(Received for publication, December 13, 1995; and in revised form, March 12, 1996)
From the
The kinetics of reaction of myosin subfragment-1 (S)
with F-actin have been monitored by the changes in light scattering and
in pyrenyl-actin fluorescence at 20 °C, pH 7.5, and physiological
ionic strength. The association rate constant of S
to
F-actin decreases about 10-fold as the molar ratio of bound S
increases from 0 to 1. This decrease in k
is most likely due to the steric hindrance of available binding
sites by initially bound S
. The apparent rate constant for
association of S
to bare filaments is 9
µM
s
, a value 1
order of magnitude higher than the one previously estimated from
experiments in which S
was in excess over F-actin. The
anticooperative binding kinetics of S
to F-actin are
consistent with the negative cooperativity displayed in the equilibrium
binding curves of S
to pyrenyl-F-actin.
Fluorescence
titration curves of partially labeled pyrenyl-F-actin by S are sigmoidal, consistent with a 4-fold higher affinity of
S
for unlabeled than for labeled actin. This conclusion is
strengthened by kinetic data of S
binding to partially
labeled F-actin, which exhibit a biphasic behavior due to the slower
dissociation of S
from unlabeled than from labeled actin.
The interaction of the myosin head (myosin subfragment-1,
S) (
)with the actin filament (F-actin) is
central to the ATP-driven vectorial movement of myosin along the
filament and the resulting production of force by muscle. The mechanism
of interaction of S
with F-actin, the nature of the
different complexes formed with or without nucleotide bound to S
and their relative stabilities have been analyzed by a variety of
kinetic and equilibrium methods. In the absence of ATP or ADP, S
forms a high affinity ``rigor'' complex (K = 10
M
) with
F-actin, with a maximal binding stoichiometry of one S
per
F-actin subunit(1, 2, 3, 4) .
Formation of the F-actin-S
rigor complex can be monitored
by the associated increase in light scattering or by the 80% quenching
of fluorescence of the pyrenyl probe covalently bound to Cys-374 of
actin. The kinetic analysis of increase in light scattering (5, 7) or of decrease in pyrenyl fluorescence (6, 7, 8, 9, 10, 11) provided
a description of the mechanism of actin-S
complex formation
in terms of 2 consecutive reactions, a rapid bimolecular reaction
followed by an isomerization process as follows, where S and A
represent myosin subfragment 1 and the actin subunit in the
filament.
The kinetic data, however, were sometimes more complex than
expected within , and simplifying assumptions were often
made in the interpretation of the data, or conditions were chosen under
which the experimental complexity appeared lower, allowing a
satisfactory description of the data by . For instance,
biphasic fluorescence changes (8) or deviations from simple
exponentials (11) have been reported under conditions where a
pseudo-first order process was expected, which could not be explained
by S microheterogeneity; agreement has not been reached on
the values found for the rate constants involved in ,
under similar ionic conditions(7, 8, 10) ;
non-linearity has been observed in binding measurements of heavy
meromyosin to F-actin(12) . Other experiments showed that
functional parameters, e.g. the K
of ATP in the actomyosin ATPase (13) and the
protection afforded by actin against proteolytic degradation of S
(14, 15) were dependent on the saturation of the
filament by S
. It was proposed recently (11) that
the existence of two rigor complexes in which S
would
interact either with one or with two adjacent F-actin subunits in the
filament would account for many of the aforementioned deviations from . This model uses the facts that the myosin head can form
a ternary complex with two G-actin molecules in a low ionic strength
buffer where actin alone remains monomeric (16, 17, 18) and that the reconstructions of
the actin-myosin interface (19) using the crystallized
structures of G-actin (20) and of S
(21) and the atomic model of the filament (22) indicate that S
can make rigor contacts with
two actin subunits interacting with each other via longitudinal bonds
along the long pitch helix.
In the present work, we have used light
scattering and pyrenyl fluorescence to monitor the kinetics of
interaction of S with F-actin in rigor. Experiments have
been performed at different actin/S
molar ratios, and both
light scattering and fluorescence kinetic data have been analyzed in a
comprehensive fashion. We find that the rate constant for association
of S
to F-actin depends on the extent of saturation of the
filament by S
. Our results also show evidence for a
5-fold lower affinity of S
for pyrenyl-labeled actin
than for unlabeled actin.
Proteins were kept on ice at a concentration of 50-80 µM and used within 2 weeks following purification.
Figure 5:
Analysis of the change in pyrenyl
fluorescence upon reaction of fully labeled pyrenyl-F-actin with an
excess of S. Typical time courses of the decrease in
pyrenyl fluorescence upon mixing 2 µM pyrenyl-F-actin with
6 µM S
(A
) or 1 µM pyrenyl-F-actin with 3, 4, or 5 µM S
(A
) (top to bottom).
Noisy curves are experimental traces (average of a minimum of four
shots). Smooth curves are calculated time courses using and .
In all
kinetic measurements, four to six consecutive shots were performed and
the traces were averaged before being analyzed. Kinetic data were
routinely analyzed within first order processes, when appropriate,
using the software attached to the instrument. When the data deviated
from the exponential behavior, a model was proposed based on the
qualitative examination of the trend shown by the data upon changing
concentrations of either actin or S, and the kinetic curves
were analyzed within the equation describing the new model. In the
absence of analytical expression of the time dependence of the
intensity of scattered light or of fluorescence, simulation of the
kinetic curves was carried out using the HOPKINSIM simulation program,
and appropriate values of the rate constants were adjusted by hand to
obtain a satisfactory superimposition of a large number of experimental
curves obtained at series of concentrations of actin and S
onto the corresponding theoretical kinetic curves.
Figure 1:
Light scattering recording of
the formation of F-actin-myosin subfragment-1 complex. Curve
a, F-actin (6 µM) was mixed with
S(A
) (2 µM). Curve b,
F-actin (2 µM) was mixed with S
(A
)
(6 µM). Experimental conditions were F
buffer
(physiological ionic conditions), 20 °C. Noisy curves are
experimental traces (average of a minimum four consecutive shots).
Smooth lines represent the monoexponential best fits (k
= 38 s
in a, 15 s
in b). Boxes represent the residuals for curves a and b as
indicated.
Figure 2:
F-actin concentration dependence of the
observed first order rate constant for the interaction of F-actin with
S. F-actin at the indicated concentrations was reacted with
1 µM S
(A
) and the first order rate
constant of the observed increase in light scattering was plotted versus the concentration of F-actin. Closed symbols (
,
) represent two independent experiments. The same experiment was
carried out under the same conditions except that fully labeled
pyrenyl-F-actin was used and the decrease in pyrenyl fluorescence was
monitored (
).
The large difference between the two time courses shown in Fig. 1arises from the two following points.
First, the increase in intensity of light scattered at 90°, R, is not a linear function of x, as
described by the following equation(28, 29) .
K is a constant proportional to the square of the
refractive index increment, P is the shape factor
of a filament containing on average x molecules of S
bound/F-actin subunit, M
and M
are the molecular masses of actin (42 kDa) and
S
(130 kDa), respectively, and F
is
the total concentration of F-actin subunits. Because the intensity of
scattered light increases cooperatively with x, as is apparent
in titration curves (5) , the final increase in light
scattering when F-actin is in excess over S
(curve a in Fig. 1) is lower than when S
is in excess
over F-actin (curve b in Fig. 1). When F-actin is in
excess over S
, x remains small (0
x
0.2) during 80% of the whole reaction and R
can be assumed to be proportional to x, but this
assumption is no longer valid when S
is in excess over
F-actin.
Second, comparison of curves a and b indicates that the rate of association of S to F-actin
declines as the density of bound S
increases. Hence, the
rate constant derived from experiments in which F-actin is in excess
over S
(Fig. 2) refers to the association rate
constant k
of S
to undecorated
or poorly decorated filaments (x < 0.2). To analyze the
kinetic curves obtained when F-actin was reacted with an excess of
S
, we took into account the non-linearity of R
with x, and we tentatively assumed a
simple linear decrease of k
from
k
to k
as x increases from 0 to 1, as follows.
Assuming the binding of S to F-actin to be
essentially irreversible, and S
to be in excess over
F-actin,
where [S] represents the total
concentration of S
, which leads to .
The time dependence of x described by was
combined with to derive the time dependence of the
intensity of scattered light upon binding of S to F-actin
under conditions where x varies from 0 to 1 during the
reaction.
Fig. 3shows the good fit of the simulated time
courses to experimental traces at three sets of F-actin and S concentrations. The value of k
was
imposed equal to 9 µM
s
as derived from Fig. 2; the fit was good for all curves
using the same value of 0.5 ± 0.5 µM
s
for k
. Values
of k
higher than 1
µM
s
did not fit the
data. Hence, the results are consistent with the view that, as the
actin filament becomes increasingly saturated by S
, steric
hindrance slows down further binding of S
to available
sites.
Figure 3:
Analysis of the increase in light
scattering upon reacting F-actin with an excess of S.
Typical time courses of the change in light scattering upon mixing: a, 0.5 µM F-actin with 3 µM S
(A
); b, 1 µM F-actin with 3 µM S
(A
); c, 2 µM F-actin with 6 µM S
(A
). Noisy curves are experimental
traces. Smooth lines are simulated time courses generated using .
The changes
in fluorescence recorded upon adding 5 µM S(A
) to 1 µM pyrenyl-F-actin,
or 5 µM pyrenyl-F-actin to 1 µM S
(A
) are shown in Fig. 4. Again the
two time courses corresponding to the formation of 1 µM pyrenyl-F-actin-S
were not superimposable. In
agreement with the light scattering results described above, the
reaction was slower when S
was in excess over F-actin, and
the time courses could be well analyzed within a first order process
only when F-actin was in excess over S
. The pseudo first
order rate constant then varied linearly with pyrenyl-F-actin
concentration and the data points superimposed with those derived from
light scattering kinetics (Fig. 2), indicating that the same
rate-limiting process was monitored by either light scattering or
pyrenyl fluorescence. In order to appreciate whether the length of the
filaments affected the kinetics of interaction with S
,
gelsolin was added to F-actin in a proportion varying between 1:500 to
1:50. The kinetics were unaffected by the length of the filaments in
this range.
Figure 4:
Kinetics of decrease in fluorescence of
fully labeled pyrenyl-F-actin upon binding S. Curve
a, 5 µM pyrenyl-F-actin was reacted with 1 µM S
(A
). Curve b, 1 µM pyrenyl-F-actin was reacted with 5 µM S
(A
). Experimental traces (noisy curves)
have been translated for the intensities at time zero to coincide. The
fluorescence of 1 µM F-actin has been normalized to 1, to
show the 85% quenching that occurs upon binding S
. Smooth
curves represent the mono-exponential best fit (k
= 32 s
in a, 20 s
in b). Residuals showing the strong deviation from a
single exponential in curve b are shown in boxes as
indicated.
When S was in excess over pyrenyl-F-actin,
the data were tentatively analyzed using and expressing
that the observed fluorescence is the weighted sum of the fluorescences
of F-actin and F-actin-S
complex, as follows, where
[F
] is the total concentration of
F-actin, and f
and f
the
intrinsic fluorescences of F-actin (taken as 1
µM
by convention) and of
F-actin-S
, respectively.
Fluorescence titration curves of 100% labeled pyrenyl-F-actin by
S showed that the fluorescence was 85% quenched at
saturation by S
(f
= 0.15). Simulated
fluorescence time courses using and were
generated under conditions where S
is in excess over
F-actin. The fit to experimental data (Fig. 5) was good using
the same values of rate parameters k
and
k
as in the light scattering experiments,
at different F-actin/S
ratios in the range 3-6. Note
that the model within which the rate constant for S
association to F-actin varies with the saturation of filaments by
S
accommodates both the light scattering kinetics (in which
the intensity of scattered light is not a linear function of bound
S
) and the fluorescence kinetics (in which the change in
fluorescence is a linear function of bound S
, as was
verified by titration curves).
Figure 6:
Fluorescence titration curves of
pyrenyl-F-actin by S show negative cooperativity.
Pyrenyl-F-actin (98% labeled) was polymerized at 10 µM in
physiological ionic conditions, in the presence of 1.2 molar
equivalents phalloidin and in the absence of ATP as described under
``Materials and Methods,'' and diluted to 2 µM (
) or 0.4 µM (
) in the samples, in the
presence of S
(A
) at the indicated
concentrations. Samples were incubated overnight before fluorescence
was assayed. Dashed line is calculated within a hyperbolic
binding using K = 0.02 µM (best fit
parameter value). Solid lines are theoretical curves
calculated using , with K
=
0.01 µM and
= -2.3. Ten other data
points, between 1 and 2 µM S
, are not shown
here but were taken into account in the fitting procedure and matched
the solid theoretical curve.
The nature of the function (x) being unknown, the
simple linear function
(x) =
x was
tried. The best fit (shown in Fig. 6) was obtained for K
= 0.01 µM and
=
-2.3 ± 0.2. Note that this value of
also corresponds to a
10-fold decrease in affinity of S
for F-actin as the
saturation of the filament by myosin heads increases from 0 to 1.
Other experiments done at higher ionic strength (0.3 M KCl) yielded binding curves that were consistent with a lower overall affinity, but showed no increased anticooperativity.
All the above
experiments have been unable to detect an appreciable difference
between unlabeled and labeled actin in reacting with S.
However, the affinity of S
for both actins is so high that
a 10-fold difference in the apparent rate constants of dissociation of
S
from F-actin or pyrenyl-F-actin would not have been
detected.
Figure 7:
Myosin subfragment-1 binds more weakly to
pyrenyl-labeled F-actin than to unlabeled F-actin. Samples of 2
µM F-actin containing different proportions (,
, 90%;
, 40%;
, 5.5%) of pyrenyl-F-actin were
titrated by S
(A
). Open and closed
circles represent two sets of independent experiments. The
fluorescence of pyrenyl-F-actin was measured at equilibrium as
described under ``Materials and Methods.'' Experimental
conditions are F
buffer, 20 °C. Symbols represent the data. Solid lines are calculated curves
within the competition scheme proposed in the text and using K
/K
= 4 and K
= 0.05
µM.
The relative change in pyrenyl fluorescence, Y,
reflected the binding of S to pyrenyl-F-actin, as described
by the following equation, where
(0),
(S
) and
(
) represent the fluorescence intensity observed in the
absence of S
, or at a given total concentration S
or at a saturating concentration of S
.
The combined equations for mass conservation lead to:
where [S] and [S] represent the
free and total concentrations of S
,
[F
] and [*F
] the total
concentrations of unlabeled and labeled F-actin, respectively. The
value of [S] is the solution of a cubic equation. A simple
fitting procedure was used by generating a series of incremented values
of [S], and calculating the corresponding values of
[S
] according to . The theoretical
curves representing Y versus [S
] were
adjusted to match the ensemble of experimental curves obtained at
different percentages of pyrenyl-actin by changing the values of K
and K
, knowing that both
values have to be lower than 0.1 µM; hence, only the ratio K
/K
can actually be derived
from these measurements. The value of K
/K
had to be in the range of
4-5 to provide a satisfactory fit to all the curves taken
together. The
value increased when values of 3.5 or
5.5 were used for K
/K
.
In
conclusion, equilibrium binding measurements of S to
mixtures of unlabeled and pyrenyl-labeled F-actin demonstrate that the
affinity of S
for labeled actin is 4-fold lower than for
unlabeled actin. In conclusion, derivatizing Cys-374 alters the
interaction of S
with F-actin. Kinetic evidence for a lower
affinity of S
for pyrenyl-actin was provided by observing
the time course of pyrenyl fluorescence upon mixing S
with
partially labeled F-actin; Fig. 8A shows that when
S
was substoichiometric with respect to total F-actin, a
biphasic change was observed, a rapid quenching of fluorescence being
followed by a slower recovery to a higher final fluorescence intensity.
This biphasic change indicates that the rates of S
association to labeled or unlabeled F-actin both are very fast,
but the rate of dissociation of the F-actin-S
complex is
lower for unlabeled actin. The extent of fluorescence recovery was
lower when the molar ratio of S
to F-actin increased; no
fluorescence recovery was observed when the concentration of S
was sufficient to bind to both labeled and unlabeled actin. As
shown in Fig. 8B, the amplitude of the rapid transient
decrease in fluorescence varied linearly with S
until the
1:1 molar ratio to total F-actin was reached, then remained constant,
giving a high affinity titration curve within which pyrenyl-F-actin
would react with S
with the same affinity as unlabeled
F-actin. The final fluorescence in turn confirmed the sigmoidal
titration curve displayed in equilibrium measurements (Fig. 7).
The slow recovery of fluorescence was a first order process of rate
constant k = 0.16 s
, which is the
rate-limiting process in the dissociation of S
from
pyrenyl-F-actin.
Figure 8:
Fluorescence monitoring of the reaction of
S with a mixture of unlabeled and pyrenyl-labeled-F-actin. Panel A, F-actin (4 µM) containing 60%
pyrenyl-actin was mixed with 0.8 µM (top curve)
or 1.5 µM (bottom curve)
S
(A
). The fluorescence scale is normalized
attributing a value of 1 to 1 µM pyrenyl-F-actin (i.e. the fluorescence at time zero of the reaction is 4
0.6
= 2.4). The inset shows an enlarged view of the initial
rapid decrease in fluorescence. Noisy curves are experimental traces.
Smooth curves are simulated time courses using KINSIM and the simple
competition scheme for binding S
to unlabeled or labeled
F-actin and the rate constants k
= k`
= 9 µM
s
, k
= 0.04
s
, k`
= 0.16
s
(k and k` are rate constants
referring to unlabeled and labeled F-actin, respectively). Panel
B, analysis of the transient and final amplitude of the
fluorescence decreases recorded upon reaction of S
at the
indicated concentrations with 4 µM F-actin containing 60%
pyrenyl-labeled actin.
, amplitude of the transient decrease
(minimum in fluorescence in panel A);
, amplitude of the
overall fluorescence decrease.
To get a more precise idea of the mechanism of F-actin-S interaction, the temperature dependence of the binding reaction
was studied under physiological ionic strength.
In a temperature
range of 4-25 °C, S was mixed with an excess of
pyrenyl-labeled F-actin. The concentration of F-actin was varied in the
range 2-10 µM. In this range k
varied linearly with F-actin at all temperatures, as shown in Fig. 9. The apparent second order rate constant k
showed a strong temperature dependence (Fig. 9, inset). The slope of the plot of ln k
versus 1/T decreased
somewhat at higher temperatures, as the diffusion became rate-limiting.
A value of 27 kcal/mol was derived from the maximum slope of the plot
(measured below 10 °C). This value is in agreement with previous
determinations derived from pressure relaxation studies of
F-actin-S
-ADP and, as proposed by Geeves and Gutfreund (31) , reflects the involvement of a conformational change in
the measured reaction. This conformation change may occur on the
rapidly preformed F-actin-S
complex, or on one of the two
reacting proteins prior to the diffusion-controlled reaction. The first
model described by has thus far been favored in the
field. Within , the present data should be accommodated as
follows. The value of k
/k
should be at
least 40 µM, to account for the strictly linear dependence
of k
on F-actin in the range 0-15
µM. The value of k
should be
appreciably faster than the observed apparent k
, i.e. k
10
µM
s
; hence, k
should be larger than 400
s
. The value of k
, which
corresponds to the rate-limiting process observed in the slow
fluorescence recovery shown in Fig. 7, is 0.16 s
for pyrenyl-actin and should be 4-fold lower (0.04
s
) for unlabeled actin. The apparent second order
rate constant represents k
/K
, and has a value of
9 µM
s
at 20 °C;
hence, k
should have a value of at least 400
s
. The isomerization of the collision complex would
therefore increase the affinity of S
for unlabeled F-actin
by 400/0.04 = 10
-fold, leading to a value of 4
nM for the global equilibrium dissociation constant of the
rigor complex. Note that this value refers to the higher affinity
binding of S
to the undecorated filament. As the filament
becomes increasingly decorated, the affinity of S
may
decrease up to 10-fold, according to the above data.
Figure 9:
Temperature dependence of the kinetics of
interaction of pyrenyl-F-actin with S. Myosin subfragment 1
(0.5 µM) was reacted with an excess of pyrenyl-F-actin as
indicated, at the following temperatures:
, 4 °C;
, 6
°C;
, 8 °C;
, 10 °C;
, 15 °C;
, 20 °C. Kinetic data were analyzed and plotted as under Fig. 2. Inset, Arrhenius plot of the data. The
logarithm of the apparent bimolecular rate constant, derived from the
slope of the lines shown in the main panel, is plotted versus the reciprocal of the temperature (in degrees Kelvin). The slope
of the dashed line corresponds to an activation energy of 27
kcal/mol.
The equilibrium and kinetics of binding of myosin
subfragment-1 to F-actin have been investigated with the aim to
understand whether the binding was a reaction described by a single
equilibrium dissociation constant, as concluded from earlier
works(1, 2, 3, 4, 5) , or
whether the equilibrium binding might be non-Michaelian, and associated
to more complex kinetics, as suggested by more recent works, which
pointed to the nonlinear dependence of physical parameters monitoring
F-actin-S interaction on the extent of S
bound
fo
F-actin(11, 32, 33, 34, 35) .
We find that the rate of S
binding to F-actin depends on
the extent of bound S
, steric hindrance of the first bound
S
molecules preventing further binding of S
to
the partially decorated filament. Both the kinetics of changes in light
scattering or pyrenyl-actin fluorescence ( Fig. 1and Fig. 3-5) and the equilibrium binding measurements (Fig. 6) can be quantitatively accounted for, assuming that the
apparent bimolecular association rate constant decreases linearly with
the molar ratio of bound S
. A good fit to the data was
obtained assuming a 10-fold decrease in the association rate constant k
of S
to F-actin as the
saturation of the filament by myosin heads increases from 0 to 1. The
corresponding 10-fold decrease in affinity of S
for
F-actin, over the whole titration curve, was confirmed in the analysis
of the equilibrium binding data (Fig. 6).
Evidence for
negative cooperativity in binding kinetics was provided by the
observation that the F-actin-S complex is formed faster
when F-actin is in excess over S
than in the opposite
situation, and that the process is not first order when S
is in excess over F-actin. Such non-exponential reactions have
been noticed in the past(8, 11) . One of the main
points of the present paper is to propose a quantitative analysis of
this phenomenon. The kinetic data were analyzed assuming a linear
dependence of k
on the stoichiometry x of S
bound per F-actin subunit. This linear dependence
was chosen, in the absence of any experimental evidence for a defined
binding scheme, because it provided a simple analytical solution () that allowed simulation and fitting of the
non-exponential binding kinetics. A more physically reasonable analysis
of the data, taking into account the polarity of the filament, would
imply to define different rate constants for association of S
to F-actin subunits having either no neighboring bound
S
, or a neighboring bound S
either on the
barbed or on the pointed end side of the subunit to which S
is binding. Consideration of the helical nature of the filament,
in which each actin subunit has in fact four neighbors, would introduce
further splitting of the different association rate constants. A
complete analysis would also require a statistical analysis of the
distribution of S
bound at each value of x, and
Monte Carlo methods would be necessary to model the binding scheme
adequately. However, we are not sure that fitting such a complex model
to the present data would provide an unambiguous set of association
rate constants.
Our results and conclusions are in partial agreement
and in partial disagreement with the recent work of Andreev et
al.(11) . While the equilibrium and kinetic data of
S association to F-actin are in agreement with the
anticooperativity in S
binding demonstrated by the
sedimentation data of Andreev et al.(11) , our kinetic
observations and our interpretation differ. Andreev et al. reported that the time course of quenching in pyrene fluorescence
upon binding S
was biphasic when F-actin was in excess over
S
, which was interpreted as a two-step binding of S
to one F-actin subunit, followed by an isomerization consistent
with binding of S
to a second actin subunit in the
filament. Our observations are somewhat different, since a simple
exponential binding process was recorded when F-actin was in excess
over S
( Fig. 1and Fig. 4), and the reaction
deviated from a monoexponential when S
was in excess over
F-actin. The data presented by Andreev et al. with S
in excess over F-actin (Fig. 2A in (11) )
actually show a very poor fit to a monoexponential (in agreement with
our data), the experimental trace intersecting the exponential best fit
several times. Although our data do not support the two-step binding
model proposed by Andreev et al., clearly the basic
observations of nonlinear kinetics and anticooperative equilibrium
binding are confirmed in the present work, with a different
interpretation.
In most kinetic studies of S binding
carried out thus far, the concentration of S
was varied, in
excess over F-actin. The data therefore referred to a high molar ratio
of bound S
, and were assumed to be well described by
monoexponentials, which now appears to be incorrect (see curves
b, residuals, in Fig. 1and Fig. 4). Within our
interpretation, the rate constant derived from such kinetic data,
analyzed within a single first order process, reflects the average rate
of binding of S
to a partially decorated filament, and is
therefore lower than the rate constant for association of S
to a bare filament, which we find equal to 9
µM
s
at 20 °C
under physiological conditions. This value is within the range expected
for a diffusion-controlled reaction; however, the high activation
energy of the reaction strongly suggests that a structural change of
the F-actin S
complex is involved, in agreement with
previous works, and also with the model derived from the kinetics of
interaction of G-actin with S
(18) .
The present
work gives the first quantitative estimate of the difference in
affinity of S for pyrenyl-labeled and unlabeled actin. This
difference was previously thought to be insignificant, because S
reacts at apparently identical rates with either unlabeled or
fully labeled F-actin. However, the reaction of S
with
mixtures of labeled and unlabeled actins clearly shows evidence, both
in the equilibrium binding curves and in the kinetics, for a
4-5-fold difference in affinity, most likely accounted for by a
difference in the rate constant k
in .
The conclusion that labeling of Cys-374 on actin
interferes with S binding is in agreement with predictions
made in the reconstruction of the actin-myosin head interface from the
crystallized structures of actin and S
(19) and
using the atomic model of the actin filament(22) . It also
agrees with the fact that in F-actin, Cys-374 can be N,N`-paraphenylenedimaleimide cross-linked to Lys-191
of the adjacent subunit along the short pitch helix(36) , and
this cross-link is inhibited by S
binding(37) ,
indicating S
binding to F-actin perturbs the region of the
C terminus of F-actin. It should be noted that the difference in
affinity of labeled and unlabeled actin for S
is not
displayed by G-actin(16, 18) . Accordingly S
can be cross-linked to Cys-374 in the G-actin-S
complexes, not in the F-actin-S
complex(38) .
Finally our results suggest that the cross-bridges might operate
differently when the thin filaments are poorly or fully saturated by
myosin heads, a conclusion in agreement with biochemical data (13) and structural evidence (39) for the interaction
between adjacent heads bound to the filament at high
S/actin ratios.