actin binding.
In contrast, whole troponin had an approximately 500-fold effect on
tropomyosin binding to actin, regardless of whether Ca
was present. The small effect of Ca
on the
energetics of thin filament assembly is not attributable to the
amino-terminal region of troponin T. The results suggest that
Ca
causes the interaction between actin and the
globular region of troponin to switch between two energetically similar
states.
The regulation of muscle contraction is accomplished by the
reversible binding of Ca to the thin filament protein
troponin. Regulation is proposed to involve a
Ca
-induced dissociation of one region of troponin
from actin and from tropomyosin (Hitchcock, et al., 1973;
Margossian and Cohen, 1973; Potter and Gergely, 1974; Pearlstone and
Smillie, 1983; Tanokura and Ohtsuki, 1984; Ishii and Lehrer, 1991),
thereby facilitating repositioning of tropomyosin on the actin filament
(Lehman, et al., 1994). In particular, Ca
weakens the actin binding of the troponin subunit, TnI, (
)and the tropomyosin binding of the carboxyl-terminal
region of TnT. In this widely held scheme of thin filament function,
troponin remains anchored to the thin filament by relatively
Ca
-insensitive binding of the amino-terminal region
of TnT to the carboxyl-terminal region of tropomyosin (Pato, et
al., 1981; Mak and Smillie, 1981; Tanokura, et al., 1983;
Cho and Hitchcock-DeGregori, 1990; Ishii and Lehrer, 1991). Indeed,
rotary-shadowed electron micrographs of troponin and of TnT (Flicker, et al., 1982) show a highly extended molecule with a narrow
tail mostly attributable to the amino-terminal portion of TnT (White et al., 1987) and a distinctly more globular head region that
is likely to include portions of all three subunits.
Consistent with
the existence of a Ca-insensitive anchor, we recently
reported that the affinity of troponin for actin-tropomyosin remains
tight (>10
M
) regardless of
whether Ca
is present (Dahiya, et al. 1994).
Perhaps more surprisingly, the troponin-tropomyosin complex bound much
more tightly to actin than did tropomyosin alone, even in the presence
of Ca
or in the absence of TnI. Parallel results were
found for the binding of troponin, troponin-Ca
, or
TnT to actin-tropomyosin. These data would appear to complicate models
of regulation in which troponin has two sites of interaction with the
thin filament: a Ca
-insensitive site primarily
between tropomyosin and the elongated subunit TnT and a
Ca
-reversible site that primarily involves actin-TnI
binding and tropomyosin-TnT binding. Rather, although Ca
may cause conformational changes in troponin that result in
substantially changed binding to the thin filament, the net energetics
of this binding are not significantly altered by Ca
.
This recent report was not the first to examine the effect of
Ca on troponin binding to the thin filament. The new
aspects included an attempt to exclude the contributions of cooperative
effects in thin filament assembly by the application of a linear
lattice model. Also, equilibrium linkage relationships were used to
calculate the affinity of troponin (±Ca
) for
actin-tropomyosin. In general this affinity has been too tight to
directly measure, with or without a linear lattice approach. Thus, the
conclusions of this report are dependent upon two caveats, that the
assumptions of the linear lattice model are valid for calculating and
excluding cooperative aspects of assembly and that the described
equilibrium linkage relationships are correct.
An alternative
approach is to examine the properties of the
Ca-sensitive region of troponin (TnI, TnC, and the
carboxyl terminus of TnT), in the absence of the apparently more
Ca
-insensitive tail region (i.e. the
elongated amino-terminal portion of TnT). Because it lacks the
Ca
-insensitive, anchoring portion of TnT, the effect
of this truncated troponin complex on thin filament assembly may be
very Ca
-sensitive. This can be tested by measuring
the Ca
dependence of the affinity constants involved
in assembling thin filaments containing either troponin or truncated
troponin. To accomplish this, troponin molecules were prepared
containing intact TnT or one of several COOH-terminal fragments of TnT.
Troponin binding to pyrene-modified tropomyosin was weaker when TnT was
truncated but was no more sensitive to Ca
. Whereas
whole troponin promoted tropomyosin binding to actin about 500-fold,
regardless of whether Ca
was present, truncated
troponin had a minimal effect on tropomyosin-actin binding, again
regardless of whether Ca
was present. These results
have significance for how Ca
regulates muscle
contraction and for the participation of different regions of troponin
in thin filament assembly and regulation.
The effects of troponin or truncated troponin on the
binding of tropomyosin to actin were measured by cosedimentation of
radiolabeled tropomyosin with actin (Hill et al., 1992).
Conditions were as follows: 25 °C, 10 mM Tris-HCl (pH
7.5), 3 mM MgCl, 0.1 mM dithiothreitol,
3-5 µM F-actin, 60 or 300 mM KCl, and
either 0.5 mM EGTA or 0.1 mM CaCl
. The
concentrations of troponin and tropomyosin varied for each experiment
as indicated below. The actin-bound tropomyosin was calculated from the
difference between the total and the supernatant
H-tropomyosin after sedimentation for 30 min at 35,000 rpm
using a TLA100 rotor in the TL100 ultracentrifuge. (In the absence of
actin, there was no sedimentation.) Troponin was added in a constant
molar excess compared with the tropomyosin concentration, and the
concentration of the troponin-tropomyosin complex was calculated as
described (Dahiya et al., 1994), using the affinity constants
determined under the same conditions in the fluorescence experiments
below. The binding of troponin-tropomyosin to actin, as a function of
the troponin-tropomyosin concentration, was analyzed as a linear
lattice problem in which the ligand spans seven equivalent sites on the
lattice (McGhee and Von Hippel, 1974; Tsuchiya and Szabo, 1982;
Willadsen et al., 1992). Binding data are fit to obtain three
parameters: the affinity of the ligand for an isolated site for the
lattice, the -fold increase in affinity when binding involves
cooperative interactions with one adjacent bound ligand, and the
concentration of bound ligand at saturation.
Figure 1:
Affinity of skeletal muscle troponin
for tropomyosin in the presence and in the absence of
Ca. Tropomyosin was labeled on Cys-190 with N-(1-pyrene)iodoacetamide, and the effect of troponin on the
pyrene monomer fluorescence is shown in this representative experiment.
= 340 nm and
= 405
nm. Conditions were as follows: 25 °C, 3 mM MgCl
, 60 mM KCl, 5 mM Tris HCl (pH
7.5), 0.1 mM dithiothreitol, 0.1 mM pyrene-tropomyosin, and either 0.5 mM EGTA (
) or
0.1 mM CaCl
(
). The solid lines are
best fit theoretical curves corresponding to affinities of 2.3
10
M
(EGTA) or 6.9
10
M
(CaCl
). The abscissa shows the total added troponin concentration based
upon the initial volume, but the theoretical curves are calculated with
dilution (<6%) of both troponin and tropomyosin taken into
consideration. The units of fluorescence intensity are
arbitrary.
On the other hand, the present data demonstrate much weaker tropomyosin binding by a further truncation of troponin, troponin-(151-259), which includes rabbit TnC, rabbit TnI, and the carboxyl-terminal portion of (recombinant rat) TnT. The affinity of troponin-(151-259) for tropomyosin was 2 orders of magnitude less than the affinity of whole troponin for tropomyosin (Table 1, Fig. 2). Because the binding was so weak, it could not be determined whether the fluorescence transition was attributable to a 1:1 association between tropomyosin and troponin-(151-259). This weak binding (in comparison to whole, nontruncated troponin) was not due to the few amino acid differences between rat and rabbit TnT, nor to the presence versus the absence of reconstitution from subunits: indistinguishable results (Table 1) were obtained using troponin-(159-259), produced by controlled chymotryptic digestion of rabbit whole troponin, which selectively removes TnT residues 1-158 from the ternary troponin complex.
Figure 2:
Tropomyosin binds weakly to troponin
lacking the amino-terminal region of TnT. Troponin was reconstituted
from skeletal muscle TnC, TnI, and a recombinant, carboxyl-terminal
fragment of skeletal muscle TnT lacking 150 NH-terminal
residues. Binding to pyrene-tropomyosin was studied under the same
conditions as in Fig. 1. Two representative curves are shown,
either in the presence of EGTA (
) or in the presence of
CaCl
(
). The fluorescence intensity increased as
increasing concentrations of troponin-(151-259) were added. The solid lines are best fit theoretical curves, corresponding to
affinities of 2.2
10
M
(EGTA) and 1.8
10
M
(CaCl
). CaCl
had
little effect on the affinity constant. Binding of
troponin-(151-259) to tropomyosin was much weaker than binding of
whole troponin to tropomyosin, but in neither case did CaCl
significantly weaken the affinity (see also Table 1). The units
of fluorescence intensity are arbitrary.
One possibility from these data is that the interaction
between troponin and the region of tropomyosin near Cys-190 (Ohtsuki,
1979; Pearlstone and Smillie, 1982; Chong and Hodges, 1982; Morris and
Lehrer, 1984) is quite weak when whole troponin is present. Several
pertinent observations support this interpretation. Electron
microscopic images of troponin-tropomyosin polymers show that the
globular head region of troponin is often distinctly separated from the
tropomyosin filament (Flicker et al., 1982). Co-crystals of
tropomyosin and troponin show that troponin is most ordered near the
carboxyl terminus of tropomyosin and less ordered near tropomyosin
residues 190-235 (White et al., 1987; White, 1988).
Also, troponin has little effect on the local thermal unfolding of
tropomyosin near Cys-190 (Ishii and Lehrer, 1991). Furthermore,
although troponin causes prolongation of the fluorescence decay of
IAEDANS attached to tropomyosin Cys-190, this prolonged component is
only 20% of the total amplitude (Lamkin et al., 1983).
Finally, the binding of the TnITnT-(159-259) complex to
immobilized tropomyosin is weakened substantially by the addition of
TnC, even in the absence of Ca
(Pearlstone and
Smillie, 1983).
Comparisons among the progressively truncated forms of troponin (Table 1) show that removal of TnT residues 70-150 had a 60-fold effect on the binding constant, whereas removal of TnT residues 1-69 or 151-158 had little effect. This suggests that TnT residues 70-150 include the major region within the TnT ``tail'' that interacts with tropomyosin. These data show that ternary troponin complexes behave similarly to isolated troponin T fragments. The CB1 fragment of TnT (residues 71-151) binds readily to immobilized tropomyosin (Pearlstone and Smillie, 1977) and also binds to glutaraldehyde-fixed Bailey crystals of tropomyosin (White et al., 1987).
Notably, there is no
detectable Ca sensitivity to the relatively weak
binding of tropomyosin to troponin-(159-259) or to
troponin-(151-259) (Table 1). In the absence of the
extended amino-terminal region of TnT, which binds to tropomyosin in a
largely Ca
-insensitive manner, the binding of the
remaining portion of troponin to tropomyosin is much reduced, yet it
does not become more dependent upon the presence or absence of
Ca
. This does not prove that Ca
has
no effect on troponin-tropomyosin interactions. However, it does imply
that the net energetics of this interaction are not significantly
altered by Ca
, even when the elongated tail region of
TnT is absent. A possible explanation for this result is that, whether
Ca
is present or not, there is only a weak
interaction between tropomyosin and this region of troponin.
Figure 3:
Effect of truncated troponin on binding to
tropomyosin to actin. The binding of H-tropomyosin to 3.5
mM F-actin was studied by cosedimentation using a tabletop
ultracentrifuge, and conditions were as in Fig. 1. A,
the concentrations of tropomyosin and troponin-(159-259) were
varied in parallel so that the total concentration of truncated
troponin was always 2 mM in excess of the total tropomyosin
concentration. Open symbols, EGTA; filled symbols,
CaCl
; circles, tropomyosin alone; triangles, tropomyosin plus troponin-(159-259). In the
absence of truncated troponin, there is no effect of CaCl
on the free (i.e. non-actin-bound) tropomyosin
concentration required for half saturation of the actin. Truncated
troponin had a small effect on the apparent affinity, most notably in
the absence of CaCl
, when the apparent K
was shifted to about 0.1 mM, as compared with
0.2-0.3 mM for tropomyosin alone. B, increasing
concentrations of truncated troponin, either troponin-(159-259) (diamonds) or troponin-(151-259) (squares),
were added to samples containing 0.3 mM
H-tropomyosin and 5 mM F-actin. A
concentration of approximately 2 mM truncated troponin was
sufficient to approach saturation of its effect on tropomyosin-actin
binding, regardless of whether CaCl
(filled
symbols) was present. This is roughly 3-fold less than would be
predicted from the data in Table 1, perhaps because of a
difference between pyrene-tropomyosin and the carboxymethylated
H-tropomyosin.
Because of the weak affinity of
troponin-(159-259) for tropomyosin (Table 1), it was
necessary to determine whether the results in Fig. 3A were attributable to inadequate concentrations of truncated
troponin. Fig. 3B shows that this was not the case,
since little change was seen at higher concentrations of truncated
troponin. Fig. 3, A and B, shows a trend for
truncated troponin to have a greater effect on tropomyosin-actin
binding in the absence of Ca. Since the free actin
concentration is nearly constant, one can estimate the effect of
Ca
on the affinity of tropomyosin-truncated troponin
for actin from the Ca
-induced change in the ratio of
bound to free tropomyosin in the presence of 6 µM
troponin-(151-259) or troponin-(159-259). (This calculation
uses the last data point of each curve in Fig. 3B.)
Removal of Ca
increased this ratio by a factor of
three in each case, suggesting a 3-fold effect of Ca
on tropomyosin-truncated troponin binding to actin.
Figure 4:
Binding of skeletal muscle troponin to
tropomyosin in the presence of high ionic strength. Increasing
concentrations of rabbit fast skeletal muscle troponin were added to
0.1 mM pyrene-tropomyosin, and a representative experiment
demonstrating the resultant fluorescence change is shown. Conditions
were as in Fig. 1, except the KCl concentration was 300 mM instead of 60 mM. The solid lines correspond to
best fit values of troponin-tropomyosin binding constants of 8.6
10
M
(
; EGTA)
and 4.2
10
M
(*;
CaCl
). See Table 1for average values of several such
determinations. Fluorescence intensity units are
arbitrary.
Fig. 5shows a
representative experiment of troponin-tropomyosin binding to actin.
Troponin was added in a constant molar excess of the tropomyosin
concentration. The tropomyosin was radioactively tagged, permitting
measurement of its free and actin-bound concentrations. Comparing the filled and open symbols in Fig. 5, it can be
seen that removal of Ca strengthens binding of
troponin-tropomyosin to actin about 2-fold, similar to the results with
truncated troponin (Fig. 3). (The Fig. 3results can only
be analyzed qualitatively because of uncertainty in the stoichiometry
of truncated troponin binding to tropomyosin and to the thin filament.)
The analysis in Fig. 5differs from previous results using
skeletal muscle troponin (Hill et al., 1992), because the
concentration of the troponin-tropomyosin complex is calculated based
upon the affinity constants in Table 1, rather than assumed to be
the same as the non-actin-bound tropomyosin concentration. (We recently
published a similar analysis for cardiac troponin (Dahiya et
al., 1994) but now present skeletal muscle troponin data so that
it may be related to the truncated skeletal muscle troponin data.) The
advantage of this analysis is that it permits calculation of the
affinity of troponin for actin-tropomyosin, using the thermodynamic
linkage relationships schematically shown in Fig. 6. It should
be noted that there is a potential error in this calculation, because
pyrene-tropomyosin may have different properties than
H-tropomyosin. Fluorescence competition studies (Dahiya et al., 1994) suggest there are no major differences between
them, at least at low ionic strength.
Figure 5:
Binding of the troponin-tropomyosin
complex to actin, analyzed by the linear lattice binding equation.
Experimental conditions were the same as in in Fig. 4. The
concentrations of troponin and tropomyosin were varied in parallel so
that the concentration of total troponin was always in excess of the
total tropomyosin concentration. Two representative data sets are
shown, either in the presence of EGTA () or in the presence of
CaCl
(
). The concentration of free
troponin-tropomyosin was calculated from the measured, non-actin-bound
concentration of
H-tropomyosin and the binding constants in Table 1. The theoretical curves correspond to K
= 1.8
10
M
and y = 29 in the presence of CaCl
and to K
= 2.2
10
M
and y = 44 in the
presence of EGTA. K
is the affinity of
troponin-tropomyosin (or troponin-tropomyosin-Ca
) for
an isolated site on F-actin in the absence of any interaction with
other troponin-tropomyosin complexes. y is a cooperativity
parameter, and the product K
y is
approximately equal to the apparent overall binding
constant.
Figure 6:
Equilibrium linkage analysis of troponin
and tropomyosin binding to actin. The figure shows association
constants, in units of mM for several steps
involved in thin filament assembly. Conditions are as in Fig. 4and Fig. 5, i.e. in the presence of 300
mM KCl. These results are for binding events at an isolated
site on the actin filament, a small portion of which is schematically
shown. Analysis of isolated site binding is essential for the
equilibrium linkage scheme to be valid. Troponin binds tightly (K > 10
M
) to
actin-tropomyosin, regardless of whether Ca
is
present. Similarly, troponin has a profound effect on tropomyosin
binding to actin, increasing its association constant about 500-fold,
regardless of whether Ca
is present. This is in
contrast to the 2- or 3-fold apparent effect of truncated troponin (Fig. 3). Replacement of Ca
by
Mg
causes an approximately 2-fold increase in the
affinity of troponin either for tropomyosin or for actin-tropomyosin.
The affinity of tropomyosin for an isolated site on F-actin, 600 M
, is from Hill et al.(1992). The
affinity of troponin for actin-tropomyosin (4
10
and 2
10
M
in the
absence and presence of Ca
, respectively) was not
directly measured but was calculated from the linkage relationships to
the other values in the figure.
Significantly, whereas
truncated troponin has a weak, difficult to quantify effect on the
binding of tropomyosin to actin, whole skeletal muscle troponin has a
very large effect (Fig. 6). The figure shows obligate
equilibrium linkage relationships in the associations of tropomyosin
and troponin for an isolated site on F-actin. Note that the actin
binding constants do not depend upon cooperative effects (this is
required for this scheme to be valid and is one of the measurements
that results from the linear lattice analysis of actin binding data)
and therefore differ from apparent binding constants
(1/K) that partially depend upon cooperative
binding of tropomyosin or troponin-tropomyosin to actin. The average
affinity of troponin-tropomyosin for an isolated site on F-actin
(determined from several experiments similar to those in Fig. 5)
is 3.4 ± 1.0
10
M
in the absence of Ca
and 2.6 ± 0.8
10
M
in the presence of
Ca
. Regardless of whether Ca
is
present, troponin binding to tropomyosin causes its affinity for actin
to increase about 500-fold. This is very different from the small
effects of truncated troponin on tropomyosin-actin binding seen in Fig. 3, despite concentrations of truncated troponin that would
be expected to produce, based upon Table 1and in the absence of
actin, significant formation of the tropomyosin-truncated troponin
complex. Fig. 6also shows the affinity of troponin for an
isolated actin-tropomyosin site, as indirectly calculated from the
equilibrium linkage relationships. This affinity is too tight to
measure directly, whether Ca
is present (2
10
M
) or absent (4
10
M
).
The data in this report suggest the following. 1) Troponin
greatly promotes the binding of individual tropomyosin molecules to
actin, regardless of the Ca concentration. This
effect depends upon the presence of TnT residues 1-150. More
narrowly, the important region for this effect consists of residues
70-150, since troponin-(70-259) promotes tropomyosin-actin
binding almost as well as does whole troponin (Hill et al.,
1992). 2) The amino-terminal region of TnT, especially residues
70-150, is required for tight association of troponin to
tropomyosin. These results are consistent with observations involving
TnT fragments, as opposed to the ternary troponin complexes in the
present work. Also, Morris and Lehrer (1984) found that
troponin-(159-259) bound more weakly to tropomyosin than did
intact troponin. In a separate study of TnT fragments, Ishii and
Lehrer(1991) concluded that it was the NH
-terminal rather
than the COOH-terminal region of TnT that binds most tightly to actin.
Our results more narrowly suggest the importance of the
TnT-(70-150) region but do not by themselves allow any conclusion
comparing this region with the troponin-(159-259) region. 3)
There is a direct relationship between the strength of troponin binding
to tropomyosin (as the amino-terminal region of TnT is progressively
deleted or included), and the ability of troponin to enhance
tropomyosin-actin binding. Binding of troponin-(151-259) or
troponin-(159-259) to tropomyosin had only a small effect on the
affinity of the resultant complex for actin, regardless of the
Ca
concentration. One possible explanation is that,
unlike whole troponin, these truncated troponins do not interact
simultaneously with both actin and tropomyosin;
troponin-(159-259) is primarily an actin-binding portion of
troponin and TnT-(1-150) is the primary tropomyosin-binding
portion. Alternatively, it is possible that the weak effect of
truncated troponin on thin filament assembly occurs because deletion of
TnT residues 1-150 has a detrimental effect on the stability and
function of the remainder of the troponin complex.
Ca binding to the thin filament results in a structural change
generally considered to include a repositioning of tropomyosin on actin
(Haselgrove, 1972; Huxley, 1972; Parry and Squire, 1973; Lehman et
al., 1994). This event rapidly follows Ca
release, even in the absence of cross-bridge binding (Kress et al., 1986). Although cross-bridges also affect thin
filament conformation and cooperative cross-bridge effects may be
important in muscle activation (Lehrer, 1994), it is clear that
Ca
alone has a major effect on thin filament
structure. Despite progress on the Ca
-induced
conformational change in TnC at high resolution (Herzberg et
al., 1986; Gagnèet al., 1994), the
absence of an atomic strucure for whole troponin has hampered
determination of how this change in TnC is transmitted to the remainder
of the thin filament. At present, the best supported idea (reviewed in
Leavis and Gergely, 1984; Zot and Potter, 1987; Chalovich, 1992) is
that TnI interacts with actin in the presence of low
Ca
concentrations and, because TnI is part of a
ternary troponin complex that is bound to tropomyosin, the position of
tropomyosin on the actin filament is thereby constrained.
Ca
binding to TnC releases this constraint by
facilitating the interaction between TnI and TnC, which disrupts the
TnI-actin binding. Indeed, fluorescence resonance energy transfer data
support a Ca
-induced movement of TnI relative to
actin within the thin filament (Tao et al., 1990).
Furthermore, a specific TnI peptide has been shown to interact with
either Ca
-TnC or with actin and to be inhibitory of
actin-myosin interactions (Syska et al., 1976).
It is
worthwhile to consider how the present work relates to this model for
regulation. Simply put, the small effect of Ca summarized in Fig. 6is inconsistent with the idea that
the action of Ca
can be understood as a release of
TnI from actin. This small energetic change in troponin-thin filament
binding is not plausibly consistent with disruption of an actin-TnI
protein-protein interface. Rather, a Ca
-induced
release of TnI-actin binding can only be occurring if some other
interactions(s) between troponin and the thin filament are strengthened
by Ca
. A recent publication (Dahiya et al.,
1994) reached a similar conclusion regarding cardiac thin filament
assembly and suggested TnT as one possibility for a
Ca
-strengthened interaction with the thin filament,
perhaps via the direct TnT-actin binding that was demonstrated by
Heeley and Smillie(1988). Also, it was shown that an increased
cooperative interaction between adjacent cardiac troponin-tropomyosin
complexes did not occur on Ca
binding, so one should
look primarily within each troponin-tropomyosin-7 actin region for the
thermodynamic changes accompanying Ca
binding to TnC.
The present work shows similar behavior for skeletal muscle troponin,
which is the protein used for most of the data supporting the model of
TnI-mediated regulation. More significantly, the present data suggest
that the small effect of Ca
on thin filament assembly (Fig. 6) is probably not attributable to compensatory effects
involving the amino-terminal region of TnT. Removal of this region
weakened troponin's interaction with tropomyosin, but did not
increase the Ca
sensitivity of thin filament
assembly.
The above arguments suggest that, within the intact thin
filament, Ca has little effect on the energetics of
the interaction between actin-tropomyosin and the TnC
TnI
TnT-(159-259) region of troponin. However, it is hard to imagine
how regulation could be accomplished without Ca
causing major alterations in the interface between this region of
troponin and actin-tropomyosin. It is likely that many specific
interactions are altered, even if the net energetics are little
changed, and that different actin and troponin residues are involved in
these interactions in the presence as opposed to the absence of
Ca
. The identification of these residues by
mutagenesis of actin and troponin may prove a useful avenue for future
studies.