(Received for publication, June 12, 1995; and in revised form, September 27, 1995)
From the
We have studied the kinetics of the structural transitions
induced by calcium binding to the single, regulatory site of cardiac
troponin C by measuring the rates of calcium-mediated fluorescence
changes with a monocysteine mutant of the protein (C35S) specifically
labeled at Cys-84 with the fluorescent probe
2-[4`-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At
4 °C, the binding kinetics determined in the presence of
Mg was resolved into two phases with positive
amplitude, which were completed in less than 100 ms. The rate of the
fast phase increased linearly with [Ca
]
reaching a maximum of
590 s
, and that of the
slow phase was approximately 100 s
and did not
depend on Ca
concentration. Dissociation of bound
Ca
from the regulatory site occurred with a rate of
102 s
, whereas the dissociation from the two high
affinity sites was about two orders of magnitude slower. These results
are consistent with the following scheme for the binding of
Ca
to the regulatory site:
where the asterisks denote
states with enhanced fluorescence. The apparent second-order rate
constant for calcium binding is Kk
= 1.4
10
M
s
. The two first-order transitions occur with
observed rates of k
+ k
590 s
and k
+ k
100 s
, and the
binding of Ca
to the regulatory site is not a simple
diffusion-controlled reaction. These transitions provide the first
information on the rates of Ca
-induced conformational
changes involving helix movements in the regulatory domain.
Troponin C (TnC) ()is the calcium-binding subunit of
the three-subunit troponin complex, which, together with tropomyosin,
constitutes the regulatory system in vertebrate skeletal and cardiac
muscle. TnC from skeletal muscle has two classes of calcium-binding
sites. The two carboxyl-terminal sites (sites 3 and 4) bind calcium
with a high affinity (K
2
10
M
) and also bind magnesium
competitively with a lower affinity (K
5
10
M
) (Leavis et
al., 1978). These two sites appear to have a structural role and
do not contribute to calcium-dependent regulation (Zot and Potter,
1987). The two amino-terminal sites (sites 1 and 2) are
calcium-specific, bind calcium with a low affinity (K
3.2
10
M
)
(Potter and Gergely, 1975; Leavis et al., 1978), and are the
sites that regulate contraction. TnC from cardiac muscle differs from
skeletal muscle TnC most significantly in the amino-terminal half,
where several critical amino acid substitutions have rendered site 1
incapable of binding calcium (Van Eerd and Takahashi, 1976; Leavis and
Kraft, 1978). The calcium affinities of the two classes of binding
sites in cTnC (Holroyde et al., 1980) are essentially the same
as the corresponding ones for skeletal muscle TnC.
The x-ray
crystallographic structures of TnC from chicken (Sundaralingam et
al., 1985) and turkey skeletal muscle (Herzberg and James, 1985)
show that the protein has an elongated, dumbbell-like shape in which
the polypeptide is folded into two globular domains at the amino- and
carboxyl-terminal ends. These domains are connected by a long
-helix, the middle of which is exposed to the solvent. The crystal
structure provides a structural basis for understanding the potential
conformational changes that may occur in the regulatory, amino-terminal
domain of TnC when calcium binds to sites 1 and 2 (Herzberg et
al., 1986; Strynadk and James, 1989). These putative
conformational changes may involve movements of helices B and C
relative to helices A and D. It has been suggested that these movements
expose a segment of hydrophobic residues in the amino-terminal domain,
which then become available for calcium-mediated interaction with TnI.
Fluorescence (Wang and Cheung, 1986; Tao et al., 1990) and
thermodynamic (Wang and Cheung, 1985; Cheung et al., 1987)
studies have suggested that the interaction between TnC and TnI may
serve as the Ca
switch for calcium-dependent
regulation of contraction.
Maximum tension in fast skeletal muscle
is observed in 10-13 ms after excitation and decays after an
additional 40-50 ms (Close, 1965). During a cycle of contraction
and relaxation, calcium must bind to the regulatory sites and induce
structural changes in both TnC and the other thin filament regulatory
proteins within 10-13 ms after excitation. Likewise, calcium
dissociation from the regulatory sites and reversal of the
calcium-induced conformational changes must occur within 40-50 ms
after excitation. These physiologic constraints place limits within
which reversible Ca binding to TnC and subsequent
conformational changes must occur. Several groups (Iio and Kondo, 1982;
Rosenfeld and Taylor, 1985a; Johnson et al., 1994) reported
the kinetics of calcium binding to both classes of sites in isolated
skeletal TnC and in regulatory complexes containing TnC. Calcium
binding to the low affinity sites of skeletal TnC has been reported to
be diffusion controlled (Johnson et al., 1994). However, given
the fact that most protein conformational changes occur more slowly
than this, it would be expected that the kinetics of calcium binding to
the regulatory sites should be saturable. Little information is
available on the kinetic mechanism of calcium binding to the regulatory
site of cardiac TnC, as most of the reported information is confined to
the dissociation kinetics (Robertson et al., 1981, 1982).
Cardiac TnC has two cysteine residues that can be readily alkylated by a variety of sulfhydryl-specific reagents. Mutants containing a single cysteine at either position 35 (C84S) or 84 (C35S) have been generated (Zhang et al., 1992). The calcium-induced fluorescence change of IAANS-labeled mutant cTnC(C35S) has been shown to be coincident with calcium-activated force development (Zhang et al., 1992; Kerrick et al., 1992) and ATPase activity (Kerrick et al., 1992) in skinned cardiac muscle fibers. An advantage of using IAANS-labeled mutant cTnC(C35S) to study calcium-induced structural changes is that the fluorophore is located at a single site, which simplifies interpretation of the observed fluorescence change in terms of structural transitions.
In the present work, we have studied the kinetics of calcium binding to the single regulatory site of cTnC using a monocysteine mutant labeled with IAANS at Cys-84. The observed kinetics suggest a three-step binding model. Because the IAANS probe is strategically located, the observed kinetics of the fluorescence change also provide insight into the time course of structural changes induced by calcium binding.
cTnC mutants were tested for their ability to support Ca regulation of force development using chemically skinned rabbit
skeletal muscle fibers. Briefly, endogenous TnC was extracted from the
fibers, and the TnC-depleted fibers were reconstituted with an
exogenous TnC to be tested. Force was measured on the reconstituted
fibers as a function of free Ca
concentration as
described previously (Martyn and Gordon, 1988).
In a typical binding experiment, one syringe
contained labeled protein in 30 mM Mops at pH 7.0, 200 mM KCl, 3.0 mM MgCl, and 5.0 µM EGTA. The other syringe contained the same solution but without
protein, plus different levels of total Ca
. After
mixing, the concentration of labeled protein was usually 1
µM, and the total concentration of Ca
was in the range of 0-500 µM. These conditions
ensured that the two carboxyl-terminal high affinity sites were
presaturated by Mg
, and the single low affinity
calcium-specific site remained calcium free prior to mixing. The free
Ca
level was controlled by the chelator. The control
was quite adequate when the total Ca
concentration
was low. This is important because the rate data obtained at low levels
of free Ca
were used to calculate the bimolecular
binding rate constant. At higher total Ca
concentrations, there was some uncertainty in the calculated free
Ca
levels. As shown in Fig. 4A, the
observed fast rate constant approached a limiting value at large free
Ca
concentration. Since it was this limiting rate
constant that was important in our kinetic model, the uncertainty in
free Ca
concentration did not affect the analysis.
For each experimental condition, 8-10 tracings were obtained. The
tracings were averaged, and the resultant tracing was fitted to a sum
of exponentials by a nonlinear least squares method (Bevington, 1969).
To measure dissociation of bound cations, a buffer containing either
EGTA or EDTA was mixed with an equal volume of protein saturated with
bound cations either at sites 3 and 4 or at all three sites.
Figure 4:
Plot
of the two observed rate constants with positive amplitudes obtained
from Ca binding experiments in the presence of
Mg
versus free
[Ca
]. The rate constants were determined at
4 °C from fluorescence transients illustrated in Fig. 3. Upper panel, the fast rate constants
are
represented by filled circles, and the slow rate constants
are represented by open circles. The solid line was obtained by fitting the data to ,
and the recovered parameters are listed in Table 1. The slow rate
constant (
) is not sensitive to
[Ca
]. Lower panel shows a plot of
at very low [Ca
] on
expanded scales. The slope of this line is 1.4
10
M
s
.
Figure 3:
A typical kinetic tracing fitted to a
one-exponential and a two-exponential function. The tracing was
obtained as described in Fig. 2. After mixing,
[protein] = 1 µM and free
[Ca] = 6.3 µM. Panel A shows the best two-exponential fit (solid
curve) with two rate constants,
= 517
s
, and
= 127
s
;
= 1.07. Panel B is the residual plot of the two-exponential fit. Panel C shows the residual plot for the best one-exponential
fit with a rate constant of 156 s
(the best fitted
curve not shown);
= 1.51. Note
the differences between the two residual plots, particularly at early
times.
Figure 2:
Stopped-flow kinetic tracings obtained at
4 °C by mixing IAANS-labeled mutant C35S in 0.2 M KCl, 3
mM Mg, 30 mM Mops at pH 7.0, and 5
µM EGTA with an equal volume of the same buffer containing
increasing concentration of free Ca
. Mutant
concentration after mixing was 1.0 µM. Each tracing shown
in the figure was obtained from signal averaging of 8-10 separate
tracings over the time interval 0-0.08 s. The full tracings from
0 to 10 s showed a second slow phase with a negative amplitude
(decreasing fluorescence), which did not start until about 1 s after
mixing. This slow phase is not shown here, as it was not related to
Ca
binding to the regulatory site. The transients
with large positive amplitudes are attributed to Ca
binding to the regulatory site.
Figure 1:
Fluorescence emission
spectra are shown of mutant C35S labeled with IAANS at Cys-84 in the
presence of Mg or different concentrations of free
Ca
at 20 °C, 5 µM protein in 2.0
mM EGTA, 0.2 M KCl, and 30 mM Mops at pH
7.0. Inset, Ca
titration of the labeled
mutant carried out in the same buffer;
= 325
nm, and
= 450 nm. The experimental points (filled circles) were analyzed by a nonlinear least squares
procedure using the following equation:
where F and F are the intensities determined at a given
[Ca
] and at
[Ca
] = 0, respectively; F
, K
, and n
are the intensity change, the binding constant,
and the Hill coefficient of the ith site, respectively. The
data were adequately fitted with one term of the equation, yielding a
single binding constant corresponding to pK
= 5.78 and n =
1.2.
Two observed rate constants were obtained above 2
µM calcium concentration. The fast rate constant,
, increased with increasing Ca
concentration, while the slow rate constant (
)
appeared to be insensitive to Ca
concentration. This
is illustrated in Fig. 4A. The amplitude of the fast
phase varied from about 60 to 30% of the total signal above 2
µM Ca
. The ratio of the amplitude of the
slow phase to that of the fast phase increased from less than 0.5 to a
limiting value of about 2.5 with increasing
[Ca
], and the shape of this plot of
amplitude ratio (not shown) resembled that shown in Fig. 4A for
. The recovered limiting value of
was close to the dead time of the instrument, and some signal of
the fast phase (about 15%) was lost at higher
[Ca
]. This loss was reflected in the larger
uncertainty for
as indicated in Fig. 4A but in no way limited the analysis. The data of
versus [Ca
] were fitted to with a maximum rate of 590 s
. The
initial slope of this plot (Fig. 4B) defines an
apparent second-order rate constant of 1.4
10
M
s
for calcium binding
to the low affinity site.
Figure 5:
Kinetics of Ca
dissociation from the amino-terminal domain site 2 of mutant C35S
labeled with IAANS at Cys-84. Labeled mutant in 0.2 M KCl, 30
mM Mops at pH 7.0, 3 mM Mg
, and 0.5
mM was mixed with an equal volume of 0.2 M KCl, 30
mM Mops at pH 7.0, and 10 mM EGTA at 4 and 20 °C.
Each tracing shown is from signal averaging of 8-10 separate
tracings, and the resultant tracings shown were fitted to a single
exponential function. At 4 °C, rate constant = 102
s
,
= 1.11; at
20 °C, rate constant = 296 s
, and
=
1.08.
Figure 6:
Kinetics of dissociation of bound
Ca and bound Mg
from
carboxyl-terminal domain sites 3 and 4 at 4 °C. Lower
tracing, a solution of
1 µM labeled protein in
0.2 M KCl, 30 mM Mops at pH 7.0, 2 mM
Mg
, and 20 µM EGTA was mixed with a
solution of 0.2 M KCl, 30 mM Mops at pH 7.0, 20
µM EGTA, and 20 mM EDTA. 20 µM EGTA
was present in both solutions to remove residual Ca
.
The trace was adequately fitted with a one-exponential function, with
= 0.83 s
,
= 1.27. Upper tracing, a
solution of
1 µM labeled protein in 0.2 M KCl, 30 mM Mops at pH 7.0, 5 µM EGTA, and pCa 7.2 was mixed with a solution of 0.2 M KCl, 30
mM Mops at pH 7.0, and 10 mM EGTA. The free
[Ca
] in the solution was sufficient to
saturate the two high affinity sites with the regulatory site
unoccupied. This tracing monitored dissociation of bound Ca
at the high affinity sites and was adequately fitted with a
single exponential function,
= 0.73
s
,
= 1.12. The
final levels of fluorescence were not the same because the protein
concentrations were slightly different in the two
samples.
Figure 7:
Arrhenius plots of the observed rate
constants obtained from experiments of Ca binding to
the regulatory sites of IAANS-labeled mutant C35S as described in Fig. 2. The rate constants were obtained at
[Ca
] = 300 µM. The
activation energy for the fast phase was 7.2 ± 0.4 kJ/mol and
for the slow phase was 26 ± 3 kJ/mol. The coefficient of
correlation was 0.99 for both lines.
In this paper, we have studied the kinetics of the binding of
Ca to the single regulatory site of cardiac TnC.
Native cTnC labeled with IAANS at both Cys-35 and Cys-84 has been
extensively used to investigate the equilibrium properties of
Ca
binding to the single regulatory site of the
isolated protein and of the protein reconstituted into myofibrils and
skinned muscle fibers. Because the label is located at two different
sites that are far separated in the x-ray structure of TnC, the
question arises as to whether the labels at both sites sense the same
events. Using bacterially produced mutants containing a single cysteine
residue, recent studies have shown that the fluorescence of IAANS
attached to Cys-84 in mutant C35S tracks Ca
binding
to the single regulatory site and produces Ca
titration curves that superimpose on those for the Ca
activation of ATPase and force development in skinned muscle
fibers (Zhang et al., 1992). As will be further elaborated
below, Cys-84 is strategically located in the amino-terminal domain,
and a probe linked to this position might sense
Ca
-activated conformational changes involving
specific helices.
To focus on the kinetics of Ca binding to the regulatory site, the binding kinetics were studied
in the presence of a large excess of Mg
so that the
two carboxyl-terminal sites were occupied throughout the binding
reaction. Since the dissociation of bound Mg
is at
least two orders of magnitude slower than the binding of Ca
to the amino-terminal domain, this protocol enables us to follow
the time course of the binding reaction directly. The kinetic
transients for calcium binding to the low affinity site were
biexponential at high Ca
concentration. The rate
constant of the fast phase (
) varies with increasing
[Ca
] in a hyperbolic manner, whereas
is insensitive to [Ca
].
We propose a three-step mechanism to account for these kinetic data:
The initial binding in the bimolecular step is assumed to be a
rapid equilibrium, which is characterized by K,
followed by two sequential first-order transitions in which
fluorescence enhancement occurs. In this scheme, if k
+ k
k
+ k
, the normalized fluorescence
transient can be described by two exponential terms (Bernasconi, 1976).
The rate constants from the fluorescence transient are identified with
and
. The relationships of the
observed rate constants to the kinetic parameters in are
as follows:
The initial slope of a plot of versus [Ca
] (Fig. 4) yields an
apparent second order association rate constant K
k
of 1.4
10
M
s
.
Extrapolation to [Ca
] = 0 yields k
= 117 s
. At
large [Ca
],
= k
+ k
=
587 s
. Thus, k
= 470
s
, k
= 117
s
, and K
= 2.95
10
M
at 4 °C. The sum of k
and k
is
approximately 100 s
. While their individual values
cannot be directly extracted from the present kinetic data, they can be
estimated from the experimental value of the equilibrium constant K
= 3.2
10
M
obtained from the Ca
titration at the same temperature: K
= K
(k
/k
)(k
/k
).
This yields k
= 20 s
and k
= 80 s
. These
parameters are listed in Table 1. Alternately, can
be treated as described previously (Benson, 1960), and similar
conclusions are obtained. The present model is the first kinetic
mechanism for cardiac TnC using any preparation of the protein.
The
kinetic data indicate that step 1 is thermodynamically favorable with
an equilibrium constant of about 4, but step 2 is unfavorable. This is
consistent with a 3.5-fold higher activation for the slow phase.
Equilibrium studies have shown that the K for
calcium binding to the regulatory site of a TnC
TnI complex is
considerably larger than that for isolated TnC. If the same kinetic
scheme holds for the complex, this enhanced affinity must arise from
increases in the equilibrium constants of one or all steps of the
model. Since step 2 is unfavorable with isolated TnC, this step may
become more favorable in the binary protein complex. This possibility
can be tested with either the cTnC
cTnI complex or reconstituted
troponin.
The dissociation kinetics of Ca from the
regulatory site are monoexponential at both 4 and 20 °C. Step 1 is
thermodynamically favorable, but step 2 is unfavorable with an
equilibrium constant of 0.25. The observed initial fluorescence change
in dissociation kinetics is expected to arise from the dominant
species, (CaTnC)*, with only a small contribution from (CaTnC). Thus,
the observed dissociation rate constant of 102 s
largely reflects k
. Furthermore, the
dissociation rate constant for the (CaTnC) state is predicted to be k
k
/(k
+ k
+ k
),
which is approximately 50-60 s
(Trybus and
Taylor, 1982). It would be unlikely that a small amplitude phase in the
dissociation transient occurring at 50-60 s
could be resolved from the main phase, occurring at 117 s
determined from the intercept in Fig. 4B.
The
x-ray structure of skeletal TnC shows that in the amino-terminal domain
Cys-84 is located on helix D and is surrounded by helices B and C. The
calcium-saturated structural model of the amino-terminal domain
suggests that Ca binding to the two amino-terminal
domain sites induces movements of helices B and C relative to helices A
and D, such that two EF hands are formed. These movements result in a
more ``open'' conformation of the amino-terminal domain
(Strynadk and James, 1989) and expose a hydrophobic patch on helix B.
This exposed patch may be a site for calcium-mediated interactions with
troponin I. In this model, helix D in the 4Ca state is stationary
relative to the 2Ca state. A recent study of cTnC suggests similar
helix movements in the amino-terminal domain when the single regulatory
site is occupied (Ovsak and Taskinen, 1991). These models suggest that
the observed Ca
-mediated fluorescence enhancement of
IAANS attached to Cys-84 on helix D may result from movements of
helices B and C away from helix D. A recent study (Dong and Cheung,
1995) with native cTnC, which was selectively modified at Cys-84 with
fluorescent probes, showed that the Ca
-mediated
enhancement of probe fluorescence was related to strong internal
quenching by interactions of a dipolar nature in the apo and 2Mg
states. This quenching is reduced or eliminated upon reorientation of
helices B and C from helix D. This lends further support to the
interpretation that the two first-order transitions that lead to
fluorescence enhancement of the IAANS probe may reflect these helix
movements. It is not known whether the movements actually occur in two
steps, but it is unlikely that they move independently of each other
because only the first fluorescence transition is Ca
dependent. At a saturating level of Ca
, these
transitions would take about 5 ms to reach a 95% completion. This rate
and the observed dissociation rate are likely overestimates of the
rates that would occur in muscle. It is well known that both the
time-to-peak-tension after excitation and the relaxation time can vary
widely with temperature, and they are species-dependent. These time
windows are generally longer in cardiac muscle than skeletal muscle,
but structural transitions occurring in 5 ms in isolated cTnC may be
too fast to be compatible with physiologic events. However, the
Ca
on- and off-rates in reconstituted troponin are
expected to be slower, as the Ca
dissociation rate
from the regulatory site in the cTnC
cTnI complex
is only 21 s
at 20 °C (Robertson et
al., 1982), a factor of 10 slower than with cTnC determined in the
present work. The on-rate in reconstituted troponin or in the regulated
thin filament can also be expected to be significantly slower
(Robertson et al., 1981). If tension transients follow
Ca
transients, this anticipated reduction in the
rates of reversible Ca
binding should still be fast
enough to support contraction.
In contrast to cardiac TnC, several
studies on the kinetics of Ca binding to skeletal TnC
have been reported. The early studies of Iio and Kondo (1980a, 1980b,
1982), based on tyrosine fluorescence, proposed a rapid
diffusion-controlled binding step followed by conformational changes.
Rosenfeld and Taylor (1985a, 1985b) investigated the binding of
Ca
to both classes of sites with isolated skeletal
TnC and reconstituted troponin, using a fluorescently labeled
preparation. A two-step binding mechanism with a second-order rate
constant of 1.3
10
M
s
was proposed for Ca
binding to
the high affinity sites. This value is about 100 times slower than that
for Ca
binding to the low affinity site in cTnC
determined in the present work. Rosenfeld and Taylor (1985a, 1985b)
also reported very fast Ca
binding rates to the
regulatory sites at low calcium concentration, and the rates at high
[Ca
] became too large to be measured. It
could not be determined whether the rate reached a maximum, and the
binding data did not establish whether the binding kinetics could be
described by a two-step or a three-step model. However, the rates
increased linearly with increasing [Ca
] at
low concentration, and an apparent second-order binding rate constant
of 5
10
M
s
was obtained. This is at least one order of
magnitude larger than the corresponding rate at the high affinity sites
determined using the same protein preparation. More recently, Johnson et al.(1994) reported the kinetics of Ca
binding to the two regulatory sites with a tryptophan mutant of
skeletal TnC(F29W). They observed monoexponential kinetic traces over a
narrow range of [Ca
] (0-6
µM) and assumed a simple one-step, diffusion-controlled
mechanism for the binding of Ca
to the two regulatory
sites. Since no data were reported at higher Ca
concentrations, it is not known whether the observed rate would
have saturated at high [Ca
]. Their apparent
second-order binding rate constant is in the range of 1
10
M
s
to 2
10
M
s
, essentially the same as that reported here for
cardiac TnC (1.4
10
M
s
). It appears that the Ca
binding
rate at the regulatory sites is one to two orders of magnitude faster
than that at the high affinity sites, regardless of isoform. The
previous interpretation of calcium binding to the regulatory sites as a
simple diffusion-controlled process (Johnson et al., 1989;
1994), however, is not consistent with the present evidence. The
reporter group of the skeletal TnC mutant is Trp-29 located on helix A
immediately adjacent to the amino-terminal end of the
Ca
-binding loop 1, whereas in the present work the
reporter group is located on helix D. The different locations of the
two probes may be a reason why first- order transitions arising from
activator Ca
-mediated movements of helices B and C
were not sensed with mutant F29W.
In summary, we have used the
fluorescence of an extrinsic probe linked to the single cysteine
residue on helix D of a monocysteine mutant to investigate the kinetics
of Ca binding to cardiac TnC. The dissociation of
Ca
from the regulatory site is two orders of
magnitude faster than the dissociation of Ca
or
Mg
from the two carboxyl-terminal domain sites. The
kinetics of Ca
binding to the regulatory sites is
consistent with a three-step mechanism in which the bimolecular binding
step is in rapid equilibrium and is followed by two sequential
first-order transitions.