From the Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294-2041
Received for publication, December 18, 2002
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ABSTRACT |
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Cardiac muscle activation is initiated by the
binding of Ca2+ to the single N-domain regulatory
site of cardiac muscle troponin C (cTnC). Ca2+ binding
causes structural changes between cTnC and two critical regions of
cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R,
residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI
affinity for actin and a heightened affinity for cTnC. Using Förster resonance energy transfer, we have measured three
intra-cTnI distances in the deactivated (Mg2+-saturated)
and Ca2+-activated (Ca2+-saturated) states in
reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin
complexes. Distance A (spanning cTnI-R) was unaltered by
Ca2+. Distances B (spanning both cTnI-R and cTnI-I) and C
(from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca2+-induced increases of >8 Å. These results
compliment our previous determination of the distance between residues
flanking cTnI-I alone. Together, the data suggest that Ca2+
activation causes residues within cTnI-I to switch from a The contractile state of cardiac muscle is regulated by a series
of Ca2+ and cross-bridge-dependent interactions
among the thin filament proteins, including the subunits of troponin
(Tn),1 tropomyosin, and
actin. Troponin is composed of the three subunits: a
Ca2+-binding subunit (TnC), an inhibitory subunit (TnI),
which when bound to actin, inhibits myosin ATPase and force generation
in relaxed muscle, and a tropomyosin-binding subunit (TnT). Cardiac muscle activation is initiated by the binding of Ca2+ to
the single N-domain regulatory site of cTnC. This binding causes
structural changes between cTnC and two critical regions of cTnI: the
regulatory region (cTnI-R, residues 150-165) and the inhibitory region
(cTnI-I, residues 130-149). During activation, cTnI-I dissociates from
actin and associates with cTnC (1), and cTnI-R associates with an
activation-exposed hydrophobic patch in the N-domain of cTnC (2). These
changes, together with movement of tropomyosin on the thin filament (3)
and changes in actin structure and dynamics (4), switch on the thin
filament, permitting actin-myosin cross-bridge cycling and
force development.
Numerous studies have elucidated Ca2+-induced structural
changes between TnI and the other thin filament proteins (5, 6). Less
is known about Ca2+-induced structural changes within TnI
itself. We recently reported that in fully reconstituted cTn,
Ca2+ binding to cTnC causes a 9-Å increase in the length
of cTnI-I (7). This large extension of the inhibitory region apparently pulls this region away from actin and facilitates movement of the
adjacent regulatory region toward the N-domain of cTnC. In the present
study, we have extended the previous work by examining the potential
for Ca2+-induced conformational changes within the
regulatory region and between the regulatory and inhibitory regions. We
conclude that internal structural changes in cTnI are confined to the
inhibitory region, but the cTnI-I internal changes cause the adjoining
regulatory region to be displaced as a rigid body.
Sample Preparation--
Wild type recombinant chicken slow
skeletal TnC (identical in sequence to mouse cTnC) and an adult rat
cTnT mutant, in which both endogenous tryptophan at positions 237 and
288 were converted into phenylalanine, were overexpressed in
Escherichia coli BL21(DE3) (Invitrogen) and purified as
previously described (7-9). Three constructs of mouse cTnI each
containing a single tryptophan and a single cysteine were generated:
129W/160C, cTnI(129W/167C), and cTnI(150W/167C). In these cTnI mutants,
the endogenous Trp192 was changed to Phe and the two
endogenous Cys81 and Cys98 were mutated to Ser
and Ile, respectively. Construction of cTnI mutant clones, protein
expression, purification, and characterization of the expressed
proteins were as described (10). The single-cysteine cTnI mutants were
labeled with 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid in
the presence of 6 M urea (11). The degree of labeling was
found to be >0.97 mol of probe/mol of protein. Binary (cTnC-cTnI) and
ternary (cTnC-cTnI-cTnT) troponin complexes were reconstituted by
incubation at 4 °C for 12 h in the presence of 6 M
urea, 30 mM MOPS, pH 7.2, 1 mM dithiothreitol,
5 mM Ca2+ at a ratio of [cTnI]:[cTnC] = 1:3
for the binary complexes and [cTnI]:[cTnT]:[cTnC] = 1:1.2:3 for
the ternary complexes. The samples were brought to working buffer
conditions (50 mM MOPS, pH 7.2, 1 mM
dithiothreitol, 1 mM EGTA, 5 mM
MgCl2, 0.15 M KCl) through a series of dialysis
steps as described (12). The protein concentrations were determined
using the Bradford method. Mutant function was assessed through
Ca2+ titrations performed on the binary complexes. The
measurements yielded pCa50 values of 6.19 and 6.12 for cTnI
mutants cTnI(129W/160C) and cTnI(129W/167C), respectively. These values
were similar to those previously reported with wild type cTnI (8).
Fluorescence Measurements--
Steady-state fluorescence
measurements were carried out at 20 ± 0.1 °C on an ISS PC1
photon-counting spectrofluorometer, using a 2-nm band pass on both the
excitation and emission monochromators (7). The emission spectra were
corrected for variation of the detector sensitivity with wavelength and
for sample scattering. The spectral measurements were collected using 5 µM protein samples. Ca2+ saturation (2 mM total Ca2+) was achieved by addition from a
0.1 M CaCl2 stock solution. Quantum yields of
tryptophan residues were obtained by comparison with known values as
described (13).
Time-resolved fluorescence intensity and anisotropy decays were
measured at 20 ± 0.1 °C with an IBH 5000 photon-counting
lifetime system equipped with a very stable flash lamp operated at 40 kHz in 0.5 atm of hydrogen. FRET measurement requires data collection from donor-only and donor-acceptor samples. For both, fluorescent emission from the donor was collected into 1,024 channels of a multichannel analyzer. Donor-only and donor-acceptor data were obtained
under identical experimental conditions. Donor-only decays were
analyzed as a sum of exponential terms. Donor-acceptor decays were
analyzed as a static Gaussian distribution of distances using the
subroutine GAUDIS in the package CFS_LS
(cfs.umbi.umd.edu/cfs/software/index.html) (11, 14). The reduced chi
squares ratio ( Structural Modeling--
Both the Mg2+ and the
Ca2+ models had a total of 14 rigid pseudoatoms. The models
were optimized using 100,000 steps of rigid body Monte Carlo with
simulated annealing from 400 to 0 K. The force field consisted of
bonds between successive residues, a volume exclusion term, and a term
to represent the FRET distances. Bond potential energies were
calculated using
E Fluorescence Properties of cTnI Mutants--
Three intra-cTnI
distances were studied (Fig. 1) in the
Mg2+- and Ca2+-saturated states: distance A
(residues 150-167), distance B (residues 129-167), and distance C
(residues 129-160). Distance A spans the cTnI-R. Distances B spans
both cTnI-R and cTnI-I. Distance C spans cTnI-I and the N-terminal 10 residues of cTnI-R. Tryptophan at residue 129 or 150 served as energy
transfer donors. Cysteines at residues 160 or 167 were selectively
labeled with the acceptor probe AEDANS. The steady-state and
time-resolved fluorescence properties of the donor tryptophan are
summarized in Table I.
Trp129 emission properties were nearly equivalent in the
two mutants cTnI(W129/C160) and cTnI(W129/C167) (Table I).
Trp129 is sensitive to both cTnT binding and to
Ca2+ addition. When denatured, the Trp129
mutants had low quantum yields (0.11), and they decayed biexponentially with mean lifetimes of 3.13-3.14 ns. In working buffer, the decays were triexponential, and the mean lifetimes increased to about 4.2 ns.
A small increase in quantum yield was also observed. Ca2+
addition to the binary complexes increased the Trp129
quantum yield by an average of 64% (0.17-0.27 for cTnI(W129,C160) and
0.16-0.27 for cTnI(W129,C167)). There were small increases in the mean
lifetimes. Trp129 decayed biexponentially in ternary cTn.
In the Mg2+-saturated state, cTnT increased both the
quantum yields (0.17-0.22 for cTnI(W129,C160) and 0.16 to 0.23 for
cTnI(W129,C167)) and the mean lifetimes (4.20-4.39 ns for
cTnI(W129,C160) and 4.18-4.41 ns for cTnI(W129,C167)). These results
suggest that cTnT either sterically or allosterically shields
Trp129 from the solvent. Ca2+ addition to
ternary cTn increased the Trp129 quantum yield to 0.31 and
increased its mean lifetime to 4.62 ns in cTnI(W129/C160) and to 4.68 ns in cTnI(W129/C167). The substantial quantum yield increases, and
blue spectral shifts suggest that Ca2+ addition causes the
transfer of residue 129 from a partially buried to a highly buried
conformation. Ca2+-induced environmental changes in cTnI
Trp150 are more difficult to interpret. Ca2+
binding to the binary and ternary complexes containing
Trp150 produced significant blue shifts in the emission
spectra. This change was in the same direction as that observed for
Trp129, suggestive of a less exposed environment. However,
the binding also induced decreases in the quantum yield and mean
lifetime. Subsequent to Ca2+ addition, Trp150
emission is likely partially quenched not by the solvent but by
adjacent residues (15).
Ca2+ addition caused AEDANS-labeled cTnI160 in binary cTn
to undergo a blue shift of 2.1 nm (484.4-482.3 nm) and experience an
8.6% enhanced peak intensity. As a member of the ternary cTn complex,
Ca2+ addition induces a 1.9-nm blue shift and a 13%
increase in maximum intensity. In contrast, AEDANS, when attached to
Cys167 as a member of the binary troponin complex,
undergoes a Ca2+-induced red shift of 1.4 nm (483.3-484.7
nm) and an intensity decrease of 12.3%. Similar features were observed
in the ternary complex. In the presence of the dipolar solvent water,
probe emission occurs from lower vibrational energy levels within the
singlet excited state. Emitted photons contain less energy on average, causing an observable red shift in the emission maximum. These data
indicate that Ca2+ addition causes cTnI residue 167 at the
C terminus of cTnI-R to migrate from a less to a more polar
environment. Ca2+ activation causes residue 160 to become buried.
Steady-state Förster Resonance Energy Transfer--
We have
examined FRET using both steady-state and time-resolved methods. The
impact of energy transfer between donor Trp129 and acceptor
AEDANS attached to Cys160 (distance C) on steady-state emission spectra
is shown in Fig. 2. Trp emission is
observed as a peak near 330 nm. A portion of the photons absorbed by
Trp is nonradiatively transferred to AEDANS. The AEDANS fluorescent emission of the transferred photons (sensitized emission) is observed as a peak near 480 nm. When denatured, donor and acceptor move apart on
average, resulting in decreased transfer efficiency (Fig. 2a), because less donor energy is lost to the acceptor. The
donor fluorescence increases (toward the level observed in the absence of acceptor), and the sensitized acceptor emission decreases. The
addition of Ca2+ elicits an effect similar to denaturation
in the binary (Fig. 2b) and ternary (Fig. 2c)
complexes. Ca2+ increases the interprobe distance. Distance
B underwent Ca2+-induced changes similar to those observed
for distance C (data not shown). In contrast to the large
Ca2+-induced distance changes observed for distances B and
C, distance A was quite insensitive to Ca2+ (Fig.
3). Distance B, which spans both cTnI-I
and cTnI-R, encompasses residues in distance A (cTnI-R). The
steady-state FRET data implicates cTnI-I as the region undergoing large
Ca2+-elicited structural changes. The steady-state spectral
data provide an initial indication of substantial
Ca2+-induced conformational change in cTnI-I. Time-resolved
measurements quantified these initial findings.
Time-resolved Förster Resonance Energy Transfer--
Fig.
4 shows the intensity decays of
Trp129 used to make the distance C measurement in ternary
cTn. Unless otherwise indicated, we shall confine our attention to the
most physiologically relevant complex, ternary cTn. In the
Mg2+-saturated state, the donor lifetime (Fig. 4,
black) is appreciably quenched (Fig. 4, red)
through FRET. Upon saturation with Ca2+, the donor lifetime
(Fig. 4, blue) is unquenched by the acceptor (Fig. 4,
magenta). Ca2+ does not appreciably alter the
donor-only lifetime (Fig. 4, black versus blue). Visual
inspection of the data indicates a large Ca2+-induced
extension of distance C. The recovered distance distribution parameters
are reported in Table II. The
distribution is parametrized by a mean distance and a full width at
half-maximum (half-width). The mean distance C in the
Mg2+-saturated ternary complex is 24.5 Å, a distance
similar to that observed in free cTnI and in the
Mg2+-saturated binary complex. When the donor-acceptor
probe mean distance is within the range Ro = 0.5-1.5, the lifetime decay data usually contain sufficient
information to resolve the first and second moments of the
donor-acceptor distance distribution. Beyond this range it may be
possible to recover the mean distance but with less precision. In the
Ca2+-saturated case shown, the transfer efficiency is very
small, and upon analysis the interprobe distance is observed to be
1.63. The half-width is unattainable. Ca2+ binding causes a
14.7 Å increase in distance C. Similar results were obtained for
distance B. Upon Ca2+ saturation, the distance increased
from 26.8 Å to 45.7 Å, a change of 18.9 Å. Distance A was not
changed by Ca2+ addition, its value remaining near 22.7 Å.
The data confirm the observations obtained from steady-state data.
Ca2+ activation induces a major structural rearrangement in
cTnI-I, whereas the cTnI-R is unaltered.
To examine the uncertainty in the mean distance values for distances B
and C, one-dimensional
To calculate the Ro, a value of 2/3 was
assumed for the orientation factor ( Model of cTnI Regulatory and Inhibitory Regions--
Full atom
models of the 39-residue cTnI fragment corresponding to residues
129-167 were built using a peptide builder (Insight 2). The
Ca2+-saturated state was modeled as a continuous native
The Ca2+-dependent interaction between TnC
and TnI is the key triggering step in striated muscle contraction.
Despite years of intense investigation in many laboratories (22-27),
the molecular details of the Ca2+-switch mechanism in
cardiac troponin remain incompletely understood. To elucidate the
switching mechanism, it is necessary to understand intersubunit
interactions within the troponin complex as well as the conformational
transitions that occur within the individual troponin subunits. We have
previously reported a large Ca2+-induced extension of
cTnI-I in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT)
complexes. We proposed that the Ca2+-induced extension of
cTnI-I accommodates the movement of the adjacent cTnI-R toward the
Ca2+-exposed hydrophobic patch on N-cTnC (8, 28). TnI-I
release from actin (26) is apparently required for its extension. The switching mechanism therefore involves movement in TnI-I, TnI-R, and
N-TnC. These structural arrangements are coupled with the transition of
tropomyosin from the blocked (B) to the Ca2+-induced (C)
states (29).
The present study has focused on Ca2+-induced changes in
cTnI-R itself and the relative movements between cTnI and cTnI-R. In particular, we were interested in whether Ca2+-induced cTnI
conformational changes were confined to cTnI-I or extended into the
adjacent cTnI-R. Three engineered FRET pairs were used to measure three
different intra-cTnI distances under Mg2+- and
Ca2+-saturating conditions: distance A (residues 150-167)
spanned only cTnI-R, distance B (residues 129-167) spanned both cTnI-R and cTnI-I, and distance C (residues 129-160) spanned cTnI-I and the
N-terminal portion of cTnI-R extending approximately to its midpoint.
Distance A was unchanged by Ca2+ addition. The FRET
measured mean interprobe distances (Mg2+, 22.7 Å;
Ca2+, 22.6 A) were within 4 Å of the C TnI-R Is Unbound in the Mg2+-saturated State--
An
NMR study of the Ca2+-saturated cTnC-TnI-R peptide complex
suggests that the central region of TnI-R binds to the N-TnC
hydrophobic patch, whereas the C terminus of TnI-R extends beyond the
patch in a solvent-exposed position (28). The spectral properties of
AEDANS-labeled cTnI residues 160 and 167, indicating that residue 160 is buried and that residue 167 is solvent-exposed under saturating Ca2+, are compatible with this model. The spectral and
quantum yield properties of AEDANS-labeled cTnI160 in the
Mg2+-saturated state are similar to the solvent-exposed
AEDANS-labeled Cys167 in the Ca2+-saturated
state. These results suggest that the central portion of cTnI-R is
unbound and solvent-exposed in the Mg2+-saturated state.
Relative Movement of the cTnI-I and cTnI-R Regions--
We have
previously reported the Ca2+-induced distance changes
between 24 residues (129-152) spanning cTnI-I (distance D). The Ca2+-saturated distance D, when crudely corrected from
probe-C Movement of TnI-I and TnI-R Relative to the Actin
Filament--
For ease of visualization in Fig. 6, we have aligned the
Mg2+- and Ca2+-saturated models along cTnI-R,
perhaps giving the impression that cTnI-R hardly moves with respect to
the actin surface. This is not the case. In the model, the transition
of cTnI-I from a
In summary, we have shown a large Ca2+-induced extended
conformation of the contiguous inhibitory and regulatory regions of cTnI in cTn. This conformational transition arises from an extension of
the inhibitory region and an orientational change of the regulatory region away from the inhibitory region. The regulatory region does not
experience a Ca2+-induced increase in its end-to-end
distance. The present results strengthen our previous proposal that the
inhibitory region in the cTnC-cTnI interface provides the main
Ca2+-induced switching mechanism between relaxation and
activation. The transition of the inhibitory region of cTnI from a
helix-loop-helix motif to a more extended conformation enables TnI-I to
span distance between the N- and C-cTnC domains. This switching event
places TnI-R near the N-domain of cTnC where it can bind to the
activation-exposed hydrophobic patch.
-turn/coil to an extended quasi-
-helical conformation as the actin-contacts are
broken, whereas cTnI-R remains
-helical in both Mg2+-
and Ca2+-saturated states. We have used the data to
construct a structural model of the cTnI inhibitory and regulatory
regions in the Mg2+- and Ca2+-saturated states.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
db)2, where kb is the
force constant (200 kcal/mol/Å2), rij
is the distance between atoms i and j, and
db is the optimal bond length (3.79 Å). The
potential energy for the volume exclusion term was expressed by
E
dnb)2, if rij < dnb, or
E
where dlow = dobs
(Eq. 1)
and dhigh = dobs +
, and kfret was set to 20 kcal/mol/Å2. dobs is the observed
FRET distance + 4 Å (to correct for probe-C
offset) (see Table
III), and
was set to 1.5 Å for distances B and C and to 0 for the
shorter and more reliably measured distance D. For the
Ca2+-saturated distance B, no upper bounds were set
(kfret = 0). Mg2+- and
Ca2+-saturated models were optimized to near zero energy.
The reported models are representative of three different
energy-minimized structures.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Partial sequence of cTnI. cTnI
inhibitory (red) and regulatory (blue) regions,
including the actin-binding segment (underbar) are shown.
FRET pairs were engineered to measure distances: A (residues 150-167),
B (residues 129-167), and C (residues 129-160). Distance D (residues
129-152) was measured previously (7).
cTnI tryptophan emission properties
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Fig. 2.
Steady-state fluorescence emission spectra of
cTnI(129W/160C) labeled with AEDANS used to measure distance C. a, free cTnI in +Mg2+ buffer (solid
line) and in +Mg2+ buffer + 4 M GdnHCl
(dotted line). b, binary complex (cTnC-cTnI) in
+Mg2+ buffer (solid line) and in
+Ca2+ buffer (dotted line). c,
ternary complex (cTnC-cTnI-cTnT) in +Mg2+ buffer
(solid line) and in +Ca2+ buffer (dotted
line). Excitation was at 295 nm. +Mg2+ buffer was 50 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM EGTA, 5 mM MgCl2, 0.15 M KCl; +Ca2+ buffer was +Mg2+
buffer with 2 mM CaCl2.
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Fig. 3.
Steady-state fluorescence emission spectra of
cTnI(150W/167C) labeled with AEDANS used to measure distance A. a, binary complex (cTnC-cTnI) in +Mg2+ buffer
(solid line) and in +Ca2+ buffer (dotted
line). b, ternary complex (cTnC-cTnI-cTnT) in
+Mg2+ buffer (solid line) and in
+Ca2+ buffer (dotted line). The conditions are
the same as for Fig. 2.
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Fig. 4.
Representative fluorescence intensity decay
curves of Trp129 of cTnI(129W/167C) used to measure distance B. a, free cTnI with unlabeled Cys167
(black) or AEDANS-labeled Cys167
(red). b, ternary complex (cTnC-cTnI-cTnT) with
unlabeled Cys167 and Mg2+-saturated
(black), unlabeled and Ca2+-saturated
(blue), AEDANS-labeled Cys167 and
Mg2+-saturated (red), and AEDANS-labeled
Cys167 and Ca2+-saturated (green).
Donor-only decays nearly superimpose with each other and with the
Ca2+-saturated donor-acceptor decay. The conditions are the
same as for Fig. 2.
cTnI FRET distances
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Fig. 5.
Confidence estimates in the recovered mean
distances. A grid search yielded the dependence of the variance
ratio (
2). This value is
based on the assumption that the donor and acceptor probes tumble
rapidly and randomly over the course of the measurement (dynamic
averaging). One way of testing this assumption is to measure the axial
depolarization factors of donor and acceptor probes (18). Time-resolved
anisotropy decays were obtained for both donor tryptophan and acceptor
AEDANS as members of the protein complexes. The limiting anisotropies
of donor and acceptor were determined from the decay profiles. These
values were used to calculate the depolarization factors shown in Table
II. The depolarization factors of both donor and acceptor in free cTnI
and in the binary and ternary complexes were similar. Ca2+
had a negligible effect on the depolarization factors. These results
suggest that Ca2+ binding had little effect on the
orientational freedom of the probes.
-helix. The Mg2+-saturated state was represented as a
-helix (residues 1-9)/
-turn (residues 10-21)/
-helix
(residues 22-39). Model residues 1-9 correspond to the N terminus of
the inhibitory region (TnI-I), believed to form a stable
-helical
coiled-coil with TnT (19, 20). Residues 10-21, corresponding to the C
terminus of cTnI-I, associate with the actin filament in the
Mg2+-saturated state. Model residues 22-39 encompass the
regulatory region (TnI-R) that our present data (distance A) suggest is
-helical. The FRET distances obtained in this and our previous study
(7) were used to set long range constraints. The starting models did not meet the FRET-derived distance criteria. Reduced representation models were constructed using the computer program YAMMP (21), by
representing amino acid residues as
-carbon-centered pseudoatoms. The models were further reduced by treating the
-helical segments 1-9 and 22-39 as rigid units. The remaining residues (residues 10-21) were treated as rigid bodies forming a random coil. Distances B, C, and D were used to impose long range energy constraints. Representative energy-minimized Mg2+- and
Ca2+-saturated models are shown in Fig.
6. The two states have been superimposed
along the regulatory region. Table III
shows the agreement between the FRET-derived and model-based
distances.
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Fig. 6.
Reduced atom models of the TnI-I and TnI-R
regions in the Mg2+- and Ca2+-saturated
states. Stereo views of the Mg2+- (blue)
and Ca2+-saturated (red) states, which are
superimposed along the regulatory region. The N-terminal portion of
TnI-I (white) is represented as an -helix. TnI-R
(green) is
-helical in both Mg2+ and
Ca2+ states (distance A). Distances between the C
of
residue 129 (white sphere) and residues 152, 160, and 167 (green spheres) were used to impose long range energy
constraints on the models. The addition of Ca2+ causes the
C terminus of TnI-I to undergo a
-turn/coil (blue) to
quasi-
-helix (red) transition.
Comparison of model and observed FRET distances
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-C
distance of
a 18-residue native
-helix (26.4 Å). The ~4-Å distance
difference can be attributed to donor and acceptor probe-C
offset
distances. NMR studies on the Ca2+-saturated complexes
formed between N-cTnC and the cTnI-R peptide indicate that the C
terminus of the peptide (residues 150-157) is unstructured (28). Our
results, based on full-length cTnI, indicate that cTnI-R is
-helical
throughout. This was observed in both cTnC-cTnI and cTnC-cTnI-cTnT
complexes under Mg2+- and Ca2+-saturating
conditions. The secondary structure of cTnI-R under Mg2+
saturation has not been previously reported.
offset by adding 4 Å to the FRET distance, (32.5 A),
closely matches that of a 24-residue
-helix (33.7 A). The present
measurements extend this work by retaining the donor position
(Trp129) and placing the acceptor at residues 160 (distance
C) and 167 (distance B). The large Ca2+-induced changes in
distances B (18.9 Å) and C (14.7 Å) corroborate our earlier results.
We have constructed a reduced representation molecular model of
cTnI-I/cTnI-R in the Mg2+- and Ca2+-saturated
states (Fig. 6) consistent with distances A-D (Fig. 1 and Table III).
According to the model, cTnI-I/cTnI-R exists as an extended nearly
continuous
-helix in the Ca2+-saturated state. The
cTnI-R portion of the helix (residues 150-166) is supported by
contacts with N-cTnC (28). Meanwhile, the N-terminal portion of cTnI-I
associates with the C-domain of cTnC. This arrangement requires that
the C-terminal portion of cTnI-I, which is accessible to solvent (19),
bridges the N- and C-terminal cTnC domains, running roughly
anti-parallel to the cTnC linker region. In the Mg2+-saturated state, cTnI-R remains
-helical. The
C-terminal portion of cTnI-I (residues 138-149) have been previously
modeled as a
-turn (30) and presently modeled as a loop. This
C-TnI-I sequence has been identified as the minimum length peptide
capable of inhibiting actin-myosin ATPase (31, 32). The N-terminal
portion of cTnI-I (residues130-137) likely remains associated with
cTnC in the Mg2+-saturated state. We propose that
Ca2+ activation causes an internal
-turn/loop to
-helix transition in the actin-binding portion of the TnI inhibitory
region. The extension in TnI-I caused by this secondary structure
transition allows the TnI inhibitory region to span the TnC linker
region, which is required for the adjacent TnI regulatory region to
access sites on N-TnC.
-turn/coil to bent
-helix is associated with a
19.7 Å increase in the intra cTnI-I/cTnI-R separation (distance B). In
the intact thin filament, sTnI133 (cTnI167) is observed to move by 23.6 Å relative to actin
filament,2 and this movement
is predominantly in the plane of the actin surface. N-cTnI-I is
believed to remain fixed to cTnT/C-cTnC (19, 20), and the proximity of
the C- and N-cTnC domains is essentially unaltered by Ca2+
addition (33). Assuming a relatively fixed position of N-TnC relative
to the actin filament (34), the 19.7 Å increase in distance B can be
attributed to the translation of cTnI-R into the N-cTnC hydrophobic
pocket. Narita et al. (34) have proposed that
Ca2+ activation induces a ~50-Å TnC C-domain
repositioning on the surface of actin, which would also reposition
N-cTnI-I on the actin surface.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Takeda for discussing Tn
C-C
distances prior publication.
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FOOTNOTES |
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* This work was supported in part by Grant HL52508 from the National Institutes of Health (to H. C. C.) and by Grant 0330170N from the American Heart Association (to W. J. D.) and by the University of Alabama at Birmingham M.D./Ph.D. program (to J. M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Alabama, Rm. 490, MCLM, 1530 3rd Ave. S., Birmingham, AL 35294-0005. Tel.: 205-934-2485; Fax: 205-975-4621; E-mail: hccheung@uab.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212886200
2 J. M. Robinson, W. J. Dong, and H. C. Cheung, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: Tn, troponin; TnC, troponin C; N-TnC, N-domain of troponin C; TnI, troponin I; TnI-I, troponin I inhibitory region; TnI-R, troponin I regulatory region; TnT, troponin T; c, cardiac muscle; cTnI(129W/160C), mutant cTnI(C81S/C98I/L129W/L160C/W192F); cTnI(129W/167C), mutant cTnI(C81S/C98I/L129W/S167C/W192F); cTnI(150W/167C), mutant cTnI(C81S/C98I/I150W/S167C/W192F); FRET, Förster resonance energy transfer; MOPS, 3-(N-mopholino)propanesulfonic acid; GdnHCl, guanidine hydrogen chloride; AEDANS, aminonaphthelene-1-sulfonic acid.
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