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ALA-SCANNING OF THE INHIBITORY REGION OF CARDIAC TROPONIN I*
Tomoyoshi Kobayashi, Stacey E. Patrick and Minae Kobayashi
Department of Physiology and Biophysics and the Center for Cardiovascular Research, University of
Illinois at Chicago.
Running Title: Inhibitory region of troponin I.
Address correspondence to: Tomoyoshi Kobayashi, Department of Physiology and Biophysics,
University of Illinois at Chicago, 835 S. Wolcott Ave. (M/C 901), Chicago, IL 60612. e-mail:
In skeletal and cardiac muscles, troponin (Tn),
which resides on the thin filament, senses a
change in intracellular Ca2+
concentration. Tn
is composed of TnC, TnI, and TnT. Ca2+
binding to the regulatory domain of TnC
removes TnI’s inhibitory effect on the
contraction. The inhibitory region of cardiac
TnI spans from residue 138 to 149. Upon Ca2+
activation, the inhibitory region is believed to
be released from actin, thus triggering actin-
activation of myosin ATPase. In this study, we
created a series of Ala-substitution mutants of
cTnI to delineate the functional contribution of
each amino acid in the inhibitory region to
myofilament regulation. We found that most
of the point-mutations in the inhibitory region
reduced the ATPase activity in the presence of
Ca2+
, which suggests the same region also acts
as an activator of the ATPase. The thin
filaments can also be activated by strong
myosin head (S1)-actin interactions. Binding
of N-ethylmaleimide-treated myosin
subfragment 1 (NEM-S1) to actin filaments
mimics such strong interactions. Interestingly,
in the absence of Ca2+
NEM-S1 induced
activation of S1 ATPase was significantly less
with the thin filaments containing TnI(T144A)
than that with the wild-type TnI. However, in
the presence of Ca2+
there was little difference
in activation of ATPase activity between these
preparations.
Striated muscle thin filaments exist in
equilibrium among multiple-states. Ca2+
-binding
to the regulatory domain of troponin C (TnC1)
along the thin filaments and strong cross-bridge
interactions with thick filaments are thought to
shift the equilibrium. Ca2+
-binding to the
regulatory domain of TnC, which regulates the
interaction of troponin I (TnI) with actin-
tropomyosin (Tm) and TnC (1-3). In the thin
filaments, the inhibitory region of TnI (residues
104-115 of rabbit fast skeletal TnI (fsTnI) or 138-
149 of mouse cardiac TnI (cTnI)) undergoes a
structural transition depending on the Ca2+
-state
of TnC (4, 5). In the absence of Ca2+
at the
regulatory site(s) of TnC, the inhibitory region
interacts with actin to prevent activation of
myosin ATPase activity. When Ca2+
binds to the
regulatory site(s) of TnC, the switch region of
TnI, which is located at the C-terminus of the
inhibitory region, interacts with the newly
exposed hydrophobic patch of the N-terminal
regulatory domain of TnC (6-8). This interaction
causes the removal of the inhibitory region and
the second actin-Tm binding region of TnI from
the actin surface and allows actin to interact with
myosin. In the presence of Ca2+
at the regulatory
sites of TnC, the inhibitory region and the central
helical region of TnC are mutually stabilized,
according to the recent X-ray crystal structure of
the core domain of fsTn complex (9). Although
the sequence variations in the N-terminal and the
C-terminal regions of TnT, another component of
the Tn complex, are known to alter the Ca2+
-
sensitivity of myofilament activity (10, 11). In
addition, TnT is involved in the Ca2+
-dependent
interaction of the Tn complex with actin-Tm (12).
Yet, the molecular mechanism whereby TnT
participates in the Ca2+
-regulation has not been
established.
There is evidence supporting the idea that
each amino acid residue in the inhibitory region
of TnI contributes differently and to a different
degree to myofilament activities. One example is
genetic mutations and phosphorylation of amino
acid residues in the inhibitory region of cardiac
http://www.jbc.org/cgi/doi/10.1074/jbc.M109.001396The latest version is at JBC Papers in Press. Published on May 29, 2009 as Manuscript M109.001396
Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
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TnI that cause the modification of myofilament
activities. In hypertrophic or restrictive
cardiomyopathy-linked mutations found in the
inhibitory region, such as R142Q, L145Q and
R146G/Q/W mutations (mouse cTnI sequence
number), induce Ca2+
-sensitization of
myofilament activities and an increase in
ATPase/tension at low [Ca2+
] (13, 14). Recently
we reported that thin filaments reconstituted with
R146G or R146W mutant cTnI bind Ca2+
tighter
than those with cTnI(wt) (15). The Ca2+
-
sensitization may occur as a result of the
destabilization of the off-state of the thin
filaments due to the mutation introduced into the
actin-Tm interacting residue, i.e. Arg-146, of
cTnI. On the other hand, Thr-144 is
phosphorylated by protein kinase C (PKC)
specifically, although the consequence of the
PKC-dependent phosphorylation of Thr-144 has
not been clearly defined yet. Pseudo-
phosphorylation of Thr-144 was shown to cause
Ca2+
-desensitization in in vitro motility assay
(16), whereas there is a report that indicates
phosphorylation of Thr-144 sensitizes skinned
cardiomyocytes to Ca2+
(17). Furthermore,
Tachampa et al. reported that Thr-144 of cTnI is
important for length-dependent activation of
skinned cardiac muscle (18). Thus in each case
presented above a specific change in a single
amino acid in the inhibitory region of TnI induced
different and divergent effects on myofilament
activities.
Our aim of this study is to assess the
functional contributions of the individual amino
acid residues in the inhibitory region to the
regulatory function. In order to assess the
functional roles of the individual amino acid
residues systematically, we used Ala-scanning
(19, 20). Ala-substitution deletes all the
interactions made by atoms beyond !-C yet does
not alter the peptide backbone conformation,
unless it is applied to Gly or Pro. Ala is one of the
most abundant amino acids and found in both
buried and exposed positions. We found that
almost the entire minimum inhibitory region of
cTnI we investigated (Figure 1) is important for
both the inhibition and activation. Our data also
indicate that the C-terminal part of the inhibitory
region de-stabilizes the active state of the thin
filaments. We also found that Thr-144 is
involved in NEM-S1-dependent activation of
ATPase activity in the absence of Ca2+
.
MATERIALS AND METHODS
Proteins.- Recombinant human wild-type and
mutant cTnCs, cTnC(C35S/T53C/C84S) and
cTnC(C35S), in pET3d vector were expressed
using BL21(DE3) cells. cTnC was extracted with
5 % sucrose, 1mM EDTA, 50 mM Tris/HCl, pH
8.0 and the protease inhibitors (AEBSF, E-64 and
Pepstatin A). After centrifugation, the
supernatant fraction was collected and CaCl2 and
MgCl2 were added to final concentrations 1 mM
and 5 mM, respectively, followed by the addition
of ammonium sulfate to 60 % saturation. After
centrifugation, the supernatant fraction was
applied to a phenyl-sepharose column equilibrated
with 1 M NaCl, 1 mM CaCl2 and 50 mM
Tris/HCl, pH 8.0. After washing with the same
solution, the column was washed further with the
same solution except that 0.2 mM of CaCl2 was
used instead of 1 mM. Finally cTnC was eluted
with 5 mM EDTA and 20 mM Tris/HCl, pH 8.0.
The cTnC-containing fraction was dialyzed
against 1 mM EDTA, 20 mM Tris/HCl, pH 8.0
and 1 mM DTT. Solid urea was added to the
protein solution to 6 M and cTnC was separated
on a QAE fast flow sepharose column
equilibrated with 6 M urea, 1 mM EDTA and 20
mM Tris/HCl, pH 8.0. cTnC was eluted with a
linear gradient of 0-0.5 M NaCl. Recombinant
mouse cTnIs were expressed and purified as
described previously (21). Recombinant mouse
cTnT with a myc-tag at the N-terminus was
expressed and purified with a combination of
ammonium sulfate fractionation and a DEAE-
sepharose column chromatography as described
(22). Previously we showed that myc-tag at the
N-terminus of cTnT has no effect on myofilament
activity (19). Tropomyosin was extracted from
bovine left ventricular ether powder with 1 M
KCl, 1 mM EGTA, 1 mM DTT and 50 mM
Tris/HCl, pH 8.0 followed by isoelectric point
precipitation. The precipitation fractions were
resuspended with 1 M KCl, 10 mM MOPS, pH7.0
and ammonium sulfate fractionation was carried
out. Forty-five to 60 % saturation fraction was
collected and Tm was further purified on a QAE
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fast flow sepharose column. Actin was prepared
from rabbit skeletal muscle acetone powder as
described by Spudich and Watt (23). After
purification, F-actin solution was stored in the
presence of phalloidin (1:1 molar ratio). Myosin
subfragment-1 (S1) was prepared by chymotryptic
digestion of rabbit psoas muscle myosin and
purified on a SP-sephadex column as described
previously (24).
Labeling of proteins.- Single Cys residues of
mutant cTnC(C35S/T53C/C84S) and
cTnC(C35S) were labeled with 2 fold amount of
IAANS over Cys in the presence of 0.1 M NaCl,
1 mM EDTA and 20 mM HEPES, pH 7.5. The
reaction was quenched by the addition of DTT
and the excess fluorescence dye was removed by
dialysis and a desalting column. Labeling yield
was determined using "326nm = 27,000 M-1 for
IAANS (15).
Reconstitution of the Tn complex and the thin
filament.- Equimolar amount of Tn components
were combined in a solution containing 6 M urea,
1 M NaCl, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM
DTT and 20 mM Tris/HCl, pH 8.0. The protein
mixture was dialyzed against the same solution
without urea. NaCl concentration was next
reduced to 0.3 M and finally to 0.1M. After
dialysis, the protein mixture was clarified by a
centrifugation and then the resulting supernatant
fractions were applied to a Resource-Q (1mL, GE
Healthcare) column equilibrated with 0.1 M
NaCl, 5 mM MgCl2 and 20 mM Tris/HCl, pH 8.0.
The Tn complex was eluted with a linear gradient
of 0.1 to 0.5 M NaCl in the same solution.
To reconstitute thin filaments, we first
mixed actin and Tm, followed by Tn. For the
Ca2+
-binding measurements, we first mixed actin
and Tm in a 8.5:1 molar ratio followed by the
addition of IAANS-labeled Tn to 8.5:1:1
(actin:Tm:Tn) molar ratio with
cTnC(C35S/T53C/C84S). A slightly high molar
ratio of actin to Tn-Tm was necessary to
minimize free Tn in the thin filament
preparations. Since free Tn shows an opposite
direction of fluorescence change upon binding
Ca2+
(25), the presence of excess free Tn may
interfere with the measurements of Ca2+
-binding
to thin filaments. In order to confirm a slightly
less molar ratio of Tn and excess amount of Tn
produce the same results, we also carried out
Ca2+
-binding measurements using an excess
amount of Tn with IAANS-labeled cTnC(C35S)
over actin and Tm.
Ca
2+-binding measurements.- Steady-state
fluorescence measurements were carried out using
a Model 2000-4 spectrofluorometer equipped with
two 814 PMT photon-counting detectors (Photon
Technology International) with a cell holder
containing a thermostat and a magnetic stirrer.
The Ca2+
-binding was monitored by fluorescence
emission of IAANS attached at Cys-53 of
cTnC(C35S/T53C/C84S) or Cys-84 of
cTnC(C35S). As mentioned above, the latter was
used in order to make sure that the data obtained
with a subsaturating amount of Tn in the thin
filaments gave the same affinity as that thin
filaments with saturated amount of Tn. The
fluorescence emission intensity change observed
was assumed to be due to the direct Ca2+
-binding
to the regulatory site of cTnC in the protein
complexes. The solution conditions were 100 mM
NaCl, 5 mM MgCl2, 1 mM EGTA and 20 mM
MOPS, pH 7.0. The titration was carried out at 25
oC and the free Ca2+
concentration was calculated
using the WEBMAXC Standard program (26).
The titration curves were analyzed as described
previously (15, 25). As a measure of Ca2+
-
sensitivity, pCa50 values were then calculated as
-logKd from the apparent dissociation constant,
Kd. The apparent dissociation constant, Kd, and
pCa50 are expressed as a mean + SEM from 4-6
experiments. The apparent coupling energy of Tn
with Ca2+
-binding and actin-Tm interaction was
calculated as follows:
where and are the Ca2+
dissociation constants for the Tn complex and the
thin filaments, respectively. Error propagation
was calculated by the following equation:
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where
!
" is a function of measurable
quantities
!
"1
and
!
"2
, and
!
" ,
!
"1 and
!
"2
are errors associated with
!
" ,
!
"1 and
!
"2,
respectively.
Acto-S1 ATPase measurements.- The reaction
conditions were 5.0 #M actin, 0.2 #M myosin S1,
1.0 #M Tm and 2.0 #M Tn in 35 mM NaCl, 5
mM MgCl2, 20 mM MOPS, pH 7.0 and either 0.1
mM CaCl2 or 2 mM EGTA at 25 oC. A reaction
was initiated by the addition of final concentration
of 1 mM ATP. ATPase activity was determined
from the time course of inorganic phosphate
liberation using a malachite green assay (27).
Steady-state ATPase activity was determined
from 5 to 6 time points. In the case of NEM-S1
activated acto-S1 ATPase activity, free actin
concentrations were held at 5.0 #M as described
(28, 29), and ATPase rates were expressed per
unmodified S1 concentration. In all figures and
tables, the rate for S1 alone has been subtracted
from the measured rates and the rate is expressed
as a mean + SEM from 4 (without NEM-S1) or 8-
12 (with NEM-S1) measurements.
Statistical evaluation.- Statistical evaluation was
carried out by one-way ANOVA followed by
Scheffe test as a post-hoc multiple comparison
test; p < 0.05 was considered significant. Note
that Scheffe test is one of the most conservative
evaluations and least likely produces “Type-1”
(false-positive) error (30).
RESULTS
Ala-scanning mutations. A segment cTnI,
residues from 143 to 149 (Figure 1), was
subjected to Ala-scanning to assess the impact of
Ala-substitution at each position. It should be
mentioned that the Ala-substitution of each of
these residues seems to perturb neither the
flexible structure of the inhibitory region nor the
alpha-helical structure of the switch region, that
follows the inhibitory region, based upon the
secondary structural prediction by the AGADIR
computer program (31).
Ca2+
-binding measurements and Ca2+
-dependent
actin-Tm interaction. We measured Ca2+
-binding
to the Tn complex with Ala-mutation of cTnI in
the inhibitory region residues 143-149 either
alone and in reconstituted thin filaments. The
Ca2+
-binding was reported by the fluorescence
emission intensity change of IAANS attached to
Cys-53 of cTnC mutant. As we reported recently,
IAANS attached to Cys-53 of mutant
cTnC(C35S/T53C/C84S) reports Ca2+
-binding
affinity, as well as Ca2+
dissociation kinetics from
the regulatory site of cTnC and of the cTn
complex similar to those measured directly with
Quin-2 using unlabeled wild-type cTnC and cTn.
(25). Furthermore, IAANS at Cys-53 of cTnC
reports Ca2+
-binding to the reconstituted thin
filaments with almost the same affinity as
expected from previous reports (15, 32, 33). Thus
this labeling can be used not only for the Ca2+
-
binding measurements for the cTn complex but
also for the thin filaments as well. This allowed us
to compare results obtained from different
regulatory complexes. Ca2+
-binding to the Tn
complex induces a decrease of the IAANS
emission intensity, whereas Ca2+
-binding to the
thin filaments enhances the fluorescence
intensities. This indicates that the micro-
environments of IAANS attached to Cys-53 are
different in the Tn complex and the thin filaments
in the presence of Ca2+
. In the thin filaments,
IAANS may face toward the actin-Tm surface so
that it experiences a less exposed environment
upon Ca2+
-binding. For the reconstituted thin
filaments, we also conducted the Ca2+
-binding
measurements using cTnC with IAANS attached
to Cys-84 as mentioned in “Materials and
Methods”. The Ca2+
-binding properties of the Tn
complexes and the reconstituted thin filaments are
summarized in Table 1. The changes in the
apparent pCa50 of the regulatory complexes with
mutant cTnI from that with wild-type ($pCa50)
were calculated from the apparent dissociation
constants (Kd) and are shown in Figure 2. These
Ca2+
-binding properties were used to evaluate the
perturbation of the interaction of the inhibitory
region with other thin filament components by
Ala-substitution at each position in the segment
residues from 143 to 149 (Figure 3).
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The effects of Ala-substitution of one of
these residues on the Ca2+
-binding to the Tn
complex are summarized in Table 1 and Figure 2
(A): P143A, T144A and L145A mutations did not
perturb Ca2+
-binding properties of the Tn
complex. Compared with the residues from
segment 143-145 of cTnI, Ala-substitution of the
amino acid residues in the segment 146-149
showed decreases in Ca2+
-affinity of the Tn
complex. The R147A mutation demonstrated the
largest decrease ($pCa50 = 0.18 + 0.01; p < 0.05
vs. each of the rest of mutations investigated).
Nonetheless there were relatively small
perturbations of the Ca2+
-binding properties of the
Tn complex caused by Ala-substitution of
individual amino acid residue in the minimum
inhibitory region.
In general, Ala-substitution had more
impact on the Ca2+
-binding properties of the thin
filaments (Table 1, Figure 2 (B)) than those of the
cTn complexes. Mutations that affected Ca2+
-
binding to the thin filaments resulted in
sensitization of the system to Ca2+
, as shown by
positive values of $pCa50 in Figure 2 (B). The
V148A mutation significantly increased the
apparent Ca2+
-binding affinity of the thin
filaments by $pCa50 = 0.34 + 0.03. R146A and
R149A, as well as T144A, also demonstrated
sensitization of the thin filaments to Ca2+
, but
these were not significantly from cTnI(wt) with
ANOVA analysis (they were significant vs.
cTnI(wt) with t-test). The P143A mutation did not
alter the Ca2+
-binding property of the thin
filaments. The thin filaments with the L145A
mutation did not demonstrate a fluorescence
intensity change when titrated with Ca2+
.
Therefore we could not measure the Ca2+
-binding
to the thin filaments with L145A mutation.
Ca2+
-binding to the Tn complex and actin-
Tm binding are negatively coupled. That is, Ca2+
-
binding to the Tn complex reduces its affinity for
actin-Tm. The apparent Ca2+
-binding constants
for the Tn complexes and the thin filaments were
used to calculate coupling energies between Ca2+
and actin-Tm ($$Gcoupl) for each Tn complex
(Figure 3). The apparent coupling energy,
$$Gcoupl, is a measure of apparent Ca2+
-
dependency of the interaction of the Tn complex
with actin-Tm (Scheme 1) and was calculated as
described in “Materials and Methods”. It should
be mentioned that the thin filaments exist in
equilibrium between at least two states, which
possess different affinities for Ca2+
; Ca2+
-binding
itself is capable to shift the thin filaments state.
Thus the scheme could be more complicated.
Ca2+
-dependent interactions of the Tn complex
with actin-Tm can be also assessed using a co-
sedimentation assay. However, it may require
much higher salt concentrations as demonstrated
previously (34).
As evident from Figure 3, the Ca2+
-dependent
interaction of the Tn complex with actin-Tm was
affected by Ala-mutations in the C-terminal half
of the inhibitory region (p < 0.05; R146A,
R147A, V148A and R149 vs. wt). The V148A
mutation showed a 0.55 + 0.04 Kcal/mol
($$Gcoupl(wt) - $$Gcoupl(V148A)) decrease in the
Ca2+
-dependent interaction. Since we could not
determine the apparent Ca2+
-binding affinity of
the thin filaments with the L145A mutation, we
could not determine the $$Gcoupl for the L145A
mutation.
Actin-activated mysoin-S1 ATPase with Ala-
substitution mutants. A summary of actin-
activated myosin S1-ATPase activities is shown
in Figure 4. The addition of the wild-type Tn to
the complex of myosin S1, actin and Tm
increased the ATPase activity to 0.9 sec-1
from
0.28 sec-1
in the presence of Ca2+
(Figure 4 (A)),
whereas it decreased the activity to 0.05 sec-1
in
the absence of Ca2+
(Figure 4 (B)). Thus the
ATPase activity was increased ~18-fold by Ca2+
in
the presence of the Tn complex. In the presence
of Ca2+
, all but the T144A mutation examined in
this study resulted in a decrease of ATPase
activity compared with cTnI(wt) (Figure 4 (A)).
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These data clearly demonstrated the involvement
of the inhibitory region in Ca2+
-activation.
L145A, R147A and V148A mutations greatly
impaired Ca2+
-activation. The ATPase activities
with one of these mutations were 0.36 - 0.40 sec-1
.
It is noteworthy that the P143A mutation impaired
Ca2+
-activation without affecting the Ca2+
-binding
property. In general, the shift of the equilibrium
of the thin filament states seems to be
accompanied by the alteration of the Ca2+
-binding
property. Therefore this observation for the
P143A mutation deserves further investigation. In
the absence of Ca2+
, most of the Ala-mutations
impaired the inhibitory action (Figure 4 (B)). The
L145A mutation showed the largest effect (p <
0.05 vs. every cTnI examined in this study,
including cTnI(wt)): it decreased the ATPase
activity only to 0.17 sec-1
, resulting in only ~2-
fold Ca2+
-sensitivity. P143A, T144A and V148A
mutations showed no significant effect on the
ATPase activity in the absence of Ca2+
.
NEM-S1 activation of ATPase activities with Ala-
substitution mutants. The thin filaments can be
activated through a strong cross-bridge
interaction. We used NEM-S1 to mimic such an
interaction. NEM-treated S1 has little ATPase
activity by itself, binds to actin strongly even in
the presence of ATP and stabilizes the active state
of the thin filaments. Thus addition of NEM-S1 to
ATPase reaction mixture activates the thin
filaments and enhances ATPase activity. Figure 5
shows the comparisons of NEM-S1 activation of
ATPase rates with Ala-mutations vs. those with
cTnI(wt). As NEM-S1 concentration increased,
ATPase rate increased. In the case of cTnI(wt),
the ATPase rate increased to nearly 2.0 sec-1
with
1 #M NEM-S1 in the presence of Ca2+
(white bars
in Figure 5 (A)~(G)) and to 1.4 sec-1
in the
absence of Ca2+
(dark grey bars in Figure 5
(A)~(G)). With higher concentrations of NEM-
S1, ATPase rates would be indistinguishable in
the presence and absence of Ca2+
. As reported
above, the T144A mutation did not affect Ca2+
-
dependent actin-activated S1-ATPase activity
(Figure 4). The addition of NEM-S1 activated S1-
ATPase activity with T144A mutation as well as
that with wild-type Tn in the presence of Ca2+
.
Surprisingly, in the absence of Ca2+
, ATPase
activity with T144A was not activated as greatly
as ATPase with wild-type TnI by NEM-S1
(Figure 5 (B)). At least up to 1 #M NEM-S1,
ATPase activities were always suppressed. With
3 uM NEM-S1, ATPase activity was
indistinguishable from that with wild-type TnI in
the absence of Ca2+
(data not shown), indicative
of the lower affinity of NEM-S1 for the thin
filaments with T144A. In the case of L145A
mutation, which introduced a reduced ATPase
activity in the presence of Ca2+
and an increased
activity in the absence of Ca2+
compared with
wild-type (Figure 4), an addition of 0.3 uM NEM-
S1 enhanced ATPase activity to almost the same
level as that with wild-type in the presence of
Ca2+
(Figure 5 (C)). On the other hand, in the
absence of Ca2+
, the ATPase activities with NEM-
S1 were higher or the same as those with wild-
type. Taken together, these results indicate the
stabilization of the intermediate state of the thin
filaments by the L145A mutation. In the case of
the Ala-mutations of the C-terminal half of the
inhibitory region (R146A to R149A mutation),
ATPase rates were almost the same as those with
cTnI(wt) with NEM-S1 in the absence of Ca2+
(Figure 5 (D)~(G)). In the presence of Ca2+
and
NEM-S1, however, ATPase rates were constantly
lower than those with cTnI(wt). This indicates the
de-stabilization of active state of the thin
filaments by these Ala-mutations and is also
consistent with the data for the actin-activated S1-
ATPase shown in Figure 4.
DISCUSSION
Experiments reported here are the first to
investigate the effects of the systematic Ala-
replacement of the inhibitory region of cTnI on
the Ca2+
-dependent and the strong cross-bridge
dependent myofilament activities using fully
reconstituted systems. We found (1) the C-
terminal half of the inhibitory region is
responsible for the Ca2+
-dependent interaction of
the Tn complex with actin-Tm, (2) almost the
entire segment that we investigated is responsible
for cTnI’s inhibitory action, (3) almost the entire
segment is responsible for Ca2+
-activation and
most notably (4) Thr-144 is involved in strong-
cross bridge-dependent activation of the thin
filaments in the presence of EGTA, although
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T144A mutation did not impair inhibitory action
in the absence of Ca2+
nor activation in the
presence of Ca2+
without NEM-S1.
The inhibitory region was shown to
interact with actin in the absence of Ca2+
and
inhibit the strong interaction between actin and
myosin. One possible molecular mechanism for
the inhibitory action was proposed by Patchell et
al. (35). Based on the competition for the actin
surface between a peptide derived from the
inhibitory region of TnI and the actin-binding
peptide derived from myosin, they concluded that
the inhibitory region interacts with actin so that
the actin monomer undergoes a conformational
change that stabilizes the actin in a way to
produce a surface less suitable for stable complex
formation with myosin. On the other hand, 3-D
reconstruction of the thin filaments from electron-
microscope images suggests steric blocking of
myosin-binding to actin by Tm with the
interaction between the inhibitory region and
actin holding Tm onto the myosin-binding site of
actin to prevent actin from interaction with
myosin (36-38). Regardless of the molecular
mechanism underlying the inhibitory action by
the inhibitory region of TnI, the equilibrium
constant between the turned-off state of the thin
filaments and the Ca2+
-induced state of the thin
filaments seems to be relatively small (39, 40).
We found that apparent free energy changes of
Ca2+
-dependent interaction of Tn to actin-Tm
caused by Ala-substitution of amino acid residues
in the inhibitory region are only up to 0.55
Kcal/mol (Figure 3). Yet ATPase activities were
substantially affected by Ala-substitution.
Assuming that the $pCa50 values observed with
the Ca2+
-binding experiments of the Tn complex
and those with the thin filament reflect the
mutational effects on the Ca2+
-bound state and
Ca2+
-free state of the thin filaments, respectively,
the changes in Ca2+
-free state are more
responsible for the loss of the apparent Ca2+
-
dependent interaction energy. This observation is
consistent with other findings that demonstrated
hypertrophic cardiomyopathy-linked mutations
found in the inhibitory region do not reduce the
affinity of Tn complex to actin-Tm significantly,
whereas myofilament activity in the absence of
Ca2+
is greatly disturbed (41-43). Also this
mutational effect of the inhibitory region is in
striking contrast to the case with the mutation in
the second actin-Tm binding domain. The second
actin-Tm binding site of TnI is located in the C-
terminal domain of TnI. The C-terminal mobile
domain appears to form a globular structure when
it binds to actin-Tm in the absence of Ca2+
both in
fsTnI and cTnI (44, 45). Murakami et al.
determined the solution structure of the C-
terminal domain of fsTnI and docked the structure
of the C-terminal mobile domain to electron-
microscope images of the thin filaments (44). In
their structural model, the second actin-Tm
binding site spans residues ~138-175 of fsTnI and
~172-207 of mouse cTnI. The 18 residue
truncation from the C-terminus of cTnI, as most
of Ala-mutations of the inhibitory region
observed in this study, resulted in a sensitization
to Ca2+
of the system, but, unlike Ala-mutations, it
did not affect the basal level of ATPase activity
(46, 47). This is true for some of the HCM-linked
mutations. Most notably in in vitro motility
assay, HCM-linked mutation from the inhibitory
region, R146G (mouse sequence), caused a
complete loss of Ca2+
-dependent control, whereas
HCM-linked mutations from the C-terminal
domain, G204S, $K184 and K207Q, retained
Ca2+
-dependent regulation over filament sliding
velocity (48, 49). Assuming the C-terminal
mobile domain of cTnI forms the same structure
as that of fsTnI proposed by Murakami et al. (44),
Lys-184 and Lys-207 of cTnI are suggested to be
involved in the direct actin-binding. Thus these
observations may reflect the functional difference
between the inhibitory region and the second
actin-Tm binding site of TnI. Interestingly the
calculated apparent free energy changes of Ca2+
-
dependent interaction of Tn to actin-Tm shown in
Figure 3 do not always reflect the degree of
impairment of the inhibitory action of ATPase
activity in the absence of Ca2+
shown in Figure 4
(B). For example, the mutation V148A, that
perturbs the Ca2+
-dependent interaction of Tn
with actin-Tm, did not impair the inhibitory
activity. Data presented in Figure 4 also
demonstrate that the full inhibition of the acto-S1
ATPase activity at low [Ca2+
] requires the nearly
entire minimum inhibitory region of TnI, although
the degree of the effects of Ala-mutations on the
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inhibitory action differs from residue to residue.
Previously van Eyk and Hodges conducted Gly-
substitution experiments for the inhibitory region
of fsTnI (50). Although their experiments were
carried out with short 12-residues peptides
corresponding to the minimum inhibitory region
and their system for the ATPase measurements
did not include TnT and TnC, they also found a
broad distribution of the amino acid residues that
affect the basal level of ATPase activity.
Whereas the physiological importance of
the interaction of the inhibitory region with actin
at low [Ca2+
] is obvious, the interaction with TnC
at high [Ca2+
] has been puzzling and
controversial. The C-terminal part of the
inhibitory region, rather than the inhibitory region
itself, was identified as the Ca2+
-dependent switch
region, which interacts with the regulatory
domain of TnC in a Ca2+
-dependent manner (6-
8). In the case of fast skeletal Tn, most of the
Ca2+
-dependent binding energy between TnC and
TnI appears to stem from the interaction between
the switch region of TnI and the N-terminal
regulatory domain of TnC. For the inhibitory
region, on the other hand, the crystal structure of
the core domain of fsTn complex in the presence
of Ca2+
showed that it interacts with the central
helical region (D/E helix) of TnC (9). For cardiac
Tn, the D/E helix of TnC was disordered and the
inhibitory region was not visible in the crystal
structure of the core domain of cTn complex (51),
whereas small-angle X-ray diffractions of the
cardiac thin filaments indicate an extended
structure of the D/E helix region of TnC (52).
These observations indicate flexibility in the
linker region of the Tn complex and suggest a
relatively weak interaction between the inhibitory
region of TnI and the central helix of TnC. Yet
Ala-substitution in the inhibitory region greatly
suppresses actin-activated S1 ATPase activity in
the presence of Ca2+
. From a study employing a
wide range of NEM-S1 concentrations to activate
the thin filaments, we recently concluded that
cardiomyopathy-linked mutations found in the
inhibitory region of cTnI, R146G and R146W
(mouse sequence number), stabilize the functional
intermediate state of the thin filaments (51). The
discrepancy between the Ca2+
-affinity and the
ATPase rate found in this study also indicates an
intermediate state with a unique activity. In our
Ala-scanning experiments, the C-terminal half
part of the inhibitory region de-stabilized the
active-state of the thin filaments when mutated to
Ala. Whether the extended structure of the central
D/E helix is involved in the activation process as
originally proposed by Herzberg and James (53)
or Ala-mutation of the C-terminal half of the
inhibitory region affects the structural opening of
the N-terminal regulatory domain of cTnC, which
may be a primary determinant of myofilament
activity (54), remains to be solved.
In this study we could not determine the
$$Gcoupl with the L145 mutation, since the thin
filaments with the L145A mutation did not show
the fluorescence intensity change when titrated
with Ca2+
. However, as shown in Figure 4 (B), the
L145A mutation increased the actin-activated S1-
ATPase activity in the absence of Ca2+
,
suggesting this mutation impaired the interaction
between the inhibitory region of cTnI and actin in
the absence of Ca2+
. Also the L145A mutation
resulted in a decrease in the actin-activated S1-
ATPase activity in the presence of Ca2+
. As we
discussed in detail elsewhere (43), a mutation that
causes a decrease and an increase of the ATPase
activity in the presence and the absence of Ca2+
,
respectively, is likely to stabilize the functional
intermediate state of the thin filaments. This is
consistent with our data on the NEM-S1
activation of ATPase activity shown in Figure 5
(C), which clearly illustrate a stabilization of the
intermediate-state of the thin filaments as
mentioned above. The thin filaments with a
mutation that stabilizes the intermediate state
show a higher affinity for Ca2+
(15), although the
Ca2+
-binding properties of the each state of the
thin filaments have not been characterized.
Therefore it is plausible that the L145A mutation
impairs the Ca2+
-dependent interaction with actin-
Tm and decreases the $$Gcoupl value.
The T144A mutation showed little effects
on Ca2+
-dependent interaction of Tn with actin-
Tm (Figure 3) and no effects on actoS1-ATPase
activities in the presence and absence of Ca2+
(Figure 4). Yet the T144A mutation impaired
NEM-S1 dependent activation and S1-binding of
the thin filaments in the absence of Ca2+
(Figure 5
(B) and (H)). This is most likely due to a reduced
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affinity of S1 for the off-state of the thin filaments
with T144A. Thus modification of Thr-144, such
as phosphorylation or replacement with another
amino acid, could result in an alteration of Ca2+
-
sensitivity and/or cooperativity of myofilament
activity, since strong cross-bridge interaction can
affect these parameters. In summary, our investigation of the
nearly complete minimum inhibitory region of
cTnI indicates that it is important for both the
inhibition and activation. This observation
strongly suggests the presence of an intermediate
state of the thin filaments that possesses the
intermediate activity. We also found that Thr-
144, which showed no effect on Ca2+
-dependent
ATPase activity when mutated to Ala, is involved
in strong cross-bridge dependent activation of
ATPase in the absence of Ca2+
.
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FOOTNOTES
*We thank Chad Warren for myosin S1 and NEM-S1. We also thank R. John Solaro and Joe Chalovich
for their critical reading of the manuscript. This work was supported by National Institute of
Health grant R01 HL082923.
1 The abbreviations used are: Tn, troponin; cTn, cardiac troponin; fsTn, fast skeletal troponin; MOPS, 4-
morpholinepropanesulfonic acid; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; IAANS,
2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid; NEM, N-ethylmaleimide; EDTA,
ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(!-aminoethylether)-N,N,N',N'-tetraacetic
acid;
FIGURE LEGENDS
Figure 1. Inhibitory region of TnI. (A) The sequence comparison of the minimum inhibitory region from
various vertebrates. The amino acid residues that are different from fsTnI are colored green in cardiac
sequences. Note the amino acid sequence of the inhibitory region is highly conserved. Also the amino
acid sequences of the minimum inhibitory region of the mutants we investigated in this study are shown.
(B) The crystal structure of the inhibitory region and its surrounding region in chicken fsTn complex in
the Ca2+
-bound form (PDB: 1YTZ). TnC, Pink; TnT, light blue; TnI, gray. The segment, corresponding
to residues 143-149 of mouse cTnI, is colored red.
Figure 2. Effect of Ala-mutation on Ca2+
-binding to the regulatory complexes. (A) the Tn complex, (B)
the reconstituted thin filaments. Differences of apparent pCa50 value from that with cTnI(wt) are shown.
Ca2+
-binding was measured in 100 mM NaCl, 5 mM MgCl2, 1 mM EGTA and 20 mM MOPS, pH 7.0 at
25 oC. * indicates p < 0.05 by one-way ANOVA followed by Scheffe test from cTnI(wt). # indicates p <
0.05 from the rest of cTnIs. Note that the scales for y-axis are different in (A) and (B).
Figure 3. Effect of Ala-mutation on apparent coupling energy (""Gcoupl) calculated from pCa50 values
from Ca2+
-binding experiments for the Tn complexes and the thin filaments as indicated in text.
* indicates p < 0.05 from cTnI(wt) by one-way ANOVA followed by Scheffe test.
Figure 4. Effect of Ala-mutation on Ca2+
-dependent actin-activated myosin S1 ATPase activity. The
reaction conditions were 5.0 µM actin, 0.2 µM myosin S1, 1.0 µM Tm and 2.0 µM Tn in 35 mM NaCl, 5
mM MgCl2, 20 mM MOPS, pH 7.0, 1.0 mM ATP and either 0.1 mM CaCl2 (A) or 2 mM EGTA (B) at 25 oC. The rate for S1 alone has been subtracted from the measured rates and the rate is expressed as a
mean + SEM from 4 measurements. In (A), * indicates p < 0.05 from cTnI(wt). # indicates p < 0.05 from
cTnI(wt), cTnI(T143A), cTnI(T144A), cTnI(R146A) and cTnI(R149A). In (B), * indicates p < 0.05 from
cTnI(wt). # indicates p < 0.05 from the rest of cTnIs. Note that the scales for y-axis are different in (A)
and (B).
Figure 5. Effect of Ala-mutation on NEM-S1 activated acto-S1 ATPase activity. The reaction conditions
were 5.0 µM actin, 0.2 µM myosin S1, 1.0 µM Tm, 1.0 µM Tn and NEM-S1 as indicated in 35 mM NaCl,
5 mM MgCl2, 20 mM MOPS, pH 7.0, 1.0 mM ATP and either 0.1 mM CaCl2 or 2 mM EGTA at 25 oC.
Free actin concentrations were held at 5.0 µM. (A)-(G) Open bars, cTnI(wt) in Ca2+
; light gray, mutant
cTnI in Ca2+
; dark gray, cTnI(wt) in EGTA; closed, mutant cTnI in EGTA. The rate is expressed as a
mean + SEM from 8-12 measurements per unmodified S1. (H) ATPase activities with the mutant cTnIs
expressed relative to that with cTnI(wt). For each mutant labeled at the bottom, S1 ATPase activities
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without NEM-S1 (in the presence (white bars) and the absence (light gray bars) of Ca2+
) and with 0.5 µM
NEM-S1 (in the presence (dark gray bars) and the absence (black bars) of Ca2+
) are shown. Those with a
significant difference (p < 0.05 by ANOVA) when compared with cTnI(wt) are marked with *.
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Table 1.
Summary for the effect of Ala-substituted mutations of cTnI on the Ca2+
-binding properties for the
different regulatory complexes. The dissociation constant, Kd, pCa50, which was calculated as -logKd,
and the Hill coefficient, nH, are expressed as the mean value + SEM from 4-5 experiments. As mentioned
in “RESULTS”, Ca2+
-binding to the thin filaments with cTnI(L145A) could not be measured. * indicates
p < 0.05 from cTnI(wt) by one-way ANOVA followed by Scheffe test.
______________________________________________________________________________
Complex TnI Kd (M) pCa50 nH
______________________________________________________________________________
Tn wt 3.03 + 0.04 x 10-7
6.52 + 0.01 1.11 + 0.02
P143A 3.45 + 0.02 x 10-7
6.46 + 0.00 1.17 + 0.04
T144A 2.90 + 0.06 x 10-7
6.54 + 0.01 1.08 + 0.02
L145A 3.08 + 0.05 x 10-7
6.52 + 0.01 1.12 + 0.02
R146A 3.54 + 0.06 x 10-7
* 6.45 + 0.01 * 1.14 + 0.02
R147A 4.57 + 0.04 x 10-7
* 6.34 + 0.01 * 1.16 + 0.01
V148A 3.56 + 0.03 x 10-7
* 6.45 + 0.00 * 1.14 + 0.01
R149A 3.89 + 0.04 x 10-7
* 6.41 + 0.01 * 1.14 + 0.01
thin filament wt 2.97 + 0.16 x 10-6
5.53 + 0.02 1.80 + 0.05
P143A 2.83 + 0.24 x 10-6
5.55 + 0.04 2.31 + 0.46
T144A 2.01 + 0.12 x 10-6
5.70 + 0.02 2.08 + 0.18
L145A n.d.
R146A 1.91 + 0.28 x 10-6
5.73 + 0.06
2.29 + 0.17
R147A 2.49 + 0.40 x 10-6
5.61 + 0.05 1.85 + 0.16
V148A 1.37 + 0.06 x 10-6
* 5.87 + 0.02 * 1.74 + 0.06
R149A 2.07 + 0.27 x 10-6
5.69 + 0.05 1.63 + 0.13
_____________________________________________________________________________
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Tomoyoshi Kobayashi, Stacey E. Patrick and Minae KobayashiAla-scanning of the inhibitory region of cardiac troponin I
published online May 29, 2009J. Biol. Chem.
10.1074/jbc.M109.001396Access the most updated version of this article at doi:
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