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INTRODUCTION |
Troponin is a striated muscle regulatory protein (see reviews in
Refs. 1-4) that is located at periodic, 38-nm spacing along muscle
thin filaments. This spacing is due to the 1:1 complex formation of
troponin with tropomyosin, an elongated coiled-coil protein that
stretches along seven actin monomers. Ca2+ binding to
troponin triggers conformational changes in the thin filament, thereby
allowing actin and myosin to interact to produce force and movement.
Troponin contains two domains: a globular region, which is composed of
subunits TnC,1 TnI, and the
COOH-terminal portion of TnT, and a highly extended region, or tail,
containing the remainder of TnT (5, 6). The globular region has a
central role in regulation, because it is the site of calcium binding.
In contrast, the tail region of troponin, which is the subject of this
report, has an uncertain role in conformational changes of the thin
filament. One possibility is that it has little direct effect on
regulation, acting instead as a calcium-insensitive anchor that holds
troponin onto tropomyosin (7, 8). However, the details of the
interactions of troponin with actin and tropomyosin are unknown, in any
of the conformations of the thin filament. Moreover, there is
increasing evidence that the structure of the troponin tail can alter
thin filament function in a complex manner (9-16). To better
understand the troponin tail region, the present study reports the
properties of a series of troponin complexes containing progressively
less of this region. Cardiac TnC plus TnI was reconstituted with either
cardiac TnT or a series of recombinant NH2-terminal
truncation mutants of TnT. The NH2-terminal region of TnT
was also examined in isolation, in the absence of TnC or TnI. The
results suggest that the anchoring function of this region involves
interactions with actin as well as with tropomyosin and that this
function is not confined to a small region of the tail, and they
suggest that a critical peptide in the tail may be particularly
important for proper function.
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MATERIALS AND METHODS |
Design of Recombinant TnT cDNAs for Bacterial
Expression--
Bovine cardiac TnT cDNA was cloned from a bovine
heart cDNA library.2 The
encoded amino acid sequence was almost completely consistent with the
predominant adult bovine cardiac TnT isoform, as previously determined
by protein sequencing (17). However, the cDNA contained no codon
for Glu-42 of the protein sequence, possibly due to splice site
variability within the alternative splicing region of TnT (18-20).
Many of the TnTs used in the present study are NH2-terminal
truncations, which therefore do not include the lone Cys (Cys-39) of
native TnT. (Numbering follows the published protein sequence.) Therefore, to facilitate labeling of all the planned TnTs by
carboxymethylation with radioactive iodoacetic acid, a single Cys
mutant was designed by performing the double mutation C39S/D266C.
Asp-266 was chosen because it would be present in all the truncation
mutants, was in a charged region unlikely to be buried on folding, was
not directly implicated in interactions with the other troponin
subunits (2), and would have its negative charge restored by the
planned carboxymethylation of the new Cys-266. The C39S mutation was
created using polymerase chain reaction, with a primer producing both the desired codon change (G170C) and a protein-silent change (A177G) that created a SacII site to facilitate identification of
recombinants. The D266C mutation was produced similarly by polymerase
chain reaction, using a primer with TnT cDNA alterations
G847T/A848G to encode the amino acid change, as well as A834T/C831A to
introduce a silent BSaAI site. Double mutant C39S/D266C was
obtained by subcloning. To remove 94 NH2-terminal residues,
D266C TnT cDNA was inserted in pSP72 and digested with
Eco0109 I and HindIII, and the TnT-encoding
3'-fragment was isolated. A similar digestion using BsaI and
HindIII yielded a 550-base pair fragment encoding the
COOH-terminal portion of TnT starting at Leu-120. cDNAs encoding TnT COOH-fragments starting with Glu-133, Ala-154, or Ala-174 were
created by polymerase chain reaction, using D266C TnT as the template.
Plasmids encoding NH2-terminal TnT fragments 1-94, 1-132,
1-153, and 1-173 were created by polymerase chain reaction and
contained Cys-39 rather than the C39S mutation. All cDNAs were
inserted into the expression plasmid pET3d (21) and confirmed by
automated chain termination sequencing.
Recombinant TnT Expression and Purification--
Recombinant TnT
was obtained by modification of published protocols (10). Bacterially
expressed and extracted TnTs were dialyzed against a denaturing buffer
consisting of 10 mM Tris-HCl (pH 7.5), 5 M
urea, 1 mM dithiothreitol, 5 µg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone, and 5 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone. The full-length TnT
molecules were applied to a Fast Flow Q Sepharose column, which was
then washed with an 80 mM NaCl buffer, and then purified
with a 0-0.6 M NaCl gradient. The truncated forms were similarly purified, but using an SP Sepharose column. An additional column purification step was performed using fast protein liquid chromatography columns, either Resource Q (full-length TnTs) or Resource S (truncated TnTs missing the negatively charged
NH2-terminal region). The fast protein liquid
chromatography step was sometimes omitted, and residual impurities
after the Sepharose step removed instead by gel filtration of the
troponin complex after reconstitution with TnI and TnC.
Reconstitution of Troponin Complexes Containing Truncated Forms
of TnT--
Recombinant TnT or TnT fragments were mixed under
denaturing conditions with bovine cardiac muscle TnI and TnC (13) in a 1:1:1 ratio. Protein molarities were calculated by combining measured absorbance with sequence-determined aromatic amino acid composition (21). However, NH2-terminal TnT fragments have weak molar
absorbance (one Tyr and no Trp), so they were measured by protein assay
ESL (Boehringer Mannheim), using a modification (22) of the biuret reaction. The mixtures were then dialyzed in several steps to remove
urea and KCl (13). This procedure was modified for the truncated
troponins, which precipitated when the protocol was tried without
modification: the pH of all dialysis buffers was raised from 7.5 to 8.8 (Tris-HCl in both cases), and the final dialysis step retained 0.1 M KCl, instead of removing all KCl as in the unmodified
procedure. This succeeded in producing ternary troponin complexes
containing bovine cardiac TnI, TnC and either TnT95-284, TnT120-284,
TnT133-284, TnT154-284, or TnT174-284. These dialyzed complexes,
designated Tn95, Tn120, Tn133, Tn154, and Tn174, respectively, were
further purified chromatographically using Sephadex G100.
Stokes Radius Determination--
The hydrodynamic properties of
the various troponin complexes were assessed by molecular sieve
chromatography (23), involving measurement of elution positions
relative to molecular weight standards on an FPLC Superdex 200 HR 10/30
column. The following standards and Stokes radius
(Rs) values were used to calibrate the column:
ribonuclease A, 16.4 Å; chymotrypsinogen A, 20.9 Å; ovalbumin, 30.5 Å; bovine serum albumin, 35.5 Å; aldolase, 48.1 Å; and catalase,
52.2 Å. To reduce any non-ideal behavior, low protein concentrations
were applied (0.3 mg/ml), and the column was run in the presence of 1 M KCl.
Binding of Troponin or Tropomyosin to the Thin
Filament--
Binding of radiolabeled tropomyosin or troponin to actin
was measured by cosedimentation (24). The tropomyosin was
stoichiometrically labeled under denaturing conditions on Cys-190 with
[3H]iodoacetic acid. Recombinant TnT or truncated TnT was
similarly labeled on Cys-266 and then reconstituted as above to form
troponin complexes. However, unless otherwise indicated, experiments
were performed with unlabeled troponin complexes. Troponin-induced binding of tropomyosin to actin was studied by varying the troponin concentration of samples containing 10 µM rabbit skeletal
muscle actin (25), 0.5 µM [3H]bovine
cardiac tropomyosin (26), 10 mM Tris-HCl (pH 7.5), 300 mM KCl, 3 mM MgCl2, 0.2 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, and
either 0.5 mM EGTA or 0.1 mM CaCl2.
Binding of the troponin-tropomyosin complex to actin was measured under the same conditions, except the actin concentration was 5 µM and both the troponin and the tropomyosin
concentrations were varied in parallel, with the total troponin
concentration maintained at 0.2 µM in excess of the total
tropomyosin concentration. Raising the concentration of the shortest of
these troponins (Tn120 and Tn133) to a 0.5 µM excess
relative to tropomyosin had no effect, and data points with both 0.2 and 0.5 µM excess were included. Similarly,
TnT1-153-tropomyosin was studied by including 0.5 µM excess relative to the tropomyosin concentration and by showing that
under such conditions, TnT1-153 was not limiting for tropomyosin binding to actin.
The relative affinities of the various troponin complexes for
actin-tropomyosin were compared by a competitive binding assay (modified from Ref. 27). All samples contained 6 µM
F-actin, 3 µM unlabeled tropomyosin, 1 µM
bovine cardiac troponin that had been 3H-labeled on TnT
Cys-39, 10 mM Tris-HCl (pH 7.5), 300 mM KCl, 3 mM MgCl2, 0.2 mM dithiothreitol,
0.3 mg/ml bovine serum albumin, and either 0.5 mM EGTA or
0.1 mM CaCl2. Displacement of the
3H-labeled troponin from the thin filament was produced by
varying the concentration of competing unlabeled troponin or truncated troponin that was added to the samples and then measured by liquid scintillation counting of the free concentration (F, the
dependent variable in Eq. 1, below) in the supernatant after
centrifugation. Control troponin binding is tight (K > 108 M
1) (9) under these
conditions, and the ratio (KR) of the affinity of
competitor troponin to the affinity of control labeled troponin was fit
using the following expression,
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(Eq. 1)
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where C (the independent variable) is the
concentration of added competitor troponin, S is the
concentration of troponin binding sites on F-actin, and T is
the total concentration of [3H]troponin.
MgATPase Rate Assays--
The actin-activated MgATPase rate of
rabbit skeletal muscle myosin S1 (28) was measured by release of
32P from
-labeled ATP (29). Skeletal muscle myosin S1
was used instead of bovine cardiac mysoin S1 because of its greater
stability, higher ATPase rates, and many functional similarities: (i)
the effects of Ca2+ on Vmax, ATPase
actin Kapp, and true myosin S1-ATP affinity for the thin filament (26, 30, 31); (ii) ATPase rate linearity with myosin
S1 concentration, cooperative ATPase activation by the free
Ca2+ concentration (30, 32, 33), and cooperative ATPase
activation by bound Ca2+ (34) (see Fig. 6). Rates were
linear over 10 min (four or five aliquiots removed and quenched with
sulfuric/silicotungstic acid) and were initiated by the addition of 10 µl of 20 mM ATP to 190-µl protein samples. Conditions
(except as indicated) were as follows: 25 °C, 5 mM
imidazole (pH 7.5), 1 mM ATP, 3.5 mM
MgCl2, 15 mM KCl, 1 mM
dithiothreitol, 0.1 mM CaCl2, 7 µM F-actin, 0.3 µM rabbit skeletal muscle
myosin S1, 1.5 µM bovine cardiac tropomyosin, and a total
of 1 µM of a variable mixture of Tn95 plus an inhibitory form of Tn95. This inhibitory Tn95 was made by reconstitution (13)
using a 1:1:1 mixture of TnI:TnT95-284:CBMII. CBMII is cardiac TnC
with an inactivating mutation (D65A/E66A) (27, 34) in the sole
regulatory binding site, site II.
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RESULTS |
Formation of Troponin Complexes with Variably Long Tail
Regions--
Although the structure of troponin is unknown, available
information aids interpretation of progressive NH2-terminal
truncations of TnT. Troponin contains a globular domain and an
elongated tail region (6). The globular domain contains TnI, TnC, and
the COOH-terminal portion of TnT. Specifically, rabbit fast skeletal muscle TnT COOH-terminal residues 159-259 (35) form a stable ternary
complex with TnI and TnC (36-39). The troponin tail is formed by an
imprecisely defined part of TnT, which is highly elongated (6, 7). Mass
attributable to TnT residues 1-71 is located far from the other
subunits, at the end of the tail (5). The corresponding
NH2-terminal region of bovine cardiac TnT, used in the
present study, contains an additional 27 amino acids (17), and this
produces a 20-Å increase in TnT length for the cardiac isoform (40).
These observations suggest that serial deletions of the cardiac TnT
NH2 terminus will qualitatively shorten the tail region,
without preventing troponin complex formation. The structure of such
complexes will depend on the original tertiary structure of the tail
region and upon any folding alterations resulting from the deletion.
A series of troponin complexes was created lacking a variable amount of
the TnT sequence at the NH2 terminus, from 0 to 173 residues. The least (94 residues) and the most extensive (173 residues)
truncations correspond to skeletal muscle TnT deletions of 67 or 146 residues, which can be removed with relatively minor or major effects
on troponin function, respectively (24, 36, 41). As predicted from
results with skeletal muscle TnT, SDS-polyacrylamide gel
electrophoresis analysis of gel filtered samples (Fig.
1A) indicated that all of
these cardiac TnT fragments could be combined with TnC and TnI to form
ternary complexes. In other words, none of the deletions caused
sufficient unfolding of the remaining TnT fragment to preclude
formation of a truncated troponin complex, nor did the deletions remove
sequences critical for complex formation.

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Fig. 1.
Effect of serial NH2-terminal TnT
truncations on the Stokes radius of ternary troponin complexes.
Truncated troponin complexes were formed from isolated subunits by
reconstitution (13), using bovine cardiac TnC, TnI, and, depending upon
the complex, TnT COOH-terminal fragments starting with Asp-95, Leu-120,
Glu-133, Ala-154, or Ala-174. A, 14% acrylamide
SDS-polyacrylamide gel electrophoresis analysis of ternary complexes
containing TnT COOH-terminal fragments (lanes 1-5) and of
cardiac muscle whole troponin (lane 6). Truncated ternary
complexes Tn174, Tn154, Tn133, Tn120, and Tn95 were loaded in
lanes 1-5, respectively. Also shown (lane 7) is
TnT NH2-terminal fragment 1-153 (used for Fig.
3C). B, the Stokes radii of these complexes were
assessed by gel filtration (see under "Materials and Methods").
There was a linear decrease in Rs as the amino
terminus of TnT was progressively removed. Errors are ± 1 Å.
Note that there is no discontinuity when 119 residues were removed
instead of 94 residues. However, removal of 119 residues had a much
greater effect on function (see below).
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The hydrodynamic properties of the various troponin complexes were
compared by gel permeation chromatography. There was a monotonic
pattern of decreasing Stokes radius as the amino terminus was
progressively shortened (Fig. 1B). Furthermore, this pattern was linear, with the change in Rs proportional to
the number of amino acids deleted. This suggests that the truncations did not lead to major unfolding of the remaining portions of TnT and is
consistent with the interpretation that the various troponins have
progressively shorter tail regions. It also suggests that none of the
truncations was dramatically altered from the preceding, next larger
form. The results are in general agreement with previous hydrodynamic
studies of cardiac troponin (52 Å Rs in Ref. 42)
and with scattering studies of the TnI-TnC complex (33 Å Rg
and 118 Å for the largest linear dimension of TnI in Ref. 43).
Effect of TnT Truncation on Troponin-induced Binding of Tropomyosin
to Actin--
In the presence of high ionic strength conditions,
tropomyosin binds weakly to actin unless troponin is added. Fig.
2A demonstrates this effect of
troponin, which increases the binding of tropomyosin to actin, with
indistinguishable results whether the troponin was isolated from heart
muscle (
) or troponin reconstituted using single Cys TnT mutant
C39S/D266C (
), a control molecule. TnT truncation potentially alters
this effect of troponin directly, by eliminating sites of
troponin-tropomyosin interactions, sites of interaction with actin, or
both. The figure shows a moderate weakening of the action of cardiac
troponin when 94 TnT residues were deleted (
), a more striking
suppression of function when 119 NH2-terminal residues were
absent (
), and virtual elimination of the action of troponin when
132 (
), 153 (
), or 173 (
) NH2-terminal residues
were absent. The results suggest that progressively larger truncations
produce progressively severe loss of function. Similar results were
obtained in the presence of calcium (data not shown).

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Fig. 2.
Troponin tail length is critical for
troponin-induced promotion of tropomyosin binding to actin. The
binding of 0.5 µM [3H]tropomyosin binding
to 10 µM actin was induced by adding varying
concentrations of native or recombinant troponin and measured by
cosedimentation in the presence of 300 mM KCl
(A) or 60 mM KCl (B). For native
( ) and reconstituted ( ) control troponins (TnT C39S/D266C), the
tropomyosin binding patterns were indistinguishable. Under both high
and low ionic strength conditions, deletion of the first 94 residues of
TnT ( ) decreased tropomyosin binding to actin moderately, and this
effect was much more profound when the TnT NH2-terminal
truncations were larger: ( ), 119 residues; ( ), 132 residues;
( ), 153 residues; ( ), 173 residues. The truncations produced a
similar pattern in the presence of lower ionic strength conditions
(B), except that binding is tighter under these conditions.
A second set of experiments gave similar results (not shown).
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The preceding experiments were conducted in the presence of high ionic
strength conditions (0.3 M KCl), a condition that we found
essential for quantitative assessment of the full-length control
molecules (24), which tend to polymerize with tropomyosin under lower
ionic strength conditions. To better study the truncated troponins,
they were also examined in the presence of 60 mM KCl. Under
these conditions, the truncated troponins lacking 119 (
) or 132 (
) TnT residues promoted tropomyosin binding to actin (Fig.
2B), implying direct and/or indirect interactions between tropomyosin and these troponins under these lower ionic strength conditions. However, the effect on tropomyosin sedimentation was much
less than the effects of full-length troponin (
) or troponin lacking
94 TnT residues (
).
Affinity of Troponin-Tropomyosin for Actin--
To explore the
phenomenon illustrated in Fig. 2 in more detail, actin binding was
assessed as the concentrations of the various troponin-tropomyosin
complexes were varied. If the tail region of troponin merely holds the
molecule onto tropomyosin, then deleting part of the tail would have
little effect on the affinity of the troponin-tropomyosin complex for
actin. However, Fig. 3A shows that progressive deletion of the tail had a large and progressive effect on this process in the absence of calcium. Removal of 94 residues (Tn95,
) caused a 2-fold shift from control results (
)
in this representative experiment. Deletion of another 25 residues
(Tn120,
) had an additional 7-fold effect, for a 14-fold effect
relative to whole troponin. Similar results were found for Tn120 and
for Tn133 (
). These last two complexes had only a small effect on
tropomyosin binding to actin, as seen by comparison to data with
tropomyosin alone (
). The truncations produced a similar pattern in
the presence of saturating calcium concentrations (Fig. 3B),
suggesting similar importance for the troponin tail in thin filament
assembly, regardless whether calcium is bound to TnC.

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Fig. 3.
Effect of the troponin tail on the affinity
of the troponin-tropomyosin complex for actin. Tropomyosin,
troponin-tropomyosin complexes, or TnT-tropomyosin complexes were added
to 5 µM actin in increasing concentrations, and binding
was measured by monitoring cosedimentation of [3H]labeled
tropomyosin. For Tn120 and Tn133, composite data from three experiments
are shown. For TnT1-153 and for the other complexes, representative
titrations are shown. A shows progressively weaker binding
of troponin-tropomyosin complexes to actin as the troponin tail is
shortened, determined in the presence of EGTA and 300 mM
KCl. Kapp values for native troponin ( ), Tn95
( ), Tn120 ( ), Tn133 ( ), and tropomyosin in the absence of
troponin ( ) were 6.9 ± 0.5, 3.4 ± 0.2, 0.48 ± 0.04, 0.36 ± 0.03 and 0.30 ± 0.01 × 106
M 1, respectively. Best fit McGhee-von Hippel
cooperativity parameters (72) were 13 ± 2 for native troponin,
40 ± 10 for Tn95, and 180 ± 98 for tropomyosin alone, and they were set at 30 for Tn120 and Tn133
because they could not be measured from these data. In the absence of
any troponin, tropomyosin binding to actin is weak but very
cooperative, as shown previously (24, 73). B shows the same
experiment performed in the presence of saturating Ca2+.
Kapp values for troponin, Tn95, Tn120, and Tn133
were 2.7 ± 0.2, 1.5 ± 0.9, 0.27 ± 0.02, 0.31 ±
0.03 × 106 M 1,
respectively. The corresponding cooperativity parameters were 19 ± 6, 25 ± 6, 20, and 20. The Kapp values
for Tn95 are weaker than those for whole troponin and stronger than
those for Tn120, both in the presence and in the absence of
Ca2+ (p < 0.05 in all cases). Tn120 and
Tn133 values are not significantly different from each other.
C compares the effects of whole troponin or of a troponin
tail peptide, TnT1-153, on binding of tropomyosin to actin. The
affinity is almost as tight for tropomyosin in complex with just the
troponin tail fragment (*, Kapp = 5.0 ± 0.2 × 106 M 1 and
y = 46 ± 14) as it is for the intact
troponin-tropomyosin complex ( , Kapp = 11 ± 1 × 106 M 1 and
y = 24 ± 5). This data in C were
obtained with protein preparations different from the other two
panels.
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Actin Binding Properties of Tropomyosin Complexed to a Troponin
Tail Peptide, TnT1-153--
An alternative means to evaluate the
function of the troponin tail is to examine it directly, rather than
studying the effects of its deletion. This was investigated with
several NH2-terminal TnT fragments, with mixed success.
TnT1-153 (Fig. 1A) was the easiest to examine in
cosedimentation experiments because, like whole troponin (24), it
caused tropomyosin to sediment in the presence of 300 mM
KCl only when actin was also present (data not shown). Fig.
3C shows the effects of this TnT NH2-terminal fragment alone (*), in the absence of TnI and TnC, on tropomyosin binding to actin. For comparison, binding of the troponin-tropomyosin complex to actin (
) was determined with the same actin and labeled tropomyosin preparation, a repeat of the titration in Fig.
3A. The TnT1-153·tropomyosin complex bound almost as
tightly to actin as did the entire troponin-tropomyosin complex (*,
Kapp = 5.0 × 106
M
1 and y = 46;
,
Kapp = 11 × 106
M
1 and y = 24). There is only
a 2.2-fold difference between the curves. A repeat experiment (not
shown) with different protein preparations showed a 3.5-fold effect.
Comparison to the tropomyosin alone data (
) in Fig. 3A
shows that addition of the troponin tail fragment increased the
Kapp approximately 17-fold. Furthermore, this
large effect is not due to increased cooperativity (the curves are
actually less steep when TnT1-153 is present than for tropomyosin alone), so the shift is due to enhanced interactions with actin and is
caused by the NH2-terminal portion of TnT.
A slightly longer tail fragment, TnT1-173, strongly promoted
tropomyosin binding to actin, but also caused tropomyosin sedimentation in the absence of actin. This precluded measurement of its effect on
binding (data not shown). Experiments with TnT fragments shorter than
153 residues were unsuccessful (visible aggregation using TnT1-94;
minimal effect on tropomyosin binding to actin exerted by TnT1-132),
possibly due to impaired folding (data not shown).
Troponin Binding to the Thin Filament--
The affinity of
troponin for actin-tropomyosin is 2-5 × 108
M
1, and a similar value is found for TnT in
the absence of the other troponin subunits (9, 44). The effect of TnT
truncation on this process was measured by a competition assay, which
permits sensitive assessment despite tight binding. A control troponin molecule, radioactively labeled on TnT Cys-39, was added to
actin-tropomyosin at saturating concentrations (a slight molar excess
relative to the concentration of binding sites on actin-tropomyosin).
Unlabeled troponin or truncated troponin was added to displace the
labeled troponin, and the pattern of displacement was monitored to
measure KR, the relative affinities of the unlabeled
and labeled troponins. The greater the displacement, the greater the
fitted value for KR (solid lines in Fig.
4A, using Equation 1). As
validation of the assay, KR is very close to 1 (KR = 0.95 ± 0.11) for the full-length
molecule (
) (Fig. 4A). Deletion of 94 TnT residues (
)
resulted in KR = 0.14 ± 0.02, a 7-fold effect
representing a greater reduction in function than the decrease in
affinity observed in Fig. 3, A and B. The 7-fold
effect (13-fold in a repeat experiment) is most simply explained by
suggesting that the deleted region directly participates in
interactions with both actin and tropomyosin, so binding to
actin-tropomyosin (Fig. 4A) is altered more than is binding
to actin (Fig. 3). However, the same result would be produced if the
tail interacted with actin only indirectly, through tropomyosin, and
does not exclude the possibility that there are sites of repulsion as
well as interaction. Similar results were found in the presence of
Ca2+, with KR = 0.14 and 0.18 in two
determinations (data not shown). Fig. 4A also shows that
removal of an additional 25 residues (Tn120,
), produces a marked
fall off in the affinity of troponin for the thin filament, with Tn120
able to displace very little of the control [3H]troponin.
This parallels the effects of the same deletion on binding of the
troponin-tropomyosin complex to actin. Truncation by 119 residues has a
severe effect on troponin function.

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Fig. 4.
Troponin binding to actin-tropomyosin is
greatly weakened by progressive shortening of the tail region.
A shows displacement of troponin from the thin filament,
monitored by the increase in the free, non-actin-bound concentration of
radiolabeled troponin as increasing concentrations of competing
unlabeled troponin are added. Full-length troponin ( ) causes more
displacement than is caused by Tn95 ( ), and Tn120 ( ) causes only
minimal displacement of troponin. Solid lines are best fit
curves using Equation 1, and they demonstrate the ratio of the
affinities of the unlabeled troponins for actin-tropomyosin, relative
to the affinity of the labeled control troponin; KR = 0.95 ± 0.11, 0.14 ± 0.02, and 0.002 ± 0.001 for
native troponin, Tn95, and Tn120, respectively. B is a
noncompetitive demonstration that radiolabeled Tn120 binds to actin
nonspecifically. In the absence of actin ( ), Tn120 does not
sediment. In the presence of actin, it cosediments with 5 µM actin to an extent that is indistinguishable whether 3 µM tropomyosin is absent ( and ) or present ( and ) or if Ca2+ is present ( and ).
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To further elucidate the properties of Tn120, it was radiolabeled on
Cys-266, and its binding to actin or actin-tropomyosin assessed by
cosedimentation, i.e. by direct measurement rather than
competition assay. Under the high ionic strength conditions of these
and the preceding experiments, removal of 119 residues from the TnT
tail was sufficient to eliminate specific positioning of the troponin
complex on actin-bound tropomyosin. This conclusion is supported by
Fig. 4B, which shows that the binding of Tn120 to actin was
unaffected by the presence of tropomyosin and was weakened to the
µM range.
Effects of Truncation of the Troponin Tail on the Energetics of
Thin Filament Assembly--
The assembly of troponin and tropomyosin
onto the actin filament can be approached as an equilibrium problem, in
which alterations in troponin structure can affect processes shown in
Fig. 5A. Fig. 5B
summarizes effects of troponin tail truncation on troponin binding to
actin-tropomyosin (
G3), calculated from
KR measurements, such as those in Fig.
4A, and on troponin-tropomyosin binding to actin
(
G2), taken from changes in Kapp,
measured as in Fig. 3, A and B. Truncation of the
troponin tail weakens both processes. Effects on
G3
(Fig. 5B,
and
) are larger than effects on
G2, (* and +), consistent with the known importance of
the troponin tail for binding to tropomyosin (5, 7, 36, 45, 46). The
results suggest that the troponin tail has a dual role in thin filament
assembly, facilitating not only troponin binding to tropomyosin (from
previous work), but also the association of these proteins to actin.
The simplest explanation would be that the troponin tail interacts
directly with actin as well as with tropomyosin. However, these data
could also be explained by an indirect effect of the tail, increasing
the strength of tropomyosin-actin interactions.

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Fig. 5.
Effects of troponin tail truncation on the
energetics of thin filament assembly. A, schematic
representation of equilibria relevant to troponin and tropomyosin
binding to actin. Cooperative aspects of these processes are not shown
in the figure but are included implicitly; measured values for G
include cooperative components. Energetic linkage among the reactions
is not obligatory unless cooperative aspects are excluded (not done in
the present analysis). B shows the effects, in kJ/mol, of
serial truncations of the troponin tail on G2, the free
energy of troponin-tropomyosin binding to actin (EGTA, *;
Ca2+, +). The dashed line is the average of all
the plotted  G2 determinations, with Ca2+
and EGTA combined because they do not differ. This panel also shows the
effects of the truncations on G3, which is the free
energy of troponin binding to actin-tropomyosin. The solid
line is the average of the plotted  G3 values,
combining EGTA ( ) and Ca2+ ( ). The effect of deleting
119 residues on G3 may be underestimated, because
 G3 is so weak that it is hard to measure (Fig.
4A).
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Regulatory Properties of Thin Filaments with Truncated TnT--
To
study meaningfully the effects of troponin tail truncation on
regulatory function, it is necessary for the truncated troponin to bind
specifically to tropomyosin on the thin filament. Otherwise, one cannot
presume that the troponin is positioned once per seven actins. As
indicated above (see especially Fig. 4B), this requirement is satisfied for Tn95 but not for the other truncations. Tn95 or
control reconstituted troponin (1-1.5 µM) were combined
with tropomyosin (1.5 µM) and actin (7 µM),
and Ca2+-sensitive regulation of the myosin S1 MgATPase
rate was examined in the presence of 20 mM imidazole (pH
7.1), 30 mM KCl, 3.5 mM MgCl2, and
0.3 µM myosin S1. Troponin tail truncation had no effect on the regulated MgATPase rates: 1.0 ± 0.1 s
1
(n = 5) for Tn95 and 0.9 ± 0.1 (n = 3) for control troponin in the presence of pCa 4, and < 0.1 s
1 for both troponins in the presence of pCa>8. These
identical results are consistent with the absence of Ca2+
sensitivity in the effects of this truncation on thin filament assembly
(Fig. 5B).
Tn95 is missing a portion of TnT that is located at the
tropomyosin-tropomyosin overlap joint (5), and aspects of cooperative thin filament activation by Ca2+ may depend upon this
missing portion. As we have shown recently (34), Ca2+
binding to the thin filament has a cooperative effect on the ATPase
rate of low myosin S1 concentrations, with activation paralleling the
fraction of adjacent troponins that both have bound Ca2+,
rather than simply matching the fraction of troponins with bound Ca2+. This observation was dependent upon a novel method
for controlling fractional Ca2+ binding: manipulating the
ratio of two forms of troponin, one that can bind Ca2+ and
the other that cannot because of an inactivating mutation in TnC
regulatory binding site II (TnC mutant CBMII) (27). If Ca2+
binding to adjacent troponins exerts no cooperative effect, then the
MgATPase rate will increase linearly with the fraction of troponins to
which Ca2+ is bound. In Fig.
6, this experiment is presented using
thin filaments that contain mixtures of Tn95 and Tn95 with CBMII.
Despite the absence of TnT residues 1-94, nonlinearity is seen. ATPase activation (79) is poorly described by the dashed line,
indicating that activation is not proportional to Ca2+
binding. The data are not adequate to measure the degree of
cooperativity, but the deviation from the dashed line indicates that
cooperativity was present. For comparison purposes, the pattern
previously reported for nontruncated troponin is shown (Fig. 6,
solid line). Removal of this portion of the troponin tail
did not eliminate this aspect of cooperative regulation, further
emphasizing that the most important functional regions of TnT lie
COOH-terminal to residue 94.

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Fig. 6.
Troponin tail truncation does not eliminate
the cooperative effect of Ca2+ binding on
activation of the myosin S1-thin filament MgATPase rate. The
myosin S1-thin filament MgATPase rate was measured as a function
of the fraction of Tn95 that had bound Ca2+. This fraction
was controlled by mixing, in variable ratios, Tn95 and a Tn95 that
contained CBMII, an inhibitory TnC with an inactivating mutation at
regulatory Ca2+ binding site II (27;34). The dashed
line, which poorly describes the data, shows the pattern expected
if activation were proportional to Ca2+ binding. The data
deviate from the dashed line, indicating cooperativity. The
solid line is not a fitted curve. It is included to indicate
the results previously observed when this experiment was performed with
troponin containing full-length TnT (34). Conditions and protein
concentrations are described under "Materials and Methods."
Normalized data from two separate preparations are shown. The
non-normalized rates using Tn95 and CBMII-Tn95 were 4.4 and 0.2 s 1 for one preparation, respectively, and 4.7 and 0.3 s 1 for the other.
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DISCUSSION |
TnT and tropomyosin are approximately co-linear, and they may
interact for at least one-third of the length of tropomyosin (5, 7,
40). Protein fragment studies and analyses of conserved sequence
regions suggest that the strongest interaction between tropomyosin and
troponin involves the 27 most COOH-terminal residues of tropomyosin and
some portion of TnT between residues 71 and 151 for the skeletal muscle
isoform (5, 36, 45-51), which corresponds to positions 98-178 in
bovine cardiac TnT analyzed in the present study. The present work
examines a similar region of TnT, with a focus on its contributions to
troponin and troponin-tropomyosin binding to the thin filament, rather
than on troponin binding to tropomyosin. We found that serial
NH2-terminal truncations of TnT resulted in troponin
complexes that were progressively smaller as judged by Stokes radius,
had a progressive loss of ability to interact with tropomyosin and
actin, and had a particularly sharp fall-off in function when a
specific 25-residue segment was deleted. The current deletion studies
suggest that a cardiac TnT region including residues 95-119 (residues
68-92 for rabbit skeletal muscle TnT) is critical for function. TnT
residues COOH-terminal to these could also be critical, particularly
residues 120-153, but may not be able to fold or function once 119 TnT
residues are deleted; this could explain the poor function of Tn120.
However, the smooth Stokes radius results (Fig. 1) suggest there is not an extensive unfolding process.
The present work emphasizes the importance of the troponin tail for
interactions with actin, both in the presence and in the absence of
calcium. An advantageous method for analyzing the binding of troponin
and tropomyosin to actin is to separate contributions from the
cooperativity of binding and from noncooperative binding of a single
troponin or tropomyosin to a bare actin filament (24, 52-55). This has
not been emphasized in the present study, because we find the
variability of such cooperativity measurements (up to 3-fold) to be
larger than the variability in the measurements of overall affinity. It
is possible that cooperativity contributes to the energetic differences
in Fig. 5B, which then would not be attributable simply to
actin binding. However, if the contributions of cooperativity are
excluded, and the noncooperative actin binding of the various
troponin-tropomyosin complexes are compared, the Fig. 3 data imply
effects of the deletions that are even larger than the values used in
Fig. 5B. Therefore the effects of the deletions must be
attributed primarily to alterations in interactions with actin.
Furthermore, because either Tn95 or TnT1-153 causes tropomyosin to
bind actin almost as tightly as does whole troponin, this implicates a
59-amino acid region of TnT in anchoring of troponin-tropomyosin to
actin: TnT residues 95-153, which correspond to skeletal muscle TnT
residues 68-126.
The TnT residues that are absent from Tn95 include "hot spots" for
dominant-negative TnT mutations causing familial hypertrophic cardiomyopathy (56-58) and also include the TnT hypervariable region that is developmentally regulated by alternative splicing (19). Deleting all or part of this region from cardiac or skeletal muscle TnT
has produced only small effects in a variety of assays, at most 2-fold
(12, 24, 41, 47, 59, 60). However, the present report is the first
measurement of the effect of such a deletion on the affinity of
troponin for the thin filament. Figs. 4A and 5B
show this to be a significant effect, at least 7-fold, implying a
greater importance for this region than established previously.
Because the COOH-terminal portion of TnT is associated with TnI and
TnC, Ca2+-sensitive changes in its interactions have
obvious significance for understanding regulation, as recently
emphasized by mutational studies of TnI and TnT (11, 15, 61-63).
Perhaps less obvious is the regulatory (as opposed to structural)
importance of the troponin tail. Nevertheless, the troponin tail may
affect regulatory function: as an isolated peptide, the troponin tail
increases the cooperativity of myosin S1 binding to the thin filament
(16) and alters the actin-tropomyosin-myosin MgATPase rate (15); point
mutations in the tail cause abnormally fast thin filament sliding and
cardiomyopathy (10, 56); Ca2+-sensitive regulation is
altered either by isoform variation in this region (13, 14, 64) or by
small NH2-terminal truncations (12, 47), although these
latter effects are not always seen (present study and Refs. 59 and 60);
in rat cardiac myofibrils, TnT NH2-terminal truncation
reduces force and MgATPase rate (65). It has been suggested that TnT
helps to activate the thin filament (9, 11, 15, 66), possibly due to
the tail region specifically (15). However, thin filament activation is
functionally (2, 34, 67-69) and structurally (70, 71) complex, and is
incompletely understood.
We suggest that the regulatory significance of the troponin tail can be
understood by considering the involvement of the tail region in the
energetics of assembly of the different conformational states of the
thin filament. (We have made a similar argument previously regarding
the entire TnT subunit (9).) Recent structural results (70), supported
by preliminary work from another group (71), imply there are at least
three quaternary structures for the regulated thin filament, defined by
Ca2+- and myosin-induced movements of tropomyosin. The free
energies of these states, and the equilibrium constants for transitions among them, include contributions from the direct and indirect interactions of troponin with actin. The present report quantitatively demonstrates the great importance of the troponin tail for these interactions and for stabilizing the various conformational states of
the thin filament. This is sufficient to explain, qualitatively, how
the troponin tail may also be important for regulation: its large
influence on assembly implies effects on tropomyosin movement. Detailed
evaluation of this suggestion will require further structural information concerning the conformation of troponin and its position(s) on the thin filament. Studies of troponin that distinguish critical and
less critical regions of the molecule, such as the present work, help
delineate what structures must be solved to achieve a compelling
understanding of regulation.