The fraction of actin in an active state determined from pyrene excimer fluoresecence agreed with that calculated from light-scattering measurements of myosin subfragment 1 S1–ADP to regu
Trang 1troponin states, determined by pyrene–tropomyosin
fluorescence
Boris Gafurov1and Joseph M Chalovich2
1 Uniformed Services University of the Health Sciences, Department of Pharmacology, Bethesda, MD, USA
2 Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA
The ATPase activity of striated muscle myosin is low
unless it is bound to actin Actin activation is inhibited
by the regulatory proteins tropomyosin, troponin T,
troponin I and troponin C, which bind along actin
fila-ments of skeletal and cardiac muscles Activation of
striated muscle contraction occurs when Ca2+binds to
troponin C, or in a Ca2+-independent manner when
rigor-type myosin binds to actin [1–3] Myosin is both
the target enzyme that hydrolyzes ATP and a potential
allosteric activator Much current work is devoted to understanding the structural and functional changes that occur in the large co-operative system consisting
of myosin, actin, troponin and tropomyosin Structural changes in troponin [4] and tropomyosin [5], in response to either Ca2+ or myosin subfragment 1 (S1) binding, have been documented The structure of actin
is plastic [6] and could also change in response to the regulatory proteins
Keywords
parallel pathway model; pyrene
iodoacetamide; regulation of contraction;
tropomyosin; troponin
Correspondence
Joseph M Chalovich, Department of
Biochemistry and Molecular Biology, Brody
School of Medicine at East Carolina
University, 5E-122 Brody Bldg, Greenville,
NC 27834, USA
Fax: +1 252 7443383
Tel: +1 252 7442973
E-mail: chalovichj@ecu.edu
Website: http://www.ecu.edu/biochemistry/
Chalov.htm
(Received 11 December 2006, revised 10
February 2007, accepted 1 March 2007)
doi:10.1111/j.1742-4658.2007.05765.x
Actin–tropomyosin–troponin has three structural states, but the functional properties of regulation can be explained with models having two func-tional states As a step towards assigning funcfunc-tional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin Tropomyosin labeled with pyrene–iodoacetamide
is thought to reflect the transition to the most active state,
where-as N-((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I is thought to monitor the transition to any state other than the inactive state The fraction of actin in an active state determined from pyrene excimer fluoresecence agreed with that calculated from light-scattering measurements of myosin subfragment 1 (S1)–ADP to regulated actin in both the presence and absence of Ca2+ over a range of ionic strength conditions The only exceptions were conditions where the binding
of S1–ADP to actin was too strong to measure accurately Pyrene–tropo-myosin excimer fluorescence was Ca2+ dependent and so reflected the change in population caused by both Ca2+ binding and S1–ADP binding Pyrene labeling of tropomyosin did not cause a large perturbation of the transition among states of regulated actin Using pyrene–tropomyosin fluorescence we were able to extend the ionic strength dependence of the parameters describing the co-operativity of binding of S1–ADP to actin as low as 0.1 m The probes on tropomyosin and troponin I had different responses to Ca2+ and S1–ADP binding These different sensitivities can
be explained by an intermediate between the inactive and active states of regulated actin
Abbreviations
IANBD, N-(((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole; S1, myosin subfragment 1.
Trang 2Tropomyosin occupies three positions on actin
(Fig 1), depending on the amount of Ca2+ bound to
troponin and to the amount of S1 bound to actin
[7–10] These three structural states are in rapid
equi-librium with each other [11–13], so that in each
condi-tion there is a distribucondi-tion of states [8] Some models
of regulation are constructed around the assumption
that each structural state has a unique function Other
models use the minimum number of states required to
simulate function An ongoing question is what are the
properties of these three states and how do they relate
to regulation
Two types of regulatory models are shown in Fig 2
In parallel pathway models (Fig 2A,B), actin exists in
two or three states, with discrete abilities to serve as
cofactors for myosin-catalyzed ATP hydrolysis The
relative populations of these actin states are
deter-mined by Ca2+and bound S1–ADP The overall
activ-ity of the system at any condition is defined by the
fraction of time that myosin is bound to each of these
actin states More detailed schemes of a parallel
path-way model, showing some steps in ATP hydrolysis,
have been published previously [14,15] The formalism
for a parallel pathway model was originally defined for
two functional states of actin, for simplicity [14,16]
Despite early concerns that a two-state model could
not explain the binding kinetics, it has been shown to
simulate equilibrium binding, binding kinetics and
regulation of ATPase activity correctly [15]
Tropomy-osin is a switch, in the parallel model, that changes the
structure of actin in some way that alters its ability to
stimulate myosin ATPase activity [17] Because the
two-state model is able to explain many features of
regulation, the properties of any intermediate state that
may be present are undefined The potential to define
the intermediate state does exist if it can be observed
in real time
In sequential models of regulation, actin passes from state AB (blocked) to AC (closed) to AO (open) In sequential models, one cannot define the activity of an individual state Only state AO supports myosin activ-ity, so it is necessary to go stepwise from the blocked
to the closed to the open states The model shown in Fig 2C is from McKillop & Geeves [18] and is based
on the multiple-step binding of myosin to actin Another model, that of Butters & Tobacman, has three states of actin with different orientations of tropomyo-sin that are in equilibrium with each other and with a fourth state, in which actin has undergone a conforma-tional change to an active state with a structure similar
to that stabilized by the binding of myosin [19] That model is not shown here, but it may be imagined as a funnel in which three states of regulated actin funnel
to an active state that supports contraction
The models in Fig 2 share the idea of multiple forms of regulated actin with different activities in equilibrium with each other Changes in the
distribu-1
2
3
4
EGTA Ca2+ rigor S1 bound
Fig 1 Cross-sections of actin–tropomyosin–troponin showing the
structural states identified in the absence of Ca 2+ , with saturating
Ca 2+ and with bound rigor-type myosin subfragment 1 (S1)
Tropo-myosin is shown in black The cross-section of an actin filament is
shown in outline and the orientation of the four subdomains is
shown The dashed line is for reference The figure is based on
Craig & Lehman [51].
AB AC AO
MAC MAO MAR
K1
K2
K1
MAi MAa
i
Ai(Ca) Aa
B
α β
Fig 2 Models of regulation of striated muscle contraction Actin is represented by the letter A with a subscript to designate its state; myosin is represented by the letter M The large differences in interactions among different myosin-nucleotide states is not shown Panels A and B show two-state and three-state parallel pathway models In the two-state version, myosin binds to actin that is either in the inactive (A i ) or active (A a ) state The distribution between Ai and Aa is determined by the fraction of troponin C (TnC) sites with bound Ca 2+ and the fraction of actin sites with bound rigor-type cross-bridges Rapid ATP hydrolysis occurs when actin is in state Aa The three-state model shown in (B) considers the possibility that regulated actin that has bound Ca 2+ , but no rigor-type cross-bridges, has an intermediate level of activity For simplicity, the binding to myosin is not shown for this case In this model, state Aais active and state Aiis inactive, but the properties
of state A i(Ca) , are uncertain Panel C shows a sequential model in which there are three states of actin namely blocked (A B ), closed (AC) and open (AO) Actin makes sequential transitions to the open state, AO, which is competent for accelerating ATP hydrolysis and proceeding into the force-producing state MA R
Trang 3tion of regulated actin states determine the activity of
actin–myosin, regardless of whether that activity
chan-ges as a normal regulatory response [14,20], or as a
result of some mutations in troponin [21,22] or in
experimentally produced mutations in tropomyosin
[23] Therefore, it is important to have reliable
meth-ods of determining the state of actin in real time This
manuscript explores, in detail, a well-known method of
monitoring the state of regulated actin
The fraction of actin in the active state can be
meas-ured in real time by fluorescence changes of probes on
troponin and tropomyosin Probes on troponin I
respond to both Ca2+binding and to S1–ADP binding
These probes give a good estimate of the changes in
dis-tribution of regulated actin as S1 or S1–ADP binds to
actin [11,12,24,25] Resonance energy transfer
measure-ments between probes on actin and troponin I [26] or
troponin T [13] have also proven to be valuable for
measuring the state of the actin filament Changes in
pyrene–tropomyosin fluorescence have been shown to
be a measure of the fraction of actin in the active state
[27] Pyrene–tropomyosin excimer fluorescence was
sensitive to activation by S1, but Ca2+had little effect
[27,28] Pyrene–tropomyosin excimer fluorescence did
give the predicted change in regulated actin distribution
as the amount of S1–ADP was altered, but its usefulness
was only demonstrated at relatively high ionic strength
The response of pyrene–tropomyosin fluorescence to S1
binding led to the idea that this probe measures entry
into the most active state of actin, but is insensitive to
transitions to states of intermediate activity
We report here a comparison of
pyrene–tropomyo-sin excimer fluorescence to predicted changes in the
actin state that occur in response to Ca2+ and
S1–ADP binding under conditions ranging from 100
to 240 mm ionic strength We also compare changes in
pyrene excimer fluorescence with
N-(((2-iodoacetoxy)-ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole
(IANBD)-labeled troponin fluorescence when both
probes are present on the same actin filament The
results can be readily explained by the presence of an
intermediate between the inactive and fully active
states of regulated actin Pyrene excimer formation did
not appreciably affect the distribution of actin states
Furthermore, pyrene excimer fluorescence gave
reason-able estimates of the distribution of actin states at
ionic strengths as low as 0.1 m, where it may be
possi-ble to correlate these changes with ATPase activities
Results
Regulated actin is predominantly in the inactive state
in the absence of Ca2+and bound S1 Both Ca2+and
S1–ADP bind more tightly to the active state of actin than to the inactive state, and stabilize the active state Increasing concentrations of free S1–ADP results in a co-operative binding curve, indicating a transition from a lower affinity to a higher affinity state of actin– tropomyosin–troponin This change in affinity is read-ily seen in the absence of Ca2+ as sigmoidal increases
in theta with increasing free S1–ADP concentrations (Fig 3A–D) Changes in pyrene–actin fluorescence are often used to measure the binding of S1 to actin (Fig 3; solid squares) In order to compare changes in pyrene–tropomyosin excimer fluorescence with changes
in occupancy of actin with S1, we utilized light scatter-ing to measure bindscatter-ing (open circles) Light scatterscatter-ing measurements gave binding patterns that were similar
to previous measurements using pyrene–actin fluores-cence (compare circles with solid squares) Theoretical curves, describing the relationship between theta and free S1–ADP, were produced by fitting the Hill model
to the data at the four ionic strength conditions shown
in Fig 3 This fitting procedure produced values of
K1, K2, L¢ and Y Those parameters were used to pro-duce theoretical curves for p2, the fraction of actin in the active state shown by solid curves in Fig 3 Figure 3A–D also shows that changes in pyrene– tropomyosin excimer fluorescence (triangles) followed the predicted changes in the fraction of actin in the active state The agreement between the theoretical curves and the measurements was particularly good at higher ionic strengths where the measurements were most accurate Deviations between the predicted values
of p2 (solid curve) and the measured value (triangles) were apparent at 0.1 m ionic strength Whereas exci-mer fluorescence (triangles) was low at zero free S1–ADP, the solid curve predicted from equilibrium binding data (circles) predicts an excess of 20% of the actin to be present in the active state Values of p2 near zero would be consistent with known activities That is, the p2 values determined from tropomyosin fluorescence are probably more reliable than those calculated from binding studies at low ionic strength Values of equilibrium binding parameters, deter-mined in the absence of Ca2+ as a function of ionic strength, are shown in Fig 4A–C The open symbols show the present results of binding of S1–ADP to actin containing troponin and pyrene-labeled tropomy-osin Equilibrium binding parameters were calculated
by fitting the Hill formalism to light scattering alone (circles), or to pyrene–tropomyosin fluorescence alone (triangles) The values of K2 shown in Fig 4A were independent of the type of fitting used and they agreed very well with earlier values determined from pyrene– actin fluorescence shown as solid squares The model
Trang 4is not particularly sensitive to values of K1, so these values are not shown
Figure 4B,C shows the parameters Y and L¢ Y decreased with increasing ionic strength, indicating a decreased tendency of adjacent regulatory units to exist
in the same functional state Values of Y, calculated from light scattering, were similar to those calculated from pyrene–tropomyosin fluorescence However, val-ues of Y tended to be slightly lower for the pyrene– tropomyosin system than for the pyrene actin system examined earlier, shown as solid squares It is unclear
if this difference is a result of the different probes used
Values of L¢ tended to increase with increasing ionic strength Therefore, high ionic strength stabilized the
Fig 3 Changes in light scattering (circles) and pyrene–tropomyosin
fluorescence (triangles) as a function of free myosin subfragment 1
(S1)–ADP concentration in the absence (A–D) and presence (E–H) of
Ca 2+ Measurements were made at 0.1 (A, E), 0.12 (B, F), 0.18
(C, G) and 0.24 (D, H) molar ionic strengths The curves shown with
a dashed line are fits of the Hill model to the fraction of actin with
bound S1, determined by light scattering Curve fitting was
per-formed simultaneously with paired data sets, in the presence and
absence of Ca 2+ , to constrain the variables Fractions of actin in the
active state, p2, were calculated from the equilibrium binding
param-eters (solid curves) Estimates of p2 determined from
pyrene–tropo-myosin fluorescence (triangles) are also shown Solid squares are
from a previous study with pyrene actin [15] to show that similar
values of theta are obtained by light scattering measurements and
earlier pyrene-actin measurements All measurements were made
using skeletal troponin and tropomyosin under the following
condi-tions: 0.3 l M phalloidin actin, 0.06 l M pyrene-labeled tropomyosin,
0.06 l M troponin, 25 C, in a buffer containing 20 m M Mops, pH 7.0,
5 m M MgCl2, 1 m M dithiothreitol, 2 m M ADP, 0.2 mgÆmL)1bovine
serum albumin, sufficient KCl to reach the target ionic strength and
either 1 m M EGTA (A–D) or 0.1 m M CaCl 2 (E–H).
Fig 4 Effect of ionic strength on equilibrium binding parameters in the absence (A–C) and presence (D–E) of Ca2+ Values of K 2 (A, D),
Y (B, E) and L¢ (C, F), determined by light scattering (circles) and pyrene-excimer fluorescence (triangles), are compared with earlier values determined from pyrene–actin fluorescence (solid squares) Values obtained from light scattering were obtained by a global fit
of the model to data obtained at zero and saturating Ca 2+ Earlier values from pyrene–actin fluorescence were the result of a global fit of data from six different free Ca2+concentrations but the same ionic strength [15] The conditions were the same as for Fig 3, with 1 m M EGTA used in the experiments with results shown in panels A–C and 0.1 m M CaCl 2 used in the experiments with results shown in panels D–F.
Trang 5inactive state of regulated actin relative to the active
state when no rigor type S1 was bound to actin L¢
values were similar when determined by S1–ADP
bind-ing or by tropomyosin–pyrene excimer fluorescence,
and the results were in general agreement with earlier
pyrene–actin fluorescence measurements
To determine the relationship of K2, Y and L¢ to
ionic strength in Ca2+, we first determined the effect
of Ca2+on fluorescence so that the initial point of p2
could be defined Figure 5 shows pyrene–tropomyosin
fluorescence measurements of regulated actin as a
function of Ca2+concentration at 180 mm ionic
strength In 0.1 mm EGTA, the free Ca2+ was below
that required for activation (open circles) The pyrene
fluorescence intensity increased to a maximum value
when Ca2+exceeded the EGTA concentration A
con-trol experiment was performed in the absence of
EGTA (solid circles) As expected, there was no
change in fluorescence with the addition of Ca2+
because the initial Ca2+concentration was already
high enough to give the full effect
We performed another control by comparing the
effects of Ca2+ on probes on both tropomyosin and
troponin Actin filaments were reconstituted with
pyrene-labeled tropomyosin and troponin containing
IANBD-labeled troponin I Figure 6A shows that the
addition of excess Ca2+ to an EGTA-containing
solu-tion caused 40% of the maximum possible change in
pyrene–tropomyosin fluorescence, but, on average,
92% of the maximum in IANBD–troponin I
fluores-cence The complete change of pyrene–tropomyosin
required the addition of nucleotide-free S1 Figure 6B
compares the effect of both probes to the addition of S1 in the absence of Ca2+ Although the changes are
in opposite directions, the sensitivities to S1 concentra-tion were similar
Knowing the value of p2 to be 0.4, in the absence of S1–ADP we were able to examine the relationship between predicted values of p2 and pyrene excimer fluorescence in the presence of Ca2+ Figure 3E–H shows light scattering and pyrene excimer fluorescence
at four ionic strength conditions at saturating Ca2+ Values of p2 reached their maximum values at subsat-urating concentrations of S1–ADP in all cases The
Fig 5 The fluorescence of actin filaments reconstituted with
pyrene-labeled tropomyosin is Ca 2+ dependent at 180 m M ionic
strength Pyrene–tropomyosin fluorescence was measured in the
presence (open circles) or absence (closed circles) of 0.1 m M
EGTA The curve obtained in the presence of EGTA shows the
increase in fluorescence as the total Ca 2+ concentration was
increased The conditions were the same as for Fig 3.
Fig 6 Fluorescence changes in pyrene-labeled tropomyosin (cir-cles, solid lines) and N-(((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD)-labeled troponin I (squares, broken lines) upon titration of actin–tropomyosin–troponin with
Ca 2+ and myosin subfragment 1 (S1) Both fluorescent probes were present in the actin filament at the same time and the fluor-escence changes of each probe were measured about 10 min after each addition of S1 (A) Effect of adding 1.2 m M Ca 2+ to the EGTA-containing solution and then subsequently adding S1 The response to Ca 2+ was more extreme for IANBD–troponin I than for pyrene-labeled tropomyosin Multiple lines are from emission measurements made at 10 nm wavelength increments (B) Titra-tion of regulated actin containing both probes with S1 in the absence of Ca 2+ The conditions were the same as for Fig 3, with
150 m M KCl.
Trang 6tropomyosin transition measured by pyrene
fluores-cence was not co-operative in the presence of Ca2+
The dashed lines are fits of the Hill model to the
val-ues of light scattering data, and the predicted curves
for p2 are shown as solid lines The measured values
of p2 were similar to the predicted values Poor fits to
light scattering data, as in Fig 3H, were, in part, a
result of the fact that these were not best fits to a
sin-gle data set, but were global fits to data in the presence
and absence of Ca2+
The ionic strength dependencies of K2, Y and L¢,
determined by fitting the Hill model to the data of
Fig 3E–H, are shown in Fig 4D–F The agreement of
values of K2, Y and L¢ was good between light
scatter-ing (circles) and pyrene–tropomyosin fluorescence
(tri-angles) measured on the same proteins Values of K2
were similar to those measured in the absence of
Ca2+ Values of Y were near 1 at low ionic strength
and decreased slightly as the ionic strength was raised
If Y was constrained to be greater than 1, the value of
Y would be 1 over the ionic strength range (data not
shown) Values of L¢, determined by both methods,
increased with increasing ionic strength as they did in
the absence of Ca2+
Values of Y and L¢ were substantially different for
actin filaments containing pyrene-labeled tropomyosin
compared with those with pyrene on the actin Fitting
was generally more difficult in the presence of Ca2+
because of the lack of features in those curves
Estima-tions of L¢ and Y are problematic because changes in
the value of Y can be compensated, to some extent,
for changes in L¢
The parameter, p2, defines the activity of the actin
filament in parallel pathway models Under conditions
where all of the S1-ATP is bound to actin, the ATPase
activity is approximately equal to p2*rmax+
(1) p2)*rmin, where rmaxand rminare the rates for the
active and inactive actin species, respectively A
correc-tion to this equacorrec-tion can be made for the small
differ-ence in affinity of S1-ATP for actin in states 1 and 2
Values of rmax and rmin can be determined from the
kcat for actin in the active and inactive states,
respect-ively Although these ATPase parameters have not
been determined at the conditions used for the binding
experiments, relative changes in ATPase activity
can be approximated by observation of changes in p2
Figure 7 shows how p2 would change if actin filaments
were activated by the attachment of an activating form
of S1, such as S1–ADP The inset shows values of p2
as a function of the square root of the ionic strength
The difference between the EGTA and Ca2+rates are
expected to be approximately constant over the range
of ionic strengths examined
Discussion
Transitions between the inactive and active states of regulated actin are important determinants of the regu-lation of striated muscle contraction The distribution
of these states determines the ATPase activity, whereas the rates of transitions among the states may affect the rate of force redevelopment [11] Some disease-causing mutations in troponin T change in the distribution between the states of regulated actin [21,22] The abil-ity to measure state transitions rapidly and relate them
to function will be helpful in studying such defects Fluorescent probes on troponin and tropomyosin have the potential to measure the distribution of states in real time
Ishii & Lehrer reported that probes on tropomyosin reflect changes in the fraction of actin in the active state resulting from S1 binding [27] Acrylodan-labeled tropomyosin was useful for actin–tropomyosin, but the signal was too small in the presence of troponin [29] Pyrene-labeled tropomyosin was the prefered probe for actin–tropomyosin and actin–tropomyosin–troponin [27,28] Pyrene–iodoacetamide labeling was preferred over pyrene–maleimide labeling because of the rapid response of its excimer fluorescence to S1 binding [27] The S1-induced increase in excimer fluorescence is caused by an increase in the fraction of pyrene mole-cules forming excimers Pyrene–iodoacetamide-labeled tropomyosin excimer fluorescence exhibited a small change with Ca2+ at low ionic strength Because of these considerations, we have examined more closely
Fig 7 Calculated probabilities of actin–tropomyosin–troponin in the active state (p2) in the presence (solid lines and solid circles) and absence (dashed line and open circles) of Ca 2+ Simulations were made from equilibrium binding parameters determined at 120 m M ionic strength The inset shows how values of p2 in the absence of added myosin subfragment 1 (S1) change as a function of the square root of the ionic strength.
Trang 7the suitability of pyrene–tropomyosin excimer
fluores-cence as a measure of regulated actin state changes
We studied tropomyosin excimer fluorescence over a
range of ionic strength conditions because ATPase
measurements and S1–ADP binding cannot be readily
measured under the same conditions and an
extrapola-tion of parameters is necessary Furthermore,
examin-ing the behavior at different conditions increases the
reliability of parameters obtained by curve fitting
[15,21] Values of the fraction of actin in the active
state, p2, were calculated from S1–ADP binding (light
scattering) Values of p2 were also directly measured
by pyrene excimer fluorescence Pyrene excimer
fluores-cence generally agreed with the predicted values of p2
Deviations occurred when S1–ADP binding became
too strong to measure accurately In those cases,
exci-mer fluorescence was a more reliable measure of p2
To determine if the energetics of formation of
tropo-myosin–pyrene excimers biased the distribution of
actin states, we compared the present results with
ear-lier studies where binding was measured with
pyrene-labeled actin and unpyrene-labeled tropomyosin Values of L¢
obtained from light scattering measurements with
pyrene-labeled tropomyosin in the absence of Ca2+
are in reasonable agreement with earlier values where
there was no excimer formation (Fig 4) Pyrene probes
on tropomyosin did not significantly alter the values of
K2, L¢ or Y at any ionic strength examined
Further-more, when troponin containing an IANBD probe on
troponin I was reconstituted with
N-(1-pyrene)iodo-acetamide (pyrene–iodoN-(1-pyrene)iodo-acetamide)-labeled tropomyosin
and actin, the IANBD probe retained its typical
responses to changes in Ca2+and S1 binding (Fig 6)
Fitting binding curves to obtain binding parameters
is difficult in the case of Ca2+ because the curves are
featureless hyperbolas Although we observed only
small differences in binding curves measured with
pyrene–actin and pyrene–tropomyosin in Ca2+ (Fig
3G–H), there was poor agreement between the values of
L¢ calculated in the two cases We also noted that at
low ionic strength the values of Y tended to be greater
in the presence of Ca2+, but this was not observed in
the present case with unlabeled actin It is also worth
pointing out that the parameters determined in our
earlier study with pyrene–actin resulted from global fits
of the data This change in fitting may contribute to
differences in the final values of the parameters
The parameters K2, L¢ and Y varied with ionic
strength, in agreement with our earlier observations
[15,21] High ionic strength decreased the fraction of
regulated actin in active states (increased L¢) This
result is consistent with in vitro motility assays where
higher Ca2+is required for full activation at high ionic
strength [30] This trend has now been observed from 0.1 to 0.24 m ionic strength The extension of this result to the lower ionic strength range is useful for extrapolation of the values for future simulation of ATPase activities under conditions where they can be readily measured
Tropomyosin excimer fluorescence was Ca2+ dependent, but it did not directly track Ca2+ binding Rather, the change was consistent with a state change, such as partial transition, to the most active state of actin or a total transition to an intermediate state
Ca2+ binding resulted in 40% of the maximum observed change of excimer fluorescence obtained with full activation by rigor-type myosin binding This agrees with the observation of Williams et al., that
Ca2+ alone provides 40% of the maximum value of
kcat[31]
In vitro motility assays support the view that Ca2+ alone does not provide full activation of regulated actin High levels of loading of filaments with myosin produced about a doubling of the rate at saturating
Ca2+[32] and a velocity 1.8 times higher than that of unregulated actin [33] Activities that exceed actin alone are probably the result of partial stabilization of the most active state of regulated actin In the case of cardiac troponin–tropomyosin, this extra activation was only evident for some disease-causing mutations
of troponin [34] Under those conditions, the velocity was increased 1.6-fold over full activation of the wild-type cardiac troponin Some mutations have the effect
of partially stabilizing the fully active state [21], so this 1.6-fold increase is probably an underestimate of the maximum level of activation These results suggest that
in the motility assay, Ca2+ alone produces 50–55% of the maximum activation The results could be closer to the 40% activation seen in solution for Ca2+ alone if the actin filaments in the in vitro studies were not max-imally activated
The ability of S1–ADP and rigor S1 to activate actin filaments raises the question of how an active muscle can relax once the free Ca2+ concentration is decreased A larger fraction of strongly bound cross-bridges is required for activation in EGTA than in
Ca2+ However, in EGTA at 0.18 m ionic strength, 30% saturation of the actin produces thin filaments that are 50% active (Fig 3C) A 90% relaxation would require less than 5% of the actin to contain strongly bound cross-bridges However, muscle may not behave identically to the proteins in solution Geometrical considerations, and the presence of other protein components or small molecules, could result in a considerable shift of the curves shown in Fig 3
Trang 8Probes on troponin I report changes in the state of
regulated actin caused by S1 binding to actin [12] and
also respond directly to changes in Ca2+ [12,24,25]
Different sensitivities of fluorescent probes to Ca2+
have been used in the past to argue for the presence of
an intermediate state of regulated actin Because the
probes can affect the behavior of the regulatory
com-plex, it is difficult to compare directly the results of
probes on separate regulatory complexes We have
now utilized IANBD on troponin I and pyrene on
tropomyosin within the same regulatory complex Both
probes responded to S1 binding in a similar way
(Fig 6A), but exhibited different responses to Ca2+
(Fig 6B) This result is consistent with the existence of
an intermediate structural state [7]
We used the two-state parallel pathway model of
Hill et al for predicting the fraction of actin in the
act-ive state That model is consistent with the measured
effects of Ca2+ on binding in the presence of ATP
[35,36], equilibrium binding in the presence of ADP
[16], binding kinetics [15,37] and the general features
of ATPase activities [14] Furthermore, in our view,
the functionally indistinguishable state is not the first
state of a series, but rather the state corresponding to
bound Ca2+and no bound rigor S1 (Ai(Ca)in Fig 2B)
That intermediate may resemble the inactive (Ai(EGTA))
or fully active (Aa) states in terms of key functional
properties
Although our results can be explained with two
functional states, there is evidence for three structural
states of regulated actin Pirani et al estimated the
dis-tributions of structural states by image reconstruction
of electron micrographs following dilution of the
pro-teins to low ionic strength [8] They predicted 22% of
the actin to be in the closed state in the absence of
Ca2+ [8] Because the actin filament is has little
activ-ity in EGTA [31], the closed state must be inactive
Pirani et al predicted the distribution in Ca2+ to be
20% blocked, 68% closed and 12% M state (active state) The 40% activation, predicted in the present study, from tropomyosin fluorescence does not agree with this distribution This could be an indication that there is not a simple correlation between observed struc-tural states and functional states of regulated actin
We also evaluated our results in terms of the three-state sequential model of regulation proposed by McKillop & Geeves [18], as shown in Fig 2C The increased rate of binding of S1–ADP to regulated actin
in Ca2+compared with EGTA was interpreted, in that model, as 75% of actin sites being blocked in the absence of Ca2+ We have an alternative explanation for that effect [37] However, for the present exercise
we forced the fit to populate the blocked state in EGTA in accordance with their model We used most of the constraints set by McKillop & Geeves, namely, 0 < KB < 10, 0 < KT < 20, 0 < N < 7,
103< K1 < 106 and K2¼ 200 We did not constrain the values of ‘n’ and we consequently obtained a dif-ferent pattern of changes in this parameter The simu-lations shown in Fig 8 demonstrate that popusimu-lations
of both the blocked and closed states decreased with increasing amounts of bound S1 in both the absence and presence of Ca2+ The population of the open state was much higher in Ca2+ than in EGTA in the absence of bound S1 Regulated actin was almost exclusively in the open state at saturating S1, irrespect-ive of the Ca2+concentration Whereas the population
of the open state does not correlate directly with our predicted p2, they do follow the same trend
Tropomyosin–pyrene excimer fluorescence gives a good estimate of the fraction of actin in the active state over a range of conditions Simultaneous mea-surements of probes on tropomyosin and troponin give evidence for an intermediate state By taking further advantage of this system, it may be possible to determine the role of this intermediate in regulation
Fig 8 Distribution of the blocked (circles), closed (triangles) and open (squares) states
in the course of myosin subfragment 1 (S1) binding (A) The predicted occupancy of the states at 0.18 M ionic strength in the pres-ence of 0.1 m M Ca2+ The diamonds are the p2 parameter that represents the transition
of the actin filament into the active state in Hill’s model (B) The same parameters in the Ca 2+ -free case.
Trang 9This is particularly important for the study of
disor-ders of the regulatory system
Experimental procedures
Protein preparation
Actin [38,39], myosin [40], troponin and tropomyosin [41]
were isolated from rabbit back muscle Myosin S1 was
made by digestion of myosin with chymotrypsin [42]
Pro-tein concentrations were determined by light absorbance at
280 nm, corrected for scattering, at 340 nm, using the
fol-lowing extinction coefficients (e0.1%) for 280 nm: actin,
)1.15; myosin-S1, )0.75; tropomyosin, )0.33; and troponin,
)0.37 The molecular masses assumed for the key proteins
were: actin, )42 000 Da; myosin-S1, )120 000 Da;
tropo-myosin,)68 000 Da; troponin, )71 000 Da
Actin was stored as a 40 lm stock in 4 mm imidazole
(pH 7.0), 1 mm dithiothreitol, 2 mm MgCl2 and 40 lm
phalloidin Actin was sometimes labeled with N-(1-pyrenyl)
iodoacetamide [43] Tropomyosin was labeled with
N-(1-pyrene)iodoacetamide (pyrene–iodoacetamide) [27] In
some cases, troponin I was labeled with IANBD [12] The
extents of labeling were 60% and 35% for tropomyosin
and troponin, respectively Reconstituted actin was
pre-pared by mixing actin with troponin and pyrene-labeled
tropomyosin in a 3 : 1 : 1 molar ratio to ensure saturation
of actin at the low concentrations used for binding studies
Equilibrium fluorescence measurements
Equilibrium fluorescence measurements were made on an
Aminco Bowman II Luminescence Spectrometer (Thermo
Electron Corp., Madison, WI, USA), having the cell
com-partment maintained at 25C with a circulating water bath
For light scattering measurements, the excitation and
emis-sion monochrometers were set at the same wavelength
Exci-tation and emission wavelengths used were 340 and 480 nm,
respectively, for tropomyosin–pyrene excimer fluorescence
and 492 and 536 nm, respectively, for IANBD–troponin
fluorescence
Equilibrium titrations of actin with S1–ADP were
per-formed by observing the light scattering,
pyrene–tropomyo-sin fluorescence [44] and by quenching of pyrene–actin
fluorescence [43,45,46] Details of the binding measurements
are described elsewhere [15] and are similar to those
des-cribed by others [46,47] Our binding solutions contained
20 mm Mops, pH 7.0, 5 mm MgCl2, 1 mm dithiothreitol,
2 mm ADP, 0.2 mgÆmL)1 bovine serum albumin, sufficient
KCl to reach the target ionic strength and 0.1 mm CaCl2or
1 mm EGTA The actin concentration in binding
experi-ments was 0.3 lm Solutions also contained 14 unitsÆmL of
hexokinase and 1 mm glucose to scavenge ATP and 20 lm
Ap5A to inhibit ATP formation through the myokinase
reac-tion Titrations were carried out by the stepwise addition of S1 to a 2 mL volume of pyrene-labeled actin–tropomyosin– troponin at 5 min intervals This time interval was important
to ensure equilibrium at each step Fluorescence intensities and protein concentrations were corrected for the volume change (< 10%) caused by adding S1 Rabbit skeletal tropo-nin and tropomyosin were used in this study for comparison with our existing data for those regulatory proteins
Values of theta (S1 bound to the actin total ratio) and the free S1 concentration from fluorescence or light scatter-ing measurements were calculated usscatter-ing the equations:
h¼ Fi Fmin
Fmax Fmin
½FreeS1 ¼ ½S1total h ½Actintotal ð1Þ
Where Fiis the fluorescence or scattering intensity at a total S1 concentration of i (lm); and Fmaxand Fminare the maxi-mum and minimaxi-mum values of intensity, respectively
Modeling experimental results
Light scattering was used to measure the binding of S1– ADP to actin and tropomyosin Pyrene excimer fluorescence was used to monitor the fraction of actin in the active state Equilibrium-binding parameters were extracted from light scattering data by using the co-operative binding model of Hill et al [16] or by the model of McKillop & Geeves [18] Fitting to the parallel pathway model of Hill was described
in detail earlier [15] Briefly, the relationship between the fraction of actin with bound S1 and the free S1 concentra-tion can be described by the following equaconcentra-tions [16]:
h¼ p1h1þ p2h2
hi¼ KiC
1þ KiC
p1¼ 1 p2
Y
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1 aÞ2þ 4a
Y
q
1 a þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1 aÞ2þ 4a
Y
q
a¼ð1 þ K2CÞ
n
ð1 þ K1CÞn
Y22ðqÞ
LY11ðqÞ
L0¼LY11ðqÞ
Y22ðqÞ
Y¼Y11ðqÞY22ðqÞ
Y12ðqÞY21ðqÞ
YijðqÞ ¼ xijþ 2kaqyijþ k2q2zij
9
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> ð2Þ
p1 and p2 are fractions of actin units in the inactive and active states, respectively; hI and h2 are fractions of actin containing bound S1 in the inactive and active states, respectively; K1 and K2 are S1-binding constants to the
Trang 10inactive and active states of actin, respectively; C is the free
S1 concentration; q is the free Ca2+ concentration; n is
the number of actin monomers in one actin–tropomyosin–
troponin unit (assumed to be seven); L is the equilibrium
constant for transition of an isolated actin–tropomyosin–
troponin unit with no neighbors, no bound Ca2+ and no
bound S1 from state 2 to state 1; L¢ is the equilibrium
con-stant defining the transition from the active state to the
inactive state for the entire actin filament, but without S1;
Y describes the co-operativity between adjacent regulatory
units of seven actin monomers; Y is the overall
co-operativ-ity parameter; Yij are individual co-operative interactions
between units in states i and j (we assumed that Yij¼ Yji);
xij, yijand zijrepresent the free energies of nearest neighbor
tropomyosin interactions (Wij) in exponential form e–Wij ⁄ kT
[16]; and kaand kbare affinities of troponin in states 1 and
2 for Ca2+ with values of 105
and 106Æm)1, respectively [48] We assumed that the values of ka,b did not change
over the ionic strength range in this study The simulated
curves were not very sensitive to the value of K1, so
simula-tions were normally carried out with the assumption that
K1¼ K2⁄ 8 [49]
All measurements were carried out in both Ca2+-free
and in Ca2+-saturated conditions Binding data obtained at
high and low Ca2+, but at the same ionic strength, were
analyzed using a global fit procedure [15] The global fit
helped to constrain the parameters Values of L¢, K2and Y
obtained from the fits were used to simulate p2, the fraction
of actin in the active state We also fitted theoretical values
of p2to the tropomyosin fluorescence to obtain L¢, K2and
Y From those values we were able to calculate curves of h
as a function of free S1–ADP
Tropomyosin fluorescence was normalized from 0 to 1 in
the absence of Ca2+ because we assumed that essentially
none of the actin was in the active state in the absence of
Ca2+and bound S1 This assumption is reasonable based on
ATPase activity measurements The flux is proportional to
the amount of S1 bound to each state multiplied by the kcat
associated with that state Ca2+ increases the kcat by
22-fold [20], whereas the binding of NEM-S1 increases the
kcatby a further 2.5-fold [31] This means that the fraction in
the active state in EGTA is 1.8% Binding studies were
car-ried out at higher ionic strength conditions than the ATPase
measurements Because the fraction of actin in the active
state decreases with increasing ionic strength [15], the value
of 1.8% is an upper limit The ATPase rates also predict that
in the presence of Ca2+alone, 40% of the regulated actin is
in the active state Again, this fraction is also likely to be an
upper limit because of ionic strength considerations
In order to define the fraction of actin in the active state
in the presence of Ca2+, but in the absence of bound S1,
we observed the changes in fluorescence that occurred
dur-ing Ca2+ titrations With measured values of the initial
value in EGTA, the change that occurred with the addition
of Ca2+ and the further change that occurred with
satur-ating S1–ADP, we were able to calculate the initial p2 in
Ca2+ The fluorescence data in Ca2+were normalized from this initial value to 1.0 for the maximum fluorescence observed in the presence of both Ca2+ and saturating S1–ADP Although the initial raw fluorescence values were higher in Ca2+than in EGTA, the values at saturating S1 were about the same in both cases
Fitting parameters and constraints were similar to the ones used in our earlier work [15] Global fitting was per-formed in the mlab Modeling System (Civilized Software, Bethesda, MD, USA) and always produced reasonable fits with correlation coefficients R2> 0.85
Analysis using the model of McKillop & Geeves
Because the original two-state parallel pathway model of Hill was able to account for the present data, the model was not expanded to include a third state We did, however, ana-lyze some of these data with the three-state sequential model
of McKillop & Geeves [18], shown in Fig 2C We fitted the model expressed in Eqn (3) to our binding isotherms and obtained key binding parameters K1, K2, KB, KTand n for each ionic strength and Ca2+concentration used:
h¼K1cðKTð1 þ K2ÞP
n1Þ þ Qn1
KTPnþ Qnþ 1=KB
ð3Þ
P¼ 1 þ K1cð1 þ K2Þ
Q¼ 1 þ K1c where K1 and K2 are S1-binding constants, KBis the equi-librium constant for proceeding from the blocked to the closed state, KTis the equilibrium constant for proceeding from the closed state to the open state, and n is a number
of actin monomers forming a co-operative unit We used constraints similar to those described elsewhere [15,50]
We determined the occupancy of the various states as a function of S1 bound by using differential equations to des-cribe the probability for each state [37] Curve fitting was carried out to our binding isotherms at 180 mm ionic strength, measured with or without Ca2+ The 3· 3 scheme
of the kinetic reactions, which take place when n¼ 1, is shown below, as derived previously [37]:
1
aB a-B
ck1 k-1
aT
a-T
ck1 k-1
aT
a-T
k2
k-2
Scheme 1.