The Flexibility of Actin Filaments as Revealed by Fluorescence
Resonance Energy Transfer
THE INFLUENCE OF DIVALENT CATIONS*
Miklós
Nyitrai
,
Gábor
Hild§,
József
Belágyi¶, and
Béla
Somogyi
§
From the
Research Group of the Hungarian Academy of
Sciences at the § Department of Biophysics, University
Medical School of Pécs, ¶ Central Research
Laboratory, University Medical School of Pécs, P. O. 99, H-7601 Pécs, Hungary
 |
ABSTRACT |
The temperature profile of the fluorescence
resonance energy transfer efficiency normalized by the fluorescence
quantum yield of the donor in the presence of acceptor, f',
was measured in a way allowing the independent investigation of (i) the
strength of interaction between the adjacent protomers (intermonomer
flexibility) and (ii) the flexibility of the protein matrix within
actin protomers (intramonomer flexibility). In both cases the relative
increase as a function of temperature in f' is larger in
calcium-F-actin than in magnesium-F-actin in the range of 5-40 °C,
which indicates that both the intramonomer and the intermonomer
flexibility of the actin filaments are larger in calcium-F-actin than
those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the
magnesium-F-actin. The distance between Gln41 and
Cys374 residues was found to be cation-independent
and did not change during polymerization at 21 °C. The steady-state
fluorescence anisotropy data of fluorophores attached to the
Gln41 or Cys374 residues suggest that the
microenvironments around these regions are more rigid in the
magnesium-loaded actin filament than in the calcium-loaded form.
 |
INTRODUCTION |
The tension generation in the striated muscle is performed through
a series of chemical reactions by cyclic interaction of myosin with ATP
and actin, and at least six intermediates are proposed for actomyosin
ATPase in solution (1-3). On a cellular level in supramolecular
complexes where stabilizing forces may modulate the hydrolysis process,
some contribution from actin flexibility and dynamics to the
contraction process cannot be excluded. This statement is supported by
earlier and recent suggestions about the role of actin during the force
development in muscle (4).
Flexural rigidity experiments suggested that the actin filament was
extensible (5). These findings were supported by electron microscopic
measurements on the sarcomere in rigor fibers (6). The extensibility of
the thin filaments was also suggested by the changes of the spacings of
the x-ray diffraction pattern during contraction (4, 7). Actin
filaments were shown to be elastic and extensible by measuring the
stiffness of the actin-tropomyosin complex with in vitro
nanomanipulation (8). Polarization studies using fluorescent
phalloidine on skinned rabbit psoas fibers have demonstrated that the
generation of force was associated with a conformational change in the
actin filament (9). The sliding of actin filaments is diminished by the
cross-linking of actin subunits (10). Egelman et al. (11)
and later the workgroup in DeRosier's laboratory (12) emphasized the
existence of variations in the twist along the axes of isolated
filaments, which increases the fluctuations of approximately 10° in
the azimuthal angle between adjacent monomers. The fluctuations,
bending and twisting motions, are modulated by myosin and actin-binding
proteins (13, 14). The tighter binding of myosin to actin reduces the
torsional motion of a small section of F-actin, as reported by standard
transfer-EPR measurements (13, 15). The change of the orientation of
spin labels on F-actin during interaction with heavy meromyosin was also reported (10, 16). It is also known that the actin monomers undergo conformational changes or slight rotation during contraction (17). The large free energy change caused by binding of the myosin head
to actin is also able to generate conformational change in actin
(18).
Experimental evidences suggest that the exchange of the bound
cation can also modify the dynamic and conformational state of the
actin filament. Cation-dependent changes in the mobility of
the N-terminal segment (first 21 amino acids) of actin were observed
performing nuclear magnetic resonance (NMR) experiments (19). The
torsional rigidity of actin filaments is sensitive to the nature of the
bound cation, since this parameter is larger in calcium-F-actin than in
magnesium-F-actin (20). Orlova and Egelman (21) have shown that the
bending flexibility of filaments polymerized from magnesium-actin is
approximately four times larger than in the case of calcium-F-actin.
Contrary to these data, other laboratories found no essential change in
the filament flexibility using dynamic light scattering measurements
(22) or various other techniques to determine the persistence length of
the filaments (23, 24). The direct measurement of the flexibility of
single actin filaments corroborates this latter conclusion (20). Using fluorescence methods Miki et al. (25) observed that the
binding of Ca2+ to actin increased the mobility of the
fluorophore attached to Cys374. The results of our
spectroscopic experiments indicated that filaments polymerized in
the presence of Ca2+ were more flexible than the filaments
of magnesium-actin (26).
The recently published actin powerstroke model was based on the length
changes in actin filaments, which require conformational transitions in
each monomer (27). During the ATP hydrolysis cycle the myosin heads can
adopt more than one conformation in interaction with actin, and the
multiple modes of binding can relate to different actin conformations.
Recently, the negative experimental results of the rotating
cross-bridge model have led to suggestions of a more complex model of
the muscle contraction. This model involves large scale conformational
changes of myosin head in the light chain-binding domain that rotates
relative to the actin-binding portion of the catalytic domain (28-30).
The closure of the cleft on the actin-binding domains, which follows the release of the Pi, results in a specific interaction
between the two proteins, and this interaction might be modulated by
the actual dynamic and conformational states of both proteins.
Although there are strong indications that actin is an active part of
the contracting system, we are still far from understanding the details
of the biological function of this abundant protein. The lack of
complete understanding of the function of the actin in the contracting
system can emphasize the importance of further investigations dealing
with this matter.
The principal aim of this study was to characterize the effect of
divalent cations on the internal flexibility and the conformational states of actin filaments using the method of fluorescence resonance energy transfer. According to the fluorescence resonance energy transfer data presented in this paper, the calcium-F-actin is proved to
be more flexible than the magnesium-F-actin in either the intermonomer
or the intramonomer protein flexibility. The intermonomer flexibility
is larger than the intramonomer one, regardless of the nature of the
bound cation. In accordance with these flexibility data the
steady-state anisotropy experiments indicate that the microenvironments
of the Gln41 and Cys374 residues are more rigid
in the Mg2+-saturated filaments than in
calcium-F-actin.
 |
MATERIALS AND METHODS |
Reagents--
KCl, MgCl2, CaCl2, Tris,
N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine
(IAEDANS),1 quinine
(hemisulfate salt), dimethylformamide, guinea pig liver transglutaminase (TGase), and EGTA were obtained from Sigma.
Iodoacetamide 5-fluorescein (IAF) and fluorescein cadaverine (FC) were
purchased from Molecular Probes (Eugene, OR); adenosine 5'-triphosphate (ATP) and
-mercaptoethanol were obtained from Merck (Darmstadt, Germany); the Bradford protein assay reagent was purchased from Bio-Rad
(München, Germany), and NaN3 was from Fluka (Buchs, Switzerland).
Protein Preparation--
Acetone-dried powder of rabbit skeletal
muscle was obtained as described by Feuer et al. (31).
Rabbit skeletal muscle actin was prepared according to the method of
Spudich and Watt (32) and stored in 2 mM Tris/HCl buffer
(pH 8.0) containing 0.2 mM ATP, 0.1 mM
CaCl2, 0.1 mM
-mercaptoethanol, and 0.02%
NaN3 (buffer A).
Labeling of the Cys374 residue (Fig. 1A) with
IAEDANS was performed as described earlier (33), and F-actin (2 mg/ml)
was incubated with 10-fold molar excess of IAEDANS for 1 h at room
temperature. Labeling of the same residue in separate samples with IAF
was carried out in the following way: monomeric actin (2 mg/ml) was mixed with the 10-fold molar excess of IAF over the protein and incubated for 3-4 h at room temperature. Then the actin was
polymerized for 12-16 h at 4 °C. After the labeling procedures, the
samples were centrifuged at 100,000 × g for 2 h
at 4 °C. The pellets were dissolved in buffer A and dialyzed
overnight against buffer A (in the case of IAF-labeled actin the
dialyzing buffer contained 1% (v/v) dimethylformamide as well). The
Gln41 residue (Fig. 1A) was modified with FC by
the use of the procedure of Takashi (34), and G-actin was incubated
with 10-fold molar excess of the dye in the presence of 1 mg/ml TGase.
The labeling was carried out for 16 h at 4 °C. Unbound FC was
removed similarly as described in the case of IAEDANS and IAF labeling procedures.
The G-actin concentration was determined with a Shimadzu UV-2100
spectrophotometer by using the absorption coefficient of 0.63 mg
ml
1 cm
1 at 290 nm (35). In the case of
IAEDANS-labeled G-actin the measured absorbance at 290 nm was corrected
for the contribution of the fluorescence label (using A (290 nm) = 0.21 × A (336 nm) for the bound IAEDANS).
Relative molecular mass of 42,300 Da was used for monomeric actin (36).
Occasionally, the actin concentration was also determined by using
Bradford (Coomassie Blue) protein assay reagent (37). The assay was
calibrated as described by the manufacturer using unlabeled monomeric
actin. The concentrations determined according to the two methods were
identical within the limits of experimental error. The
concentration of the IAEDANS, IAF, and FC in the protein solution was
determined by using the absorption coefficient of 6100 M
1 cm
1 at 336 nm (38), 77,000 M
1 cm
1 at 496 nm (39), and
75,500 M
1 cm
1 at 493 nm (40),
respectively. The extent of labeling for IAEDANS, IAF, and FC was
determined to be 0.83 ± 0.02, 0.55 ± 0.04, and 0.8 ± 0.03, respectively.
Sample Preparation and Polymerization--
Studying the
intermonomer flexibility, part of the actin sample was labeled with the
donor molecule (IAEDANS), and the remaining part was modified
separately with the acceptor (IAF). After the labeling procedure, the
two samples were mixed to obtain the molar ratio of the donor labeled
and not donor labeled (acceptor labeled and unlabeled) monomers of
minimum 1:10 (Fig. 1B). In order to measure the intramonomer
flexibility, the actin monomer was double-labeled (i.e.
labeled with both the donor (IAEDANS) and the acceptor (FC)). Then the
solution of labeled actin was mixed with a solution of unlabeled actin
monomers to adjust the labeled:unlabeled actin ratio to a minimum
of 1:10 (Fig. 1C). After the appropriate mixture of labeled
and unlabeled actin monomers the polymerization process was initiated.
Magnesium-G-actin was obtained according to the method of
Strzelecka-Golaszewska et al. (41). The solution of
calcium-G-actin was dialyzed exhaustively against buffer A in which the
concentration of CaCl2 was decreased to 50 µM. EGTA and MgCl2 were added and adjusted to
final concentrations of 0.2 and 0.1 mM, respectively. The
sample was stirred at room temperature for 10 min.
Polymerization of either calcium-G-actin or magnesium-G-actin was
initiated by the addition of 100 mM KCl and 2 mM of the appropriate cation (CaCl2 or
MgCl2) to the solutions of calcium-G-actin or
magnesium-G-actin, respectively. The samples were incubated at room
temperature for 2 h, then dialyzed overnight against the appropriate polymerization buffer (buffer A supplemented with 100 mM KCl and 2 mM divalent cation, while in the
case of magnesium-actin, the buffer also contained 0.1 mM EGTA).
Fluorescence Experiments--
The concentration of actin was
between 30 and 40 µM, unless stated otherwise. The
fluorescence emission spectra of the donor were recorded at
temperatures ranging between 5 and 40 °C with a Perkin-Elmer LS50B
luminescence spectrometer in the presence and the absence of the
appropriate acceptor (FC in the experiments dealing with intramonomer
flexibility and IAF in the study of intermonomer flexibility). The
excitation wavelength for the IAEDANS was 360 nm. The slits were set to
3 nm in both the excitation and emission paths. The spectra were
corrected for the inner filter effect as described earlier (42). To
calculate fluorescence resonance energy transfer efficiency (see
Equation 2), the under-curve areas of these emission spectra were
calculated between 380 and 460 nm. In this wavelength range, the
contribution of the acceptor (either the FC or the IAF) to the measured
fluorescence is negligible.
The steady-state fluorescence anisotropy of the donor and acceptor
molecules was calculated from the polarized emission components (FVV, FVH,
FHV, and FHH, where the
subscripts indicate the orientation of the excitation and emission
polarizers) as follows,
|
(Eq. 1)
|
where G = FHV/FHH. In the case of
actin-bound IAEDANS, the excitation wavelength was 360 nm, and the
emission wavelength was 460 nm, while for the fluorescein derivatives
the excitation monochromator was set to 493 nm, and the emission was
measured at 520 nm. The slits were set to 3 nm. In these experiments
the concentration of the actin was decreased to 5 µM
after the polymerization by diluting the sample with the appropriate
buffer. In this way the depolarizing effect of light scattering was
reduced to a negligible level.
The corrected fluorescence emission spectra of IAEDANS-F-actin was
recorded in the absence of the acceptor at an excitation wavelength of
360 nm to obtain the fluorescence quantum yield of the donor molecule.
The quantum yield of quinine sulfate (0.53 in 0.1 N
H2SO4) was used as a reference (43).
To test the reversibility of the temperature-induced changes in
fluorescence, the samples were re-measured by cooling back the solution
to the initial low temperature (7 °C) or repeating the measurements
after overnight dialysis. The errors of the measured data presented in
this paper are mean ± S.E. calculated from the results of three
to five independent experiments.
Donor-Acceptor Distance--
The transfer efficiency of the
fluorescence resonance energy transfer occurring between a single donor
and single acceptor can be calculated from the fluorescence intensities
as follows,
|
(Eq. 2)
|
where FDA and FD
are the fluorescence intensities of the donor molecule in the presence
and the absence of the acceptor, respectively;
symbolizes the
acceptor/monomer molar ratio. cD and
cDA are the concentrations of the donor molecule in
the samples indicated by the subscripts. By knowing the fluorescence
energy transfer efficiency (E), it is possible to determine
the distance (R) between the donor and acceptor molecules
from the following equation:
|
(Eq. 3)
|
where Ro is the Förster's critical
distance defined as the donor-acceptor distance where the fluorescence
resonance energy transfer efficiency is 50%. The use of Equation 3
requires the calculation of Ro as follows,
|
(Eq. 4)
|
where n is the refractive index of the medium,
2 characterizes the relative orientation of the donor
and acceptor molecules,
D is the fluorescence quantum
yield of the IAEDANS in the absence of acceptor, and J is
the overlap integral given in M
1
cm
1 nm4. The overlap integral (J) is
defined as follows,
|
(Eq. 5)
|
where FD(
) is the corrected
fluorescence emission spectra of the donor, and
A(
)
is the molar extinction coefficient of the acceptor.
Normalized Transfer Efficiency--
The temperature profile of
the normalized energy transfer parameter f (defined as the
ratio of the transfer efficiency and the fluorescence quantum yield of
the donor in the presence of acceptor) is proportional to the mean
value of the energy transfer rate constant,
<kti>, which has been shown to be an
appropriate parameter for monitoring intramolecular fluctuations and/or
conformational changes of a macromolecule (44),
|
(Eq. 6)
|
where
DA is the fluorescence quantum yield of the
donor in the presence of acceptor, and kf is the
rate constant of the fluorescence emission. According to earlier
publications the value of kf is fairly constant
under a wide variety of experimental conditions (see e.g.
Ref. 45), therefore here its value is taken as constant. The subscript
"i" indicates the value of the given parameter for the
ith population, taking a momentary picture, and
C is a constant involving the refractive index
(n) and the overlap integral (J), which were assumed to be constant (44). The sensitivity of this parameter to
temperature is able to provide information regarding the flexibility of
the protein matrix between the two fluorophores. It should be noted
that f is sensitive to changes in the donor-acceptor distance originating from any kind of intramolecular motions. Thus, the
temperature profile of this parameter provides information about the
average flexibility of the protein matrix located between the two labels.
The method was developed for systems where the energy transfer occurred
between a single donor and a single acceptor (44). However, in the
present experiments dealing with intermonomer energy transfer, one
should take into account that the donor can transfer energy to
acceptors located on more than one neighboring actin protomers,
i.e. the transfer is directed to a multiple acceptor system.
Considering the helical structure of the actin filament, it seems
reasonable that the acceptor population can be divided into two
characteristically different groups: 1) acceptors on the closest
protomer in the single-started genetic helix and 2) acceptors affecting
the fluorescence of the donor from the double-started long-pitch helix.
It is also assumed that acceptors on more distant protomers are not
efficient in the reduction of the donor fluorescence. Accordingly, the
donor-acceptor system can be described with two different equilibrium
donor-acceptor distance distributions. It could be easily shown by
using simple mathematical transformations that the measured normalized
energy transfer parameter of the system having a single donor
interacting with two different groups of acceptor molecules is the sum
of the normalized energy transfer efficiencies characterized by the
individual donor-acceptor systems (see also Ref. 46),
|
(Eq. 7)
|
Considering that the value of the fluorescence quantum yield is
proportional to the fluorescence intensity measured at a given wavelength, it is usually more convenient to determine the value of the
f', which is defined as follows (44),
|
(Eq. 8)
|
where FDA is the fluorescence
intensity of the donor in the presence of acceptor, and C'
is a constant that is proportional to the C used in Equation 6.
 |
RESULTS AND DISCUSSION |
The actin monomer has one high-affinity and three or more
lower-affinity (i.e. intermediate- and low-affinity)
cation-binding sites (see Ref. 47 for review). It is very likely that
in vivo the high-affinity site is occupied by
Mg2+, and the Mg2+ and K+ ions
compete for the lower-affinity binding sites (47). The ion composition
of the buffer that was used in this study to prepare magnesium-F-actin
can be considered as a reasonable model for the free ion concentrations
of Mg2+ and K+ in the cytosol (47). This
preparation resulted in a magnesium-F-actin that contains
Mg2+ at the high-affinity binding site and probably either
Mg2+ or K+ at the lower-affinity sites.
According to earlier publications the type of the cation at the
lower-affinity binding sites might have an important biological effect
(48). The calcium-actin filaments were polymerized in the presence of
millimolar concentration (2 mM) of CaCl2.
Following this procedure the Ca2+ in calcium-F-actin,
similar to the Mg2+ in magnesium-F-actin samples, occupies
the high-affinity binding site and probably competes with the
K+ for the lower-affinity binding sites.
In the present work we explored the differences between flexibilities
of filaments polymerized from calcium-actin and magnesium-actin by
investigating separately the intermonomer and the intramonomer flexibilities. To examine intermonomer flexibilities the donor IAEDANS
and the acceptor IAF are attached to different actin protomers within
the filament. The relatively low donor ratio in these samples (compared
with that of actin without the donor) assures that there is no acceptor
in the actin filament, which is in resonance transfer with two donor
molecules (see Fig. 1B).
Accordingly, in these experiments one is dealing with a single
donor-multiple acceptor system (see "Materials and Methods"). In a
different experimental setup, the double labeling of the actin monomer
makes it possible to study intramonomer flexibility within the actin
filament. In this case it was necessary to dilute the samples with
unlabeled actin to exclude the possibility of interaction between donor and acceptor molecules located on neighboring protomers. Considering the atomic model of the actin filament (49), it is very likely that the 10-fold dilution of the double labeled actin monomers with
unlabeled monomers accurately separates the labeled monomers within the
double helix of actin filaments (Fig. 1C). The experiments designed to monitor the reversibility of the temperature-induced changes in the fluorescence parameters gave evidence that the changes
were reversible.

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Fig. 1.
A, the schematic representation of the
atomic model of monomeric actin reconstructed according to the results
of Kabsch et al. (60). The subdomains are labeled with
numbers 1-4, and the positions of the amino acids, which were labeled
to carry out the intramonomer or intermonomer fluorescence energy
transfer experiments (i.e. Gln41 and
Cys374), are marked with dark surfaces.
B and C show the simplified picture of the actin
filament. The approximated position of the labeled monomers is shown
within the actin filament in the case of intermonomer (B)
and intramonomer energy transfer experiments (C). The
circles are representing monomers within the polymer.
Capital letters within the circles stand for the
monomer with covalently attached donor (D) or acceptor
(A) molecules in the intermonomer energy transfer
measurements (B), and the doubly labeled monomers are also
marked (DA) in the case of intramonomer measurements
(C). The ratio of the labeled and unlabeled monomer
populations in the pictures is approximately the same as it is after
the preparation procedure described under "Materials and
Methods."
|
|
The distance between the donor (IAEDANS at
Cys374) and acceptor (FC at Gln41) molecules is
4.46 ± 0.07 nm and 4.49 ± 0.06 nm in the Ca2+-
and Mg2+-loaded forms of the monomer, respectively,
indicating that the exchange of the bound cation does not influence the
relative position of the Gln41 and Cys374
residues in the actin monomer. The data are in good accordance with the
results of Moraczewska et al. (50), who found that the
replacement of Ca2+ with Mg2+ produced no
essential change in the distance between Gln41 and
Cys374. These results are also in agreement with our recent
observation that the distance between Lys61 and
Cys374 of the actin monomer is cation-independent (51). The
distance between Gln41 (C
) and Cys374 (S
)
residues is 4.1 nm according to the x-ray diffraction experiments (52).
The value of this parameter resolved in our experiments is somewhat
longer. The relatively small difference between the x-ray and the
fluorescence data might be due to the size of the applied fluorescent probes.
The donor-acceptor distances (between residues Cys374 and
Gln41) in the filament at room temperature are 4.45 ± 0.08 nm and 4.59 ± 0.09 nm in calcium-F-actin and
magnesium-F-actin, respectively (Table
I.), which indicates that the
polymerization does not affect significantly the donor-acceptor
distance. This is in agreement with Miki's conclusion (53) that the
small domain in the actin monomer is substantially rigid and compact
and only slightly sensitive to the binding of DNase I or myosin
subfragment 1 or tropomyosin-troponin or polymerization. Above we
speculate on the basis of the filament model (49) that the 10-fold
dilution of doubly labeled actin samples with unlabeled actin
diminishes the intermonomer resonance energy transfer. The lack of the
effect of polymerization on the donor-acceptor distance implies that
this assumption was correct. The distance between the two labeled
residues of the small domain was temperature-independent in
magnesium-F-actin (Table I). This statement is apparently not true for
the calcium-F-actin (Table I), since the value of the donor-acceptor
distance shows a decreasing tendency with increasing temperature (for
discussion, see below).
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Table I
The temperature dependence of the fluorescence quantum yield of the
IAEDANS ( D), the Förster's critical distance of the
IAEDANS-FC pair (Ro), the transfer efficiency measured in the
intramonomer transfer experiments (E), and the calculated
donor-acceptor distances (R) in calcium-F-actin and magnesium-F-actin
The S.E. of the mean are given in parentheses, except for the quantum
yield where the error appears in the third digit.
|
|
The cation dependence of the flexibility of the actin protomer within
the filament can be characterized by measuring the temperature profile
of the normalized transfer efficiency (Equations 6 and 8). In
experiments dealing with intraprotomer interactions the temperature
dependence of the relative f' is proved to be substantially larger in calcium-F-actin than in magnesium-F-actin between 5 and
40 °C (Fig. 2A). The total
change of 5% in the Mg2+-saturated form faces the 30%
increase in the Ca2+-saturated form. The data set suggests
that the protomer structure is more flexible in the
Ca2+-loaded form of the actin filament than that in the
magnesium-loaded form. The change in the relative f' is very
similar in calcium-F-actin to what was observed in the case of actin
monomer by using a similar donor-acceptor pair (51). Accordingly, the
flexibility of the small domain does not seem to be sensitive to
polymerization in calcium-actin. Contrary to this, the relative change
of f' is smaller in magnesium-F-actin than that in
magnesium-G-actin (51), indicating that in the Mg2+-loaded
form this protein segment is more rigid in the filament than it is in
the monomer.

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Fig. 2.
A, the cation dependence of the
temperature profile of the relative f' in F-actin resolved
in the experiments dealing with intra-monomer flexibility. The donor
was IAEDANS, and FC served as an acceptor. The actin concentration was
30-40 µM, while the labeled actin was present at 1-3
µM. B, the temperature profile of the relative
f' in calcium-F-actin and magnesium-F-actin resolved in the
experiments dealing with intermonomer flexibility. The value of this
parameter was calculated from the results of experiments with the
IAEDANS-IAF donor-acceptor pair. The actin concentration was 30-40
µM.
|
|
According to the results of intermonomer transfer experiments, the
change of the relative f' is larger in the calcium-F-actin than in the magnesium-F-actin (Fig. 2B), which suggests that
the strength of the intermonomer interaction is stronger in the
Mg2+-saturated filament. By comparing the data obtained in
the experiments addressing intramonomer and intermonomer fluorescence
energy transfer, one can conclude that the intramonomer flexibility is
smaller than the intermonomer flexibility for both the calcium-F-actin and magnesium-F-actin (Fig. 2, A and B).
Considering that in intermonomer energy transfer the contributions of
the two kinds of acceptor populations (see also "Materials and
Methods") to the measured fluorescence energy transfer efficiency are
probably similar (49), in these experiments it is not possible to
separate the flexural properties of the genetic helix and the
two-started long-pitch helix. The increase in the amplitude of the
relative fluctuation of the donor and acceptor molecules should result
in an increase of the mean value of the energy transfer rate constant,
<kti> and therefore the measurable
donor-acceptor distance, even if the equilibrium distance between
the two labels remains unchanged (44). In the light of our present data
regarding the cation-dependent flexural properties of the
filament, it seems possible that the slight temperature dependence of
the donor-acceptor distance measured in the calcium-F-actin is partly
the result of a temperature-induced increase in the amplitude of the
relative fluctuation of the donor and acceptor molecules.
The interpretation of the results described above requires further
spectral considerations. Both the temperature- and cation-induced changes in the shape of the emission spectra of the donor and the
absorption spectra of the acceptor are negligible (data are not shown).
Accordingly, the value of the overlap integral (Equation 5.) depends on
neither the temperature nor the nature of the bound cation. Therefore
it cannot contribute to the observed changes of f' in the
filaments. However, the value of the f', and hence the
relative f', might depend on the orientation factor
(
2). Although this is the only parameter in the
fluorescence energy transfer experiments which cannot be measured
properly, the measurements of the steady-state anisotropy of both the
donor and the acceptor molecules might provide information regarding
the behavior of
2. The anisotropy of IAEDANS and FC is
cation-dependent in the actin filament (Fig.
3, A and B). The
measured anisotropy values are larger in the Mg2+-saturated
form than in the Ca2+-saturated one for both IAEDANS and
FC, which can be taken as an indication of conformational differences
between the calcium-F-actin and magnesium-F-actin. Interestingly,
similar cation-induced change was not observed in the case of IAF (Fig.
3C). Taking into account that both IAEDANS and IAF are
connected to the same amino acid (Cys374), the different
cation sensitivity possibly originates from the application of
different fluorophores. According to the results of Orlova and Egelman
(54), there is a high-density bridge between the two strands of
filament when the high-affinity cation-binding sites are occupied by
Ca2+. This density bridge was not observed in
magnesium-F-actin. They proposed that the presence of this bridge could
be the result of the shift in the position of the C terminus.
Therefore, the cation dependence of the fluorescence anisotropy in the
case of IAEDANS and FC might reflect the cation-induced intramolecular rearrangement of the C-terminal segment within the actin filament. Although the exact nature of this rearrangement is not known, it seems
to be possible that the formation of the density bridge in
calcium-F-actin involves the modification of some of the connections between the C-terminal segment and the small domain of either the same
or the neighboring protomers. The subdomain 1 (involving the
Cys374 residue) is in close contact with the subdomain 2 (which contains the Gln41 residue) of the subsequent
protomer within the long-pitch helix (49). Accordingly, the formation
of the high-density bridge in calcium-F-actin can result in a
conformation where the microenvironments of the Cys374 and
the Gln41 residues are more flexible than these
microenvironments in the magnesium-F-actin.

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Fig. 3.
The temperature dependence of the
steady-state fluorescence anisotropy of IAEDANS (A),
FC (B), and IAF (C) in calcium-F-actin
(filled circles) and magnesium-F-actin (open
circles). The concentration of actin was 5 µM in these experiments (see "Materials and
Methods").
|
|
The temperature sensitivity of the fluorescence anisotropy of all
fluorophores is similar (Fig. 3, A-C), which suggests that the temperature-induced change in the value of the orientation factor
is also similar in these cases. Accordingly, the change in the
2 is probably not the source of the apparent
cation-dependent variation in the value of the relative
f' in either the intermonomer or the intramonomer energy
transfer experiments. All these data allow the conclusion that both the
intramonomer and intermonomer flexibilities are larger in
calcium-F-actin than in magnesium-F-actin. Furthermore, the
results of the steady-state anisotropy measurements support the
conclusion that the microenvironments of the Gln41 and
Cys374 residues are more rigid in the magnesium-F-actin
than in the calcium-F-actin.
The bending and torsional flexibility of calcium-F-actin was found to
be smaller (21) or similar (28-31) to that of magnesium-F-actin. However, we have shown here and in our previous work (26) that the
flexibility characteristic for intramolecular motions on a nanosecond
time scale is larger in calcium-F-actin than in the Mg2+-saturated form of the filament. We have
suggested (26) that the apparent conflict could be resolved considering
that the methods applied in our experiments and those used in the cited
articles (21, 28-31) provide information about intramolecular motions on a substantially different time scales.
The structure of the magnesium-F-actin can be taken as a model of the
thin filament in the relaxed state. The changes in the actin-associated
layer lines in x-ray diffraction pattern during muscle activation (56,
57) and the differences of the layer lines observed between
magnesium-F-actin and calcium-F-actin (54) are similar. Relying on
these data Egelman and Orlova (58) proposed that the structure of the
calcium-F-actin was corresponding to the thin filaments in the
activated state. It is very likely that due to the slow exchange of the
tightly bound divalent cation in actin the replacement of
Mg2+ by Ca2+ does not occur under physiological
conditions (47). Accordingly, Egelman and Orlova (58) concluded that
the activated state of the thin filament was probably induced by the
binding of myosin. One might assume that the similarity of the
structure of calcium-F-actin and the structure of the F-actin in the
activated thin filaments can extend to intramolecular dynamic events
occurring on a nanosecond timescale. Thus, considering that
actin-myosin interaction can possibly utilize the strain energy stored
in actin filaments (59), the divalent cation-dependent
changes in the intramolecular flexibility described in this study might
be important in the efficient energy transduction of the muscle contraction.
 |
FOOTNOTES |
*
This work was supported by the Hungarian Academy of Sciences
and grants from the National Research Foundation (OTKA Grants T017727,
T020117, T023209, and F020174).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:/Fax:
36-72-314-017; E-mail: sombel{at}apacs.pote.hu.
 |
ABBREVIATIONS |
The abbreviations used are:
IAEDANS, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine;
FC, fluorescein cadaverine;
IAF, 5-iodoacetamidofluorescein;
G-actin, monomeric actin;
F-actin, actin filament.
 |
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