(Received for publication, October 19, 1994; and in revised form, December 19, 1994)
From the
The kinetics of interaction of monomeric pyrenyllabeled G-actin
with myosin subfragment-1 (S (A
) and
S
(A
) isomers) has been examined in the
stopped-flow at low ionic strength. The data confirm the previously
reported existence of binary GS and ternary G
S complexes.
The increase in pyrenyl-actin fluorescence which monitors the
G-actin-S1 interactions is linked to the isomerization of these
complexes following rapid equilibrium binding steps. The rates of
isomerization are
200 s
for GS and
50
s
for G
S at 4 °C and in the absence
of ATP. DNaseI and S
bind G-actin essentially in a mutually
exclusive fashion. Both GS and G
S are dissociated by MgATP
and MgADP. The kinetics and mechanism of ATP-induced dissociation of
G
S are quantitatively close to the ATP-induced dissociation
of F-actin-S
, which indicates the G
S is a good
model for the F-actin-S
interface. GS and G
S
display different kinetic behaviors in response to nucleotides, GS
being less efficiently dissociated than G
S by MgATP. This
result suggests that different mechanical properties of the
cross-bridge might correlate with different orientations of the myosin
head and different actin/myosin binding ratios.
Kinetic studies of the contractile cycle of the actomyosin ATPase have shown that the energy of ATP hydrolysis is transduced into movement via changes in affinity of myosin for F-actin, modulated by the nucleotide bound to myosin(1, 2, 3, 4, 5) . A complete understanding of the mechanism of transduction of chemical energy into mechanical energy necessitates a description of the structural changes in the actomyosin interface in connection with the biochemical steps. The recent determinations of the three-dimensional structures of actin(6) , of the myosin head(7) , and the combination of these crystallographic data with the low resolution electron microscopy structure of the decorated filament (8) have led to a structural model for the F-actin-myosin subfragment-1 rigor complex(9) .
An alternative approach to
the functional changes in the actomyosin interface consists in
characterizing the small size complexes formed between the myosin head
and monomeric actin. These complexes, which are precursors in the
myosin subfragment-1 (S)-induced polymerization of G-actin
into decorated filaments, are helpful to define the minimum
actin-S
contractile unit. It was found (10) that
S
forms binary (GS) and ternary (G
S) tight
complexes with G-actin at low ionic strength and in the absence of ATP.
Both nucleotide effects on the stability of GS and G
S and
cross-linking studies (11) showed that the G-actin-S
interface is very similar to the F-actin-S
interface
in the rigor state. The second step in the polymerization process of
G-actin induced by S
appears to be the condensation of
G
S units in small oligomers (12) , which emphasizes
the functional relevance of the interaction of the myosin head with two
actin molecules. The interaction of the myosin head with two actin
subunits, adjacent along the long pitch helix of the filament, is also
an essential feature of the structural model of the actin-myosin rigor
complex(9) , which may have important implications in the
mechanism of force generation. The present paper is a kinetic analysis
of the interaction of monomeric pyrenyl-G-actin with the two myosin
subfragment-1 isoforms carrying either the A
or the A
light chain. The pyrene probe has previously been used to
characterize the interaction of S
with F-actin (13, 14, 15) and G-actin(10) . The
kinetics of G-actin interaction with S
(A
) and
S
(A
) presented here confirm the existence of GS
and G
S complexes with both S
isoforms and show
that G
S is more easily dissociated by MgATP than GS, which
may have implications in muscle contraction.
Bovine pancreatic DNaseI grade II was purchased from Boehringer and purified by hydroxylapatite chromatography(23) . The concentration of DNaseI was determined by titration of an aliquot of the purified preparation by G-actin using the DNase inhibition assay(24, 25) . The DNA substrate was bought from Sigma.
Actin and S concentrations were
determined spectrophotometrically as described(12) .
Dissociation of G-actin S complexes by nucleotides was carried out as follows. One of the
drive syringes contained either calcium nucleotide (prepared as an
equimolar CaCl
-nucleotide mixture diluted to the desired
concentrations in buffer G
), or magnesium nucleotide
(prepared in the same way but diluted in buffer G
containing 100 µM MgCl
instead of 100
µM CaCl
). The other drive syringe contained
the G-actin-S
complex rapidly preformed at 0 °C
immediately before being brought in the syringe. For experiments with
magnesium nucleotide, G-actin-S
was in buffer G
containing only 10 µM CaCl
so that after
mixing the nucleotide was 90% magnesium nucleotide.
Figure 1:
Rapid kinetics of complex formation
between pyrenyl G-actin and myosin subfragment 1. a, pyrenyl
G-actin-ATP 1:1 complex (2 µM) in G buffer was
mixed at 4 °C with S
(A
) at the following
concentrations (in µM, top to bottom): 4, 1, 0.2. The
fluorescence of 2 µM pyrenyl-G-actin is normalized to 1. b, S
(A
) (0.4 µM) was
mixed with pyrenyl-G-actin at the following concentrations in
µM (top to bottom): 2, 0.6, 0.2. The fluorescence of
pyrenyl G-actin is subtracted. Noisy curves are experimental traces
(average of four consecutive shots); solid lines represent the
monoexponential best fit; dashed lines represent the simulated
time courses using KINSIM and the fluorescence, equilibrium and rate
parameters given in Table 1.
In a second series of experiments, the concentration of S was kept constant (0.4 µM), and the concentration of
pyrenyl-G-actin was varied. In the whole range of actin concentrations
(0-2 µM), the time courses of the increase in pyrene
fluorescence were again well fitted by monoexponentials (Fig. 1b).
The actin and S concentration
dependences of the amplitudes and k
were
analyzed as follows. When the concentration of pyrenyl-G-actin was
constant, the amplitude of the fluorescence change showed a saturation
behavior upon increasing S
(A
) or
S
(A
) concentration. As illustrated in Fig. 2, and in agreement with previous static fluorescence
titration curves(12) , all data points fell above the
stoichiometric line corresponding to the formation of a 1:1 GS complex
of infinitely high affinity and were close below the stoichiometric
line corresponding to a 2:1 G
S complex of infinitely high
affinity. Similar data were obtained with S
(A
)
and S
(A
), S
(A
)
displaying an apparently slightly higher affinity than
S
(A
). The amplitude of the fluorescence change
was maximum at 2-3 µM S
and did not show
any further increase with up to 20 µM S
. The
maximum increase in pyrenyl G-actin fluorescence at saturation by
S
was 2.1-fold in satisfactory agreement with the 3.3-fold
enhancement observed (10) in equilibrium measurements performed
with higher spectral resolution of excitation and emission wavelengths.
Figure 2:
Fluorescence titration curves of pyrenyl
G-actin by S (stopped-flow amplitude data). Pyrenyl G-actin
(2 µM) was mixed with S
(A
)
(
) or S
(A
) (
) at the indicated
concentrations under the conditions described in Fig. 1a. Thin lines represent the theoretical
titration curves of infinitely high affinity G
S (left) and GS (right) complexes, respectively. Thick lines are calculated curves within using
values of parameters in Table 1.
When the concentration of S was constant the dependence
of the amplitude of the fluorescence change on G-actin concentration,
shown in Fig. 3, was consistent with the formation of a tight
G
S complex when S
was saturated by G-actin.
Note that interpretation of the fluorescence titration curve of S
by G-actin within the formation of a single 1:1 GS complex would
imply that the affinity is low (K
0.3
µM), and this interpretation would be totally inconsistent
with the titration of G-actin by S
shown in Fig. 2.
Solid curves in Fig. 2and Fig. 3are calculated within , (see
``Appendix''), and values of parameters shown in Table 1.
Figure 3:
Fluorescence titration curves of S by pyrenyl G-actin (stopped-flow amplitude data). Pyrenyl G-actin
at the indicated concentrations was rapidly mixed with 0.4 µM S
(A
) (
) or
S
(A
) (
), under conditions described in Fig. 1B. The thin lines represent the
theoretical titration curves corresponding to infinitely high affinity
GS (left) and G
S (right) complexes,
respectively. Thick lines are calculated curves within using values of parameters given in Table 1.
Analysis of the rate constants showed the following.
When G-actin was constant, the observed first-order rate constant for
the fluorescence increase varied with S in a
sigmoïdal fashion, as shown in Fig. 4, between two limits of 50 s
at low
S
to 200 s
at saturating S
(20-30 µM). On the other hand, as shown in Fig. 5, when S
was constant, the observed
first-order rate constant decreased upon increasing G-actin from
100 s
to a lower limit of 55-60
s
at high G-actin. The fact that the rates reach a
limit indicates that the change in fluorescence monitors a conformation
change following the formation of rapid equilibrium complexes. However,
the complex formed when G-actin is saturated by S
(k = 200 s
) is not identical to the one
obtained when S
is saturated by G-actin (k = 55 s
). Hence the kinetic data confirm
the interpretation of previous (10) and present amplitude data
and lead to the conclusion that the rate constant of 200 s
represents the sum of the rate constants for isomerization of a
1:1 GS complex, while the rate constant of 55-60 s
represents the sum of the rate constants for isomerization of a
2:1 G
S complex. The sigmoidal appearance of the change in k
versus S
(Fig. 4)
reflects the fact that, as illustrated in Fig. 10a under ``Appendix,'' the formation of
G
S
is the predominant reaction (k = 55 s
) at low S
, and the
formation of GS* (k = 200 s
) becomes
increasingly predominant upon increasing S
. The converse
argument holds for the decrease in k
upon
increasing G-actin at constant S
(compare Fig. 5and
10b).
Figure 4:
S concentration dependence of
the first-order rate constant for interaction of pyrenyl G-actin with
S
. The first-order rate constant for the change in
fluorescence observed upon reaction of pyrenyl G-actin (squares, 2 µM; circles, 1
µM) with S
(A
) (
,
) or
S
(A
) (
,
) is plotted as a function
of [S
]. Closed triangles represent the
monoexponential best fit of simulated time courses using KINSIM and the
parameter values given in Table 1. Inset, expanded view
of the data at low
[S
].
Figure 5:
G-actin concentration dependence of the
first-order rate constant for interaction of pyrenyl G-actin with
S. Pyrenyl-G-actin at the indicated concentrations was
reacted with 0.4 µM S
(A
) (
,
) or S
(A
) (
,
). Squares and circles refer to two independent experiments. Large squares represent the rate constant of the exponential
best fit of simulated curves (e.g.dashed lines in Fig. 1a) using KINSIM and values of parameters in Table 1.
Figure 10:
Distribution of the different fluorescent
and non-fluorescent G-actin-S complexes in a range of
G-actin and S
concentrations. a, molar fraction of
actin in different species at 2 µM total actin and
different concentrations of S
(A
). Dashed
line, free G-actin;
, GS;
, GS
(fluorescent);
, G
S;
,
G
S
(fluorescent). b, molar fraction of
S
(A
) in different species at 0.4 µM total S
and different G-actin concentrations. Dashed line, free S
(A
); other symbols
as in panela.
The above results lead us to propose the simple
following model () for binding G-actin to S.
In the above scheme, GS and GS are rapid equilibrium
complexes. The fluorescence change is associated to the isomerization
steps leading to GS
and G
S
. This
scheme therefore represents an extension of the one previously proposed (10) in which no isomerization of GS and G
S was
included. The details of the procedure used to derive the analytical
expression of the fluorescence change at equilibrium within , to fit the values of the equilibrium dissociation
constants to the data, to derive the rate constants, and simulate the
kinetic curves are all given under ``Appendix.''
Fig. 1, a and b, show the superimposed
experimental and calculated time courses at a constant G-actin (resp.
S) concentration and varying S
(resp. G-actin)
in a 10-20-fold concentration range. The simulated time courses
were consistent with monoexponentials. The calculated rate constants
varied with either S
or G-actin in a manner quantitatively
consistent with experimental data (large symbols in Fig. 4and Fig. 5), which confirmed the validity of .
In all experiments presented in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5,
G-actin has CaATP as bound nucleotide. Since under physiological
conditions actin has MgATP as bound nucleotide, supplementary
experiments were carried out in which CaATP-pyrenyl-G-actin 1:1 complex
at a concentration 4 µM was rapidly converted into
MgATP-G-actin by addition of 15 µM MgCl
and
0.2 mM EGTA 3 min before being rapidly mixed with
S
. The amplitude of the pyrene fluorescence change upon
addition of S
gave titration curves (data not shown)
superimposable with those obtained in Fig. 2and Fig. 3(controls with CaATP-G-actin from the same preparation
were run in parallel in the same experiment). The rate constants for
the isomerizations, k
+ k
and k
+ k
, were 80 s
for GS
GS* and 30 s
for G
S
G
S
transitions, respectively, i.e. about twice lower than the corresponding values obtained with
calcium G-actin. Hence all conclusions drawn here with calcium-actin
also apply to the physiological magnesium-actin species. For technical
reasons the work was done with calcium-actin which is more stable and
does not give rise to oligomers with concomitant hydrolysis of
actin-bound ATP.
The decrease in fluorescence was a first-order process at all nucleotide concentrations. The amplitudes of the fluorescence decrease showed a saturation behavior as a function of nucleotide concentration, and the rate constants increased linearly with nucleotide concentration up to at least 400 µM, i.e. above the range shown in the figures.
The minimum kinetic scheme accounting for this process is the following.
where n = 1 or 2, and N represents the
nucleotide (MgATP or CaATP or MgADP). K and K are rapid equilibrium dissociation constants and k
and k
the
isomerization rate constants corresponding to the conformational
change, which is monitored by the change in fluorescence and through
which the affinity of S
for G-actin decreases.
1) The
dissociation of G S by nucleotides was first examined.
Pyrenyl G-actin (3 µM) and S
A
(0.5
µM) were premixed and reacted with nucleotide at different
concentrations. The results are shown in Fig. 6. Given a total
concentration of 0.5 µM ATP-binding sites, the amplitude
data (Fig. 6a) showed that the equilibrium dissociation
constant for MgATP, K
k
/k
was too
low (
0.1 µM) to be measured. In other words, with
MgATP, the value of K
is very high, and the
reaction is quasi irreversible. From the slope of the linear increase
of k
with MgATP (Fig. 6b), a
value of 1.6 µM
s
was derived for k+/K
. The
value of k
, as derived from the ordinate
origin, was
0.2 s
. Only a lower estimate of 400
µM can be proposed for K
.
Figure 6:
ATP and ADP induced dissociation of
(G-actin)-S
ternary complex. Preformed
G
S complex (3 µM pyrenyl-G-actin, 0.5
µM S
(A
) was rapidly mixed with
either MgATP (
) or MgADP (
) or ATP-Ca (
) at the
indicated concentrations. The decrease in pyrenyl fluorescence was
consistent with the complete dissociation of S
from G-actin
at saturation by all nucleotides. Panel a, amplitude data. Panel b, observed dissociation rate constant. Inset,
expanded view of the data at low [ATP] or
[ADP].
When the
dissociating nucleotide was CaATP or MgADP, the values found for k
k
/k
were 2.8 and 1
µM, respectively. The slopes of k
versus CaATP or MgADP were similar to those obtained
with MgATP, however, the values of k
were
definitely higher (
2 s
), indicating that the
reaction is reversible and that the value of K
must be lower than with MgATP.
2) The dissociation of GS by
nucleotides was investigated in a similar fashion, and qualitatively
similar data were obtained, pointing out to the same mechanism for
nucleotide-induced dissociation of GS. However, a large quantitative
difference between GS and GS was observed regarding the
efficiency of MgATP to promote dissociation of S
from
G-actin. In the experiment shown in Fig. 7, the GS
complex was preformed and rapidly mixed with different amounts of
MgATP. Essentially GS
was present, with S
/G
ratios of 5 and 16, respectively. The decrease in fluorescence linked
to dissociation of GS
varied with MgATP in an identical
fashion at the two S
/G ratios, consistent with a much lower
affinity of MgATP for GS than for G
S. In this experiment,
MgATP binds to GS
and free S
. The fact that
identical amplitude patterns were observed although the concentration
of S
was different in the two assays (hence some
competition between GS and S
for binding ATP was expected),
suggests that the binding of MgATP to GS
is completed
before its binding to S
. The analysis of the rate
constants, gave k
= 0.4 ± 0.1
s
and k
/K
= 0.08 ± .01 µM
s
(Fig. 7, inset), a value
20-fold lower than the one (1.6 µM
s
, Fig. 6b) obtained for
G
S. Using these two numbers, a value of 5 ± 1.8
µM was derived for K`
. This estimate
is consistent with the amplitude data which can be well fitted using an
equilibrium dissociation constant K`
of 3
µM, a value again 30-fold higher than the one found for
the binding of MgATP to G
S. An alternative model implying
dissociation of GS as a consequence of the initial binding of MgATP to
free S
has not been considered here.
Figure 7:
MgATP dissociates S more
efficiently from the G
S than from the GS complex. The
preformed GS
complex was rapidly mixed with MgATP at the
indicated concentrations. The amplitude of the fluorescence decrease
linked to the dissociation of S
-MgATP is plotted versus the concentration of MgATP.
,
, 1 µM pyrenyl-G-actin, 5 µM S
(A
);
, 1 µM pyrenyl-G-actin, 5 µM S
(A
);
, 0.5 µM pyrenyl-G-actin, 8 µM S
(A
)
(normalized to the same
F
, i.e. to
1 µM G-actin). Continuous lines are calculated within , and with k
/K
=
0.08 µM
s
, k
= 0.4 s
, values
coming from the k
data (
) shown in the inset. The data obtained for dissociation of G
S
(
) coming from Fig. 6a are replotted, for
comparison, in the main frame and in the inset.
In conclusion both
amplitude and kinetic data indicate that MgATP is much more efficient
to promote dissociation of GS than to promote dissociation
of GS.
A comparison of these data, with the ATP-induced dissociation
of F-actin-S(26) is interesting. At 0.5 °C and
in the presence of 0.1 M KCl, values of K`
< 0.01 µM, and k
/K
= 0.6
µM
s
have been
reported, which compare well with the data obtained here for the
dissociation of G
S by MgATP, but differ significantly from
the data obtained for the dissociation of GS.
All equilibrium and
rate parameters for nucleotide-induced dissociation of GS and
GS are summarized in Table 2.
The binding of S to pyrenyl G-actin in the
presence of DNaseI was investigated in the stopped-flow as follows. A
first experiment showed that upon mixing DNaseI with the preformed
fluorescent pyrenyl-G-actin-S
complex (0.5 µM G-actin, 3 µM S
(A
)), a
decrease in pyrene fluorescence was observed. At saturation by DNaseI,
the final fluorescence level was identical to that of G-actin. At all
concentrations of DNaseI, the decrease in fluorescence was a
first-order process whose rate constant k
increased with the concentration of DNaseI and reached a higher
limit of 60 s
at saturation by DNaseI. The DNaseI
dependences of the amplitudes as well as of the k
were both described by the same hyperbolic binding isotherm.
In another experiment shown in Fig. 8, pyrenyl G-actin in the
presence of different amounts of DNaseI was mixed with
S(A
) at a constant concentration. Three series
of experiments were performed concentrations of
S
(A
). The extent of fluorescence change
reflecting binding of S
decreased upon increasing DNaseI
concentration. The process was first-order with a rate constant of 200
s
at all DNaseI concentrations. The data were
tentatively analyzed within a general square model described below () within which binary DNaseIG-actin (DG) and
G-actin-S
(GS) complexes and a ternary
DNaseI-G-actin-S
complex (DGS) can be formed, with
equilibrium dissociation K
and K
in the binary complexes, K`
and K`
in the ternary complexes, with K
K`
= K
K`
(detailed balance). It was assumed from
experimental evidence that the fluorescence of pyrenyl G-actin was
enhanced only in the GS complex.
Figure 8:
Mutual exclusion binding of DNaseI and
S to G-actin. Pyrenyl-G-actin (1 µM) in the
presence of the indicated concentrations of DNaseI was rapidly mixed
with S
(A
) at 3 µM (
,
), 8 µM (
), and 15 µM (
).
The increase in pyrenyl fluorescence was monitored. Solid lines are calculated within the general square model ()
using the following values of equilibrium parameters: K
= 3 nM; K
= 63 nM; K`
= 5 µM; K`
= 105 µM. Inset, concentration of DNaseI,
[DNaseI]
, at which 50% of the maximum
fluorescent change was observed, as a function of
S
(A
) concentration.
The amplitude data in Fig. 8were analyzed within , using K = 3 nM, and K
= 63 nM. The data could be
fitted by only if K`
/K
(= K`
/K
) was higher than
10
, which essentially reduces to a mutual exclusion binding
behavior of DNaseI and S
to G-actin, the ternary complex
DGS being negligible in solution in the range of concentrations of
S
and DNaseI investigated. Accordingly, the concentration
of DNaseI causing half-dissociation of G-actin-S
varies
linearly with S
(Fig. 8, inset) consistent
with the apparent competitive binding of DNaseI and S
to
G-actin. Kinetic data indicate that S
and DNase dissociate
from the ternary complex at rates 60 s
and >200
s
, respectively.
If S and DNaseI bind
G-actin in competition with each other as indicated by the above
result, the inhibition of DNaseI activity by G-actin should be relieved
by S
. Surprisingly, the opposite result was obtained, and
we confirmed the data obtained by Chen et al. ((30) , Fig. 5) when we performed an identical experiment. However, a
control experiment displayed in Fig. 9demonstrated that the
presence of 40 µg/ml DNA (the substrate concentration used in the
DNase activity assay) very potently inhibits the interaction between
G-actin and S
, presumably due to the binding of the
polyanionic DNA to the lysines of S
which are involved in
the G-actin-S
contact. In conclusion, the result obtained
by Chen et al.(30) , and confirmed here, is explained
by the failure of S
to bind to G-actin in the
DNA-containing DNase activity test and should not be taken as a proof
that S
does not significantly inhibit DNaseI binding to
G-actin. The DNase activity assay simply appears inappropriate to
examine the effect of S
on DNaseI binding to G-actin.
Figure 9:
The interaction between G-actin and
S is inhibited by DNA. Pyrenyl-G-actin (2 µM)
was mixed with S
(A
) at the indicated
concentrations, in the absence (
) or in the presence (
) of
40 µg/ml DNA. Note that the affinity of S
for G-actin
decreases by
3 orders of magnitude (K
3 µM) in the presence of 40 µg/ml
DNA.
The kinetic results presented here bring new information
about the mechanism of interaction of the myosin head (myosin
subfragment-1 S(A
) and
S
(A
) isomers) with monomeric actin using the
fluorescence of pyrenyl-actin as a probe. In agreement with previous
reports (10, 12, 32) but at variance with
others(31, 33) , S
(A
) and
S
(A
) can both interact with two molecules of
monomeric actin, hence binary (GS) and ternary (G
S)
complexes exist in solution. Kinetic analysis of the interaction of
pyrenyl-G-actin with S
further shows that the increase in
pyrene fluorescence which monitors the formation of GS and
G
S is linked to an isomerization of these complexes
following the rapid bimolecular reversible reactions of G-actin with
S
. Protein-protein interactions are tightened about one
order of magnitude by this isomerization, hence the fluorescent
GS
and G
S* complexes are the major
G-actin-S
complexes at equilibrium. The isomerization rate
constants of GS and G
S are appreciably different (
200
s
and 50 s
at 4 °C) which,
added to the analysis of the fluorescence titration curves of pyrenyl
G-actin by S
and of S
by pyrenyl G-actin
(stopped-flow amplitude data) leaves no doubt about the existence of
the two types of G-actin-S
complexes. One should note that
although a recent report (31) states that G-actin interacts
with S
with a 1:1 stoichiometry, the data shown ( Fig. 5in Ref.31) cannot be interpreted within a 1:1 binding
scheme.
The kinetics of S(A
) and
S
(A
) association to G-actin were practically
identical, especially regarding the isomerization reactions. Hence the
quantitative difference in ability of the two isomers to promote actin
polymerization (10, 12, 33) is linked to the
different stabilities of short oligomers, resulting from the
condensation of the G
S complex, which are kinetic
intermediates in the formation of arrowhead-decorated
filaments(12) . Since hydrophobic actin-actin contacts,
plausibly corresponding to the lateral bonds along the genetic helix of
the filament, are involved in oligomers, the results suggest that the
40 amino acid extension of light chain A
somehow stabilizes
actin-actin bonds along the genetic helix, in decorated
F-actin-S
(A
) filaments, rather than
longitudinal actin-actin bonds.
The kinetic scheme which is
presented here is the simplest which can account for the data. This
does not eliminate the possibility that other more complex schemes, in
particular containing a larger number of kinetic steps connecting the
different complexes, may be relevant. The simple scheme, however,
provides an easy comparison of the characteristics of the interaction
of the myosin head with monomeric actin and with filamentous actin. The
changes in pyrenyl F-actin fluorescence upon binding S have
also shown evidence for a similar two-step binding mechanism, the
change in fluorescence being linked to the second isomerization
step(14, 15) . The rate constant for isomerization of
pyrenyl-F-actin-S
was 200 s
at 20 °C
and 20 s
at 6 °C and low ionic
strength(15) . This latter value is close to the isomerization
rate constant found here for G
S, which supports the view
that the G
S complex may be a good model of the
F-actin-S
interface. The possibility that S
interacts with two actin subunits in the filament, adjacent along
the long pitch helix, in a geometry similar to that of the
G
S complex, has been raised by the image reconstruction of
the decorated filament (8) and is an intrinsic feature of the
current atomic model of the F-actin-myosin complex(9) ,
supported by available biochemical cross-linking
studies(34, 35, 36) . Within this model the
two F-actin subunits, called actin 1 and actin 2, which are part of the
F-actin-S
rigor complex, interact longitudinally, the
barbed end (subdomains 1 and 3) of actin 1 being in contact with the
pointed end (subdomains 2 and 4) of actin 2. The myosin head interacts
mainly with the N- and C-terminal regions of actin 1 (in subdomain 1),
and makes a secondary contact with the top of subdomain-1 of actin 2.
In this ternary complex, S
bridges subdomain 2 of the actin
2 subunit. In support of this model, changes in subdomain 2 of actin
such as covalent modification of lysine 61 (25, 37, 38) or subtilisin cleavage between
Met
and Gly
(39) all affect S
binding to F-actin. Similarly, limited proteolytic digestion
studies (30, 40) showed that S
binding to
G-actin induces changes in subdomain 2 (loop 38-69) of G-actin,
which supports a structure of the G
S complex similar to the
F-actin-S
complex.
In a recent report (41) equilibrium and kinetic data of S binding to
F-actin were interpreted in terms of two different rigor complexes,
actin-S
and (actin)
-S
, depending on
the F-actin/S
ratio. More work is needed, to correlate the
possible different F-actin-S
complexes to the GS and
G
S complexes studied in the present work. An important
difference between G-actin-S
and F-actin-S
interaction is that while G-actin monomers are dispersed in
solution, actin subunits interact with each other in the filament, so
that at all F-actin/S
ratios, the two adjacent actin
subunits which may form the regular (actin)
-myosin
interface are preassociated to interact with S
in a ternary
rigor complex. Hence we suggest that the G
S structure is a
good model of the F-actin-S
interface.
Both GS and
GS complexes are dissociated by ATP and ADP with relative
efficiencies that compare well with F-actin-S
records.
Kinetic data show that the mechanism of the ATP or ADP-induced
dissociation involves binding of nucleotide to the G-actin-S
complexes followed by an isomerization of the
(G-actin)
-S
-nucleotide complexes which
kinetically limits the dissociation of S
-nucleotide from
G-actin. Again the change in pyrene fluorescence monitors the
isomerization step rather than the dissociation of S
. All
these features are qualitatively similar to the mechanism of
ATP-induced dissociation of S
from F-actin (42, 43) at low ionic strength, which emphasizes that
the G-actin-S
interaction is functional in terms of
regulation by nucleotides.
One of the interests of the
G-actin-S complexes resides in the opportunity to study the
regulation of the interaction of the myosin head with one actin
molecule or two actin molecules separately, since experimental
conditions can be designed under which essentially either GS
or G
S
exist in solution. The different
behavior of GS and G
S in response to ATP and ADP is
striking. Both the high affinity derived from the amplitude data and
the dependence of k
on ATP concentration show
that G
S is more similar to F-actin-S
in
solution than GS, which supports the view of the ternary G
S
complex as the minimum actomyosin unit. On the other hand, the fact
that the myosin head can interact with either one or two actin subunits
with different mechanical properties (i.e. different use of
ATP) should be considered in cross-bridge function. Whether a change in
the actin/S
binding stoichiometry is involved in the
cross-bridge ATPase cycle and in force development requires further
investigation. Nevertheless, recent measurements of the mechanics of
single myosin (44) molecules have shown that the time during
which myosin interacts with the actin filament displays different ATP
concentration dependences at low load and high load and varies with the
load when ATP is not limiting (see (45) for a recent review).
Our data, showing a different ATP dependence for GS and G
S,
suggest that the different actin-myosin interaction times may correlate
with different orientations of the myosin head, for instance the myosin
head would interact predominantly with 2 actin subunits at high load
(high ATP affinity) and with 1 actin subunit at low load (low ATP
affinity). These possible different orientations would be compatible
with the noticed (46) large variability in the measurements of
displacement and forces of single myosin molecules along the actin
filament. In solution studies of F-actin-S
interaction, it
has also been reported (47) that the binding of ATP to
F-actin-S
varied with the actin/S
molar ratio.
The interaction of G-actin with S appears potently
inhibited by DNaseI. At variance with previous reports (29, 30, 31) the present results can be
quantitatively accounted for by a simple mutual exclusion binding
scheme of S
and DNaseI to G-actin, the affinity of either
S
or DNaseI for G-actin being reduced about
10
-fold in the potential ternary complex. In other words,
in the presence of micromolar amounts of G-actin and DNaseI, a
concentration as high as
100 µM S
would
be necessary to observe a ternary DNaseI-G-actin-S
complex.
Our conclusion is consistent with the inhibition by DNaseI of the
S
-induced polymerization of G-actin, as well as with
previous results indicating competition between DNaseI and S
for binding to actin (37, 38, 39, 48, 49) , and
with data showing a decrease in binding strength of DNase I by at least
one order of magnitude to the covalent MBS actin-S
complex(29) .
In conclusion, the interaction of
G-actin with the myosin head may be a useful system to investigate the
change in actin-myosin interaction during the ATPase cycle, and the
GS complex is a good model of the F-actin-S
interface.
In , f represents the intrinsic
pyrene fluorescence in G-actin, GS, and G
S complexes, while f
and f
represent the
intrinsic pyrene fluorescences of actin in GS* and G
S*,
respectively. As discussed previously(12) , f
represents the average fluorescence of the two actins in
G
S*, but the specific fluorescences of each actin molecule
in G
S*, f`
and f``
, are unknown, and f
= 1/2(f`
+ f``
).
The amplitude F of the
fluorescence change is the difference between the fluorescences
observed at equilibrium (t
) and at time 0:
Quantitative analysis of the amplitude data ( Fig. 2and Fig. 3) is complex since a total of six parameters (four
equilibrium dissociation constants K, K
, K
, and K
and two fluorescence parameters f
and f
) are involved. Theoretical curves describing the
amplitude of the fluorescence change at different total G-actin and
S
concentrations were generated by computer using and and the following mass conservation
equations:
The concentration of free G-actin was the solution of the
quadratic as a function of [S]. A series of
incremented values of [S] were generated, from which the
corresponding values of [G], [S], and
the concentrations of all G-actin-S
complexes were
calculated. To simplify the search for equilibrium parameter values,
useful hints were derived from visual inspection of the data obtained
under extreme conditions where only binary or ternary complexes exist,
as follows. In Fig. 4, the change in k
with [S
] in a range of high values (
4
µM) of [S
] reflects the formation of
GS and GS* complexes essentially, in which only the constants K
and K
are involved.
Examination of the data suggests that K
is of the
order of 1 µM. The value of k
reached at saturation by S
represents k
+ k
= 210 ± 20s
. The shape of the
curve k
versus [S
] above 5 µM S
imposed a range of possible values of k
between 5 and 30 s
, corresponding to a range
of K
of 6-40. Note that if K
was much larger than 1 µM, the value of K
0.1 would impose K
K
0.1 µM,
a value too high to be compatible with the dependences of the
amplitudes on S
and G-actin, which impose K
K
< 0.1 µM.
Similarly, the dependence of k
on G-actin (Fig. 5)
in a range of high concentrations, and the shape of the titration
curves (amplitude data, Fig. 2and Fig. 3) required that
the value of K
be at most 0.1 µM, and K
between 3 and 6. With these constraints the best
fit to the titration curves (amplitude data, Fig. 2and Fig. 3) was sought varying K
, K
, f
, and f
. In parallel, the time courses of the change in
fluorescence were simulated, in a large range of G-actin and S
concentrations, using KINSIM, searching for the best fit to
experimental traces by refining the values of k
, k
, k
, k
keeping
with the imposed constraints. The resulting best values of equilibrium
and rate parameters are given in Table 1. From these values, it
appears that a large increase in affinity of G-actin for S
is linked to the isomerization steps k
and k
, and the fluorescent complexes
GS* and G
S* are largely predominant over their
non-fluorescent counterparts. Setting f
= 1
by convention, a value of f
= f
= 1.6 ± 0.1 was found to fit all
data adequately. In a search for the best fit to the data, it was found
that fluctuations of more than 20% in the values of each of the
parameters taken separately yielded sets of theoretical curves that
could not fit the set of amplitude data adequately even upon varying
the values of other parameters in a compensatory fashion. In other
words, the two complementary experimental saturation curves in Fig. 2and Fig. 3together impose serious constraints in
the choice of equilibrium parameters, and we trust that the proposed
set of values is reasonably robust. The solid lines in Fig. 2and Fig. 3are theoretical curves calculated within using the values of K
, K
, K
, K
, f
, and f
given in Table 1for the two isomers of S
.
Finally the
distribution of the different actin-S complexes present in
solution in a broad range of actin and S
concentrations,
calculated within and consistent with all data shown, is
displayed in Fig. 10.