(Received for publication, March 26, 1997, and in revised form, May 6, 1997)
From the Department of Biochemistry and Molecular
Genetics and
Department of Neurology, University of Alabama
at Birmingham, Birmingham, Alabama 35294 and the ¶ Department
of Physiology and Biophysics, University of Washington,
Seattle, Washington 89195
The kinetics of the binding of
Ca2+ to the single regulatory site of cardiac muscle
troponin was investigated by using troponin reconstituted from the
three subunits, using a monocysteine mutant of troponin C (cTnC)
labeled with the fluorescent probe
2-[(4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at
Cys-35. The kinetic tracings of binding experiments for troponin
determined at free [Ca2+] > 1 µM were
resolved into two phases. The rate of the fast phase increased with
increasing [Ca2+], reaching a maximum of about 35 s
1 at 4 °C, and the rate of the slow phase was
approximately 5 s
1 and did not depend on
[Ca2+]. Dissociation of bound Ca2+ occurred
in two phases, with rates of about 23 and 4 s
1. The
binding and dissociation results obtained with the binary complex
formed between cardiac troponin I and the IAANS-labeled cTnC mutant
were very similar to those obtained from reconstituted troponin. The
kinetic data are consistent with a three-step sequential model similar
to the previously reported mechanism for the binding of
Ca2+ to a cTnC mutant labeled with the same probe at Cys-84
(Dong et al. (1996) J. Biol. Chem. 271, 688-694). In this model, the initial binding in the bimolecular step
to form the Ca2+-troponin complex is assumed to be a rapid
equilibrium, followed by two sequential first-order transitions. The
apparent bimolecular rate constant is 5.1 × 107
M
1 s
1, a factor of 3 smaller
than that for cTnC. The rates of the first-order transitions are an
order of magnitude smaller for troponin than for cTnC. These kinetic
differences form a basis for the enhanced Ca2+ affinity of
troponin relative to the Ca2+ affinity of isolated cTnC.
Phosphorylation of the monocysteine mutant of troponin I by protein
kinase A resulted in a 3-fold decrease in the bimolecular rate constant
but a 2-fold increase in the two observed Ca2+ dissociation
rates. These changes in the kinetic parameters are responsible for a
5-fold reduction in Ca2+ affinity of phosphorylated
troponin for the specific site.
Muscle contraction consists of a cascade of events involving several protein structural changes and protein-protein interactions within the thick and thin filaments (1). For contraction to occur, the N-terminal domain of the myosin heavy chain in the thick filament must first bind to actin in the thin filament. The formation of this active actomyosin complex is, however, inhibited by troponin I. This inhibitory action is regulated in vertebrate skeletal and cardiac muscle through the binding of calcium to another troponin subunit, troponin C. This Ca2+ binding releases the inhibition of formation of the actomyosin complex.
The crystal structures of sTnC1 from turkey
(2, 3) and chicken (4, 5) reveal a dumbbell-shaped molecule with two globular domains connected by a long central helix. Each domain contains two metal ion binding sites, designated as sites I and II in
the N-domain and sites III and IV in the C-domain. Sites III and IV
have a relatively high affinity for Ca2+
(Ka 107
M
1) and also bind Mg2+
competitively (Ka
103
M
1), and sites I and II have a lower
Ca2+ affinity and are specific for Ca2+
(Ka
105
M
1). Current evidence indicates that the two
Ca/Mg sites in the C-domain most likely play a structural role, and the
Ca2+-specific sites in the N-domain carry out a regulatory
function.
Cardiac muscle troponin C differs from the skeletal muscle isoform in that site I cannot bind Ca2+ due to several amino acid substitutions in critical positions within the 12-residue Ca2+-binding loop. Although the three-dimensional structure of cTnC has not been determined, it is reasonable to assume, on the basis of sequence homology and similarities in physiologic functions, that its structure is similar to that of sTnC. Reversible Ca2+ binding to the single Ca2+-specific site is believed to induce conformational changes in the N-domain, and these changes appear to modulate the troponin C-troponin I interaction. The precise nature of these conformational changes are still obscure. A useful biophysical approach to delineate Ca2+-induced global structural changes is the use of extrinsic fluorescent probes that are attached to TnC. cTnC contains two cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 located in the C-terminal end of helix D where the cTnC-cTnI interaction occurs. In a previous study of two monocysteine mutants of cTnC (6), we showed that the probe IAANS covalently linked to Cys-84 is partially buried and sensitive to Ca2+ binding to the single Ca2+-specific site. The same probe attached to Cys-35, however, is highly exposed to solvent and not sensitive to Ca2+ binding. Once incorporated into troponin, the probe attached to the two cysteine residues have very different properties. The fluorescence of IAANS attached to Cys-84 of cTnC within the troponin complex is insensitive to Ca2+ binding, but the fluorescence of the probe attached to Cys-35 within the complex decreases by a factor of 3 in response to Ca2+ binding to the Ca2+-specific site. Similar fluorescence properties of IAANS attached to Cys-35 are observed with the cTnC-cTnI complex. These results provide evidence of involvement of the non-functional binding loop I of cTnC in the Ca2+-induced interaction between cTnC and cTnI.
Several previous studies (7-10) reported the dissociation and association kinetics of calcium binding to both classes of sites in isolated sTnC and in regulatory complexes containing sTnC. The binding of Ca2+ to the two regulatory sites was suggested to be diffusion controlled (10) on the basis of binding data obtained over a narrow range of [Ca2+]. Our recent study of a monocysteine cTnC mutant labeled at Cys-84 with IAANS showed that the Ca2+ binding mechanism is not diffusion controlled but must be described by a three-step sequential model (11). In the present work, we have extended this kinetic study of Ca2+ binding to the Ca2+-specific site of cTnC in reconstituted cardiac troponin. For this work, we used the monocysteine cTnC mutant cTnC(C84S) labeled at Cys-35 with IAANS. The overall kinetic mechanism is similar to that for Ca2+ binding to isolated cTnC, but there are significant differences in the rates of individual steps. Phosphorylation of cTnI is found to affect the rates of both Ca2+ binding to and dissociation from the Ca2+-specific site in troponin.
cTn was extracted from an
ether powder that was prepared from the left ventricles of fresh bovine
hearts (12). Troponin subunits were initially separated on a
CM-Sephadex C-50-120 column in the presence of 6 M urea,
50 mM citrate, pH 6.0, 1 mM EDTA, and 1 mM DTT. Crude cTnC, cTnI, and cTnT were pooled and
subsequently purified, separately, on a DEAE-Sephadex A-50 column in
the presence of 6 M urea. cTnC was eluted with a gradient
from 0 to 0.5 M KCl at pH 7.0, while cTnI and cTnT were
eluted at pH 8.0 with a gradient from 0 to 0.5 M KCl. The
purity of the proteins was monitored by SDS-polyacrylamide gel
electrophoresis. The purified proteins were lyophilized in the presence
of 0.1 M KCl, 0.5 mM EGTA, 0.5 mM
DTT, 20 mM imidazole, pH 7.2, and stored at 20 °C.
Cardiac TnC mutant, cTnC(C84S), was genetically generated and characterized as previously reported (11). The mutant was labeled at Cys-35, as described previously (6), with the fluorescent probe IAANS under denatured conditions, and the degree of labeling was found to be >95%.
Troponin ReconstitutionThe binary complex cTnC-cTnI and fully reconstituted cTn were prepared by incubating the IAANS-labeled cTnC mutant with a large excess of the other troponin subunits in buffer A (30 mM MOPS, pH 7.2, 1 mM DTT, and 50 mM Ca2+) containing 6 M urea. After 30 min at room temperature, the solutions were dialyzed against buffer A, which also contained 3 M urea and 1 M KCl. The urea and KCl concentrations were then reduced by changing the dialysate to solutions of buffer A containing no urea and a decreasing concentration of KCl in five steps: 1, 0.7, 0.5, 0.3, and 0.1 M KCl. Uncomplexed cTnI and cTnT precipitated during the dialysis in decreasing [KCl] and the precipitate were removed by centrifugation. The samples were then dialyzed against a solution containing 30 mM MOPS, pH 7.2, 1 mM DTT, 1 mM EGTA, and 0.3 M KCl (standard buffer).
Phosphorylation of cTnIcTnI was phosphorylated by protein kinase A in a medium containing 50 mM KH2PO4, pH 7.0, 0.5 mM EGTA, 0.5 mM DTT using 125 units of the catalytic subunit/mg of cTnI. The reaction was started by adding ATP to a final concentration of 0.5 mM, followed by incubation at 30 °C for 20 min. The solution was then dialysed exhaustively at 4 °C against the standard buffer. Previous studies have shown that this protocol produces about 90% phosphorylation of PKA sites in cTnI (13).
Fluorescence MeasurementsSteady-state fluorescence measurements were carried out at 20 ± 0.1 °C on an SLM 8000C spectrofluorometer. The bandpass of both the excitation and emission monochromators was set at 3 nm, and the measurements were made in the ratio mode. Emission spectra were corrected for variations of the detector system with wavelengths. Fluorescence quantum yields of IAANS were measured by the comparative method as in previous work (6). A standard calcium solution (Orion) was used in Ca2+ titration experiments as described previously (11). EGTA was used to control the level of free calcium, and the free calcium concentrations were calculated by using known stability constants of the chelator for cations and proton as given in Fabiator's algorithm (14). The same procedure was used to calculate free calcium concentration for the kinetic measurements described below.
Kinetic MeasurementsTransient kinetic measurements were performed at 4 °C on a Hi-Tech Scientific PQ/SF-53 stopped-flow spectrometer equipped with a 150 watt xenon lamp. The dead time of the instrument was determined to be 1.8 ms. For measurements of IAANS fluorescence, the excitation monochromator was set at 325 nm, and the emitted light was isolated at a right angle by a cut-off filter (OG 380) and detected by an EMI9798QB photomultiplier. To measure Quin-2 fluorescence, the excitation wavelength was 339 nm, and the emission was measured with a 445-nm cut-off filter.
In a typical binding experiment, one syringe contained a labeled
protein complex in the standard buffer containing 5 µM
EGTA and 3 mM MgCl2. The other syringe contained
the same solution, but without protein, plus different levels of free
Ca2+ (0 to -500 µM). The concentration of the
protein was about 1 µM after mixing. These conditions
ensured that the two C-terminal high affinity Ca/Mg sites were
presaturated by Mg2+ and that the single low affinity
Ca2+-specific site remained free prior to mixing. The free
[Ca2+] was controlled by EGTA. This control was adequate
when the total Ca2+ concentration was low, and this allowed
calculation of the second-order binding rate constant from rate data
obtained at low free [Ca2+] (Fig. 4). There was some
uncertainty in the calculated free [Ca2+] at higher total
[Ca2+], but this uncertainty was not important because
one of the binding rates approached a limiting value with increasing
free [Ca2+], and the other rate was insensitive to
[Ca2+] (Fig. 4). Dissociation of bound Ca2+
was measured by mixing a buffer containing EGTA with an equal volume of
labeled protein saturated with Ca2+ as described previously
(11). The second-order rate constant of Ca2+ binding to
EGTA is about 106 M1
s
1 (8). This rate is considerably slower than the
second-order rate constant of Ca2+ binding to cTnC. The
rate constant of Ca2+ dissociation from EGTA is 0.3-0.4
s
1 (8), much slower than the binding rate of
Ca2+ binding to cTnC. The rates involving EGTA would not
contribute to the observed cTnC rates. A number of control experiments
were carried out using Quin-2 to directly measure Ca2+
dissociation (8, 15). In these measurements, Ca2+
dissociation was monitored by the increase of Quin-2 fluorescence. The
final concentrations were 30 mM MOPS, pH 7.2, 0.3 M KCl, 3 mM MgCl2, 6 µM protein, 50 µM Ca2+, and 150 µM Quin-2. For each experimental condition, multiple tracings (8-10) were obtained, the tracings were averaged, and the
resulting tracing was fitted to a sum of exponentials by a nonlinear
least-squares method (16).
Fig. 1 shows the
fluorescence emission spectra of cTn reconstituted with the
IAANS-labeled cTnC mutant. In the presence of Mg2+, the
intensity at the emission maximum decreased by about 5% with a
negligible spectral shift. Ca2+ red-shifted the spectrum by
9 nm and decreased the quantum yield by a factor of 3. Similar
Mg2+ and Ca2+-induced spectral changes were
observed for the binary complex cTnC-cTnI prepared with the
IAANS-labeled cTnC mutant. Table I summarizes the
spectral properties of these samples and the corresponding samples
prepared with cTnI phosphorylated by PKA.
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The binding of Ca2+ to cTn and cTnC-cTnI was monitored by the increase in the fluorescence intensity of the IAANS-labeled cTnC. The apparent Ca2+ binding constants were determined from the half-maximum increase in the fluorescence (data not shown). These equilibrium constants are listed in Table II.
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The kinetics of Ca2+ binding to the
single regulatory site was studied with IAANS-labeled cTnC
reconstituted into cTn. Fluorescence transients showing negative
amplitude produced by mixing the cTn with Ca2+ in the
presence of Mg2+ are depicted in Fig. 2.
Below 1 µM Ca2+, the tracings could be fitted
to a single exponential, but above 1 µM, two exponential
terms were required. Fig. 3 shows a tracing obtained at
1 µM Ca2+. A one-exponential fit clearly was
not acceptable, but an improved and acceptable fit was obtained with
two exponential terms. The faster rate constant (f)
increased with [Ca2+], whereas the slower rate constant
(
s) did not depend on [Ca2+]. The
Ca2+ dependence of these two rate constants is shown in
Fig. 4A. The value of
s was
about 3-5 s
1 over the entire range of
[Ca2+]. The rate of the fast phase reached a maximum
value of about 34-37 s
1 at large [Ca2+],
and the initial slope of this plot (Fig. 4B) was 5.1 × 107 M
1 s
1. The
amplitude of
f varied from 85 to 60% of the total
signal above 1 µM Ca2+. The ratio of the
amplitude of
s to
f increased from less
than 0.2 to a limiting value of about 0.55 with increasing
Ca2+ concentration, and the shape of a plot of this
amplitude ratio versus [Ca2+] resembled the
f versus [Ca2+] plot shown in
Fig. 4A (data not shown). When cTn was reconstituted with
phosphorylated cTnI, the kinetics was qualitatively unchanged. The
fluorescence transients were also biphasic above 1 µM
Ca2+, with the slower rate constant of 5-8
s
1 that was insensitive to [Ca2+]. The
faster rate increased with increasing [Ca2+], reaching a
maximum value (about 80 s
1) that is about a factor of 2 faster than that of the nonphosphorylated cTn. The initial slope of
this increase of
f was reduced to 1.7 × 107 M
1 s
1. These
experiments were also carried out with the binary cTnC-cTnI complex,
using both nonphosphorylated and phosphorylated cTnI. The results were
very similar to those obtained with the fully reconstituted cTn,
indicating that cTnT in cTn had negligible effects on the rate of
Ca2+ binding to the regulatory site.
Kinetics of Calcium Dissociation from the Regulatory Domain
The kinetics of Ca2+ dissociation from the
Ca2+-specific site was measured with the cTnC-cTnI complex
and reconstituted cTn, using IAANS-labeled cTnC. Upon mixing the
Ca2+-saturated proteins in the presence of Mg2+
with an EGTA buffer, the kinetic tracings showed a large positive amplitude change. The fluorescence increase was completed in about 60 ms when the troponin was reconstituted with nonphosphorylated cTnI,
and 30 ms when reconstituted with phosphorylated cTnI (Figs. 5A and 6A). The
fluorescence transients from the cTn reconstituted with
nonphosphorylated cTnI could be fitted with a two-exponential function
with two rate constants: d1 = 20.6 s
1 with
a 70% of the total fluorescence change, and
d2 = 4.7 s
1 with a 30% of the fluorescence change. When the cTn
was reconstituted with phosphorylated cTnI, the rates of both phases
increased by a factor of 2:
d1 = 46.9 s
1
(66% fluorescence change), and
d2 = 8.9 s
1 (34% fluorescence change). The one-exponential fits
of these transients were not satisfactory as shown in Figs.
5B and 6B. Very similar results were obtained
with the cTnC-cTnI complexes. These observed dissociation rates are
listed in Table II. The present Ca2+ dissociation results
are consistent with those of a previous study (13) in that cTnI
phosphorylation by PKA enhanced the rate of Ca2+ release
from cTn reconstituted with native cTnC that was doubly labeled with
IAANS at Cys-35 and Cys-84. However, the previous study reported a
single dissociation rate, in contrast to the biphasic dissociation
reported here. The difference is unlikely due to the probe located at
two positions in the previous study because IAANS attached to Cys-84 is
Ca2+-insensitive in the troponin complex (6).
To establish whether Ca2+ binding to the two high affinity Ca/Mg sites in the carboxyl-terminal domain contributed to the observed dissociation rates, a dissociation experiment was performed by mixing EDTA with IAANS-labeled cTnC-cTnI and cTn saturated with either Mg2+ or Ca2+ at the two Ca/Mg sites (11). No fluorescence change was detected, indicating that IAANS attached to Cys-35 was not sensitive to Ca2+ binding to the Ca/Mg sites. These results are in agreement with the equilibrium fluorescence results.
Experiments were performed using native proteins and Quin-2 as the
fluorescent Ca2+ chelator to establish whether the IAANS
fluorescence transients accurately reflected removal of bound
Ca2+ from the Ca2+-specific site rather than
conformational changes. The fluorescence of Quin-2 is expected to
increase upon chelation of Ca2+ (8). When Quin-2 was mixed
with cTnC previously incubated with an excess of Ca2+
sufficient to saturate all three sites, biphasic dissociation kinetic
tracings with a positive amplitude were observed (data not shown). The
two rate constants were 133 s1 (35% amplitude) and 7 s
1 (65% amplitude). These amplitudes suggested that the
fast phase reflected removal of bound Ca2+ from the single
Ca2+-specific site, and the slow phase was associated with
Ca2+ removal from the two Ca/Mg sites. These results were
in agreement with previous Ca2+ dissociation rate data
obtained from cTnC with Quin-2 as the chelator (15, 17). The assignment
of the fast phase to Ca2+ removal from the specific site
supports our previous conclusion that the single-exponential IAANS
transient observed with the cTnC mutant monitors Ca2+
dissociation from the specific site (11). With the cTnC-cTnI complex
and reconstituted cTn, the Quin-2 transients were also biphasic: the
fast rate constant (
df) being in the range
of 33-38 s
1 (amplitude
A
df ~ 26%), and the slow rate
constant (
ds) in the range of 2-4
s
1 (amplitude A
ds ~ 74%). If Ca2+ dissociation from the specific site was in
fact biphasic as the IAANS transients suggested, the Quin-2 amplitude
change associated with this dissociation would be partitioned into two
phases, with amplitudes Adf and
Ads for the fast and slow phases, respectively. The sum of these amplitudes (Adf + Ads) should be about one-third of the total
amplitude. Thus, the amplitude Adf should be
less than 33%, approximately 20-25% on the basis of the biphasic
IAANS amplitudes. The observed amplitude of the fast Quin-2 phase
(A
df) was in agreement with this
expectation. The rate of the slow phase associated with
Ca2+ removal from the specific site and the rate of removal
of Ca2+ from the Ca/Mg sites would be expected to be
similar and not easily resolvable, and a single composite slow rate
would be expected. The observed slow Quin-2 rate reflected this
composite rate, and the assoicated amplitude would be larger than
two-thirds because it contained contributions from the slow phase
associated with the specific site and removal of Ca2+ from
the other two sites. The observed amplitude
A
ds is consistent with this
prediction. Thus, the biphasic IAANS kinetics observed with cTn and the
cTnC-cTnI complex was not limited by slow conformational changes and
reflected closely Ca2+ removal from the specific site.
The effect of temperature on the two Ca2+
binding rates was studied with phosphorylated and nonphosphorylated
cTn. From the Arrhenius plots of these data (data not shown), the
following activation energies were obtained for the fast and slow
phase: Ef = 55.3 ± 3.2 kJ/mol and
Es = 46.4 ± 2.7 kJ/mol for
nonphosphorylated cTn, and
Ef = 33.2 ± 2.5 kJ/mol and
Es = 31.9 kJ/mol for
phosphorylated cTn.
Native cTnC has two cysteine residues at positions 35 and 84. These two residues are readily alkylated by the fluorescent probe IAANS. The doubly labeled cTnC has been extensively used to study the equilibrium properties of Ca2+ binding to the regulatory site of both isolated cTnC and cTnC reconstituted into myofibrils and skinned muscle fibers. In a previous study (6), we reported that the fluorescence of IAANS attached to Cys-84 of the monocysteine mutant cTnC(C35S) tracked Ca2+ binding to the regulatory site in isolated cTnC. The probe attached to Cys-84 became insensitive to Ca2+ upon complex formation with either cTnI or cTnI plus cTnT. The fluorescence of the probe attached to Cys-35 in the mutant cTnC(C84S), however, was found to be sensitive to Ca2+ binding to the regulatory site only upon reconstitution with cTnI or with both cTnI and cTnT. We report here the kinetics of Ca2+ binding to and dissociation from the single Ca2+-specific site of cTnC reconstituted into the cTnI-cTnC and the troponin complex, using the mutant cTnC(C84S) labeled at Cys-35 with IAANS.
Cys-35 is located at the y coordinate of the 12-residue
inactive Ca2+ binding loop of site I. The low florescence
quantum yield of IAANS attached to this residue in the cTnC mutant
clearly indicates a highly exposed environment. This property is little
affected by Ca2+ binding to the regulatory site in the
isolated cTnC, but is significantly changed when the labeled mutant is
reconstituted with the other troponin subunits. This change reflects
the interaction between cTnI and the inactive binding loop I, which
alters the local conformation of Cys-35. The presence of
Mg2+ has little effect on this new conformation, but
Ca2+ binding to the regulatory site of cTnC in the
nonphosphorylated reconstituted complexes causes a significant
conformational change which leads to a large reduction of fluorescence
quantum yield. In phosphorylated troponin, this Ca2+
induces a 2-fold reduction in quantum yield, indicating that the
phosphorylation of cTnI at Ser-23 and Ser-24 affects
Ca2+-induced conformational changes in the N-domain of cTnC
in the complexes. These large Ca2+-induced changes in IAANS
fluorescence provide a convenient signal to study the kinetics of
Ca2+ binding to troponin.
The Ca2+ dependence of the two observed Ca2+ binding rates is similar to that previously observed with uncomplexed cTnC (11) and suggests a similar kinetic scheme for the binding of Ca2+ to both isolated cTnC and cardiac troponin (cTn).
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(Eq. 1) |
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(Eq. 2) |
The overall kinetic mechanism for the binding of Ca2+ to the regulatory site of cTnC is similar for isolated cTnC and reconstituted cTn, but there are significant quantitative differences. With cTn, both the limiting value of the observed fast binding rate and the slow Ca2+-insensitive binding rate are reduced by more than 1 order of magnitude. These lower rates may be related to the higher activation energies of the two phases when compared with the activation energies previously observed for isolated cTnC (11). These differences lead to a second-order binding rate constant (Kok1) that is about 3-fold reduced and a large reduction in the derived rate constants of the first-order transitions. With isolated cTnC, step 1 is thermodynamically favorable with an equilibrium constant of 4, but step 2 is unfavorable. With cTn, step 1 remains thermodynamically favorable but with a smaller equilibrium constant (1.6). Step 2 is now thermodynamically favorable with an equilibrium constant of 1.2, and the equilibrium constant of the initial step (Ko) increases by a factor of 9. Similar conclusions apply to the binary cTnC-cTnI complex. This is because the presence of cTnT in troponin has negligible effects on the observed rate constants. The stabilization of the bimolecular step and the second first-order step by cTnI results in a large increase in the overall Ca2+ binding constant for both cTn and the cTnC-cTnI complex relative to the Ca2+ affinity of isolated cTnC. The Ca2+ affinity of cTnC is known to be considerably enhanced when cTnC is complexed with cTnI or reconstituted into cTn. The proposed kinetic scheme is consistent with these previous equilibrium results, and the present kinetic results show that the positive free energy coupling of Ca2+ binding to cTnC by cTnI is related to alterations of the rate constants of the elementary kinetic steps of Ca2+ binding.
The single-exponential IAANS transient in Ca2+ dissociation
kinetics previously observed with isolated cTnC likely arises from the
dominant equilibrium species, (Ca-cTnC)*, and this transient largely
reflects k1 (11). This is likely the case
because step 2 is thermodynamically unfavorable for cTnC. In contrast, in reconstituted troponin, steps 1 and 2 are both thermodynamically favorable with comparable equilibrium constants. The IAANS fluorescence transient observed in dissociation kinetics is expected to arise from
both Ca2+-bound species, (Ca-cTn)* and (Ca-cTn)**. Thus,
the biphasic dissociation kinetics with cTn likely reflects
dissociation from both species. The fast phase (
d1 = 20.6 s
1) may be related to dissociation from (Ca-cTn)*.
The dissociation rate constant for (Ca-cTn)** is predicted to be
k
1k
2/(k
1 + k2 + k
2) (18), which
is < 2 s
1. This predicted value is considerably
smaller than the k
1 value (13.3 s
1) determined from Fig. 4B, and the large
separation makes resolution of the two kinetic phases possible.
We previously suggested that two first-order fluorescence transitions observed in the kinetics of Ca2+ binding to cTnC were related to reorientations of helices B and C in the regulatory domain (11). This interpretation was based on the modeled structure of the 4Ca2+ state of sTnC (19) and cTnC (20). These helix reorientations are believed to be the basis for Ca2+-induced interactions between troponin C and troponin I as part of the Ca2+ activation mechanism. Helix movements within the regulatory domain are expected to be modified when troponin C is bound to the other troponin subunits. Qualitatively, these movements within cTn appear unmodified because the same kinetic model is adequate to describe the binding of Ca2+ to both cTnC and cTn. The rates of the first-order transitions, however, are more than 10-fold reduced. Our previous spectroscopic study suggested that these movements may be less extensive in cTn than as suggested by the 4Ca2+ model of isolated troponin C (6). At a saturating level of Ca2+ and 4 °C, the binding transitions in cTn would take about 40 ms to reach a 95% completion, and the dissociation would take about 50 ms. At elevated temperatures, these events could be faster. These time constants are likely more compatible with physiologic events than those previously determined with cTnC.
Having elucidated the kinetic mechanism of Ca2+ binding to cTn, we then examined the effect of phosphorylation of cTnI by PKA on the various steps of the mechanism and the contributions of the kinetic steps to the equilibrium Ca2+ binding constant. The phosphorylation results in a 2-3-fold increase of the bimolecular binding rate constant and the two first-order rate constants associated with step 1. The rate constants of step 2 are increased by a factor of 7-8. The net effect of these changes is a substantial decrease in Ko and reduction of the overall equilibrium binding constant. The latter result is consistent with a similar decrease in the equilibrium constant for formation of the cTnC-cTnI complex resulting from phosphorylation of cTnI (21). It is also in agreement with previous reports of decreased Ca2+ affinity of cTnC upon complex formation with phosphorylated cTnI (13). The present results also corroborate the previous report that cTnI phosphorylation results in an increase in the observed rate of Ca2+ removal from cTn. This phosphorylation effect, however, is not limited to the rate of Ca2+ dissociation; it also reduces the bimolecular Ca2+ binding rate. These kinetic effects may be related to a new conformation of cTnI. The hydrodynamic shape of cTnI is less asymmetric in the phosphorylated state than in the nonphophorylated state, as suggested by a smaller rotational correlation time of the phosphorylated protein (22). Consistent with this finding, phosphorylation of a mutant cTnI by PKA has recently been shown to shorten the mean distance between Trp-192 and Cys-5 by 8-9 Å, which is accompanied by a considerable narrowing of the distribution of the distances between the two sites (23). Similar phosphorylation-induced decreases in the distance parameters of cTnI also occur in the cTnC-cTnI complex in the presence of Mg2+ and Ca2+. This substantial decrease in distance likely arises from a folding of the N-terminal extension of cTnI that contains the two unique PKA target sites (Ser-23 and Ser-24). The functional role of PKA phosphorylation of cTnI has been amply demonstrated (24). Since this phosphorylation is thought to mediate the dynamics of Ca2+ binding to the regulatory site, it is important to delineate the kinetic mechanism by which the dynamics of Ca2 binding is modulated. The present study provides this information. What is unresolved at this time is whether the folded N-terminal segment is sufficient to bring about the observed changes in both the binding and dissociation kinetics.
In conclusion, we have used the fluorescence of an extrinsic probe linked to Cys-35 of a monocysteine TnC mutant to study the kinetics of Ca2+ binding to cardiac muscle troponin complexes. The kinetics is consistent with a three-step sequential mechanism. Phosphorylation of cTnI by PKA alters both the binding and dissociation rates but not the overall kinetic mechanism.