Báo cáo Y học: Effect of adenosine 5¢-[b,c-imido]triphosphate on myosin head domain movements Saturation transfer EPR measurements without low-power phase setting ppt

Effect of adenosine 5¢-[b,c-imido]triphosphate on myosin
head domain movements
Saturation transfer EPR measurements without low-power phase setting
No
´
ra Hartvig
1
,De
´
nes Lo
˜
rinczy
2
, Nelli Farkas
1
and Joseph Belagyi
1
1
Central Research Laboratory and
2
Institute of Biophysics, School of Medicine, University of Pe
´
cs, Hungary
Conventional and saturation transfer electron paramagnetic
resonance spectroscopy (EPR and ST EPR) was used to
study the orientation of probe molecules in muscle fibers in
different intermediate states of the ATP hydrolysis cycle. A
separate procedure was used to obtain ST EPR spectra with
precise phase settings even in the case of samples with low
spectral intensity.
Fibers prepared from rabbit psoas muscle were labeled

with isothiocyanate spin labels at the reactive thiol sites of
the catalytic domain of myosin. In comparison with rigor, a
significant difference was detected in the orientation-
dependence of spin labels in the ADP and adenosine
5¢-[b,c-imido]triphosphate (AdoPP[CH
2
]P) states, indica-
ting changes in the internal dynamics and domain orienta-
tion of myosin. In the AdoPP[CH
2
]P state, approximately
half of the myosin heads reflected the motional state of
ADP–myosin, and the other half showed a different dynamic
state with greater mobility.
Keywords: adenosine 5¢-[b, c-imido]triphosphate (AdoPP-
[CH
2
]P); myosin; saturation transfer EPR; spin-labeling.
It is generally accepted that domain movements in the
myosin head play a decisive role in the energy-transduction
process of muscle contraction [1–5]. It is a multistep process
which can produce several conformational states of myosin
[6–9]. Extensive studies using different techniques have
indicated that the nucleotide-binding pocket does not
experience large conformational changes during the hydro-
lytic cycle [10–12]. However, small conformational changes
induced by nucleotides in the motor domain should be
converted into larger movements. The data show that,
whereas the structure of the motor domain remains similar
to rigor, the regulatory domain swings around a point at the

distal end of the motor domain [8,13–17]. The changes in the
50-kDa domain affect the segment of the 20-kDa domain
that contains the essential thiol groups, SH1 (Cys707) and
SH2 (Cys697). This part may be involved in the transducing
of small conformational changes [18,21,22].
Spectroscopic probes are widely used in muscle
research. Paramagnetic probes provide a direct method
by which dynamic changes and the rotation and orien-
tation of specifically labeled proteins can be followed. In
muscle fiber studies, the probe molecules, particularly the
maleimide-based nitroxides and iodoacetamide spin labels,
are usually attached to the reactive thiol site Cys707 of the
motor domain [21–23], or to the regulatory light chain
[9,24,25]. The main problems that limit interpretation of
spectroscopic measurements are the location of the probe
molecules on the proteins, the relative orientation of the
spin labels with respect to the magnetic field, and the
perturbing effect of probes on structure and function. We
have previously observed that an isothiocyanate-based
spin label is more sensitive to the domain orientation in
the myosin head than the widely used maleimide spin
label [26]. Selective modification of Cys707 strongly affects
MgATP hydrolysis, and the motor function of myosin
heads in the in vitro motility assay is blocked [27,28]. The
most pronounced effect was observed in the intermediate
complex ADP–P
i
[19,20].
Spin label molecules bound to myosin are able to detect
nucleotide binding and conformational changes in the

myosin head related to the hydrolytic cycle of ATP [29]:
AM þ ATP $ A þ MÁATP $ A þ M
Ã
ÁATP $
AM
ÃÃ
ÁADPÁP
i
$ AM
Ã
ÁADP þ P
i
$ AM þ ADP þ P
i
where M denotes myosin, A stands for actin, and the
asterisk (*) distinguishes intermediate conformational chan-
ges. From consideration of the crystal structure of the
myosin head [3,4], and from fluorescence measurements, it
was concluded that the so-called open-closed transition does
not require hydrolysis of ATP [30]. It was also suggested
that the nonhydrolyzable analogue adenosine 5¢-[b,c-imi-
do]triphosphate (AdoPP[CH
2
]P) was able to induce the
transition. The intermediate states have different spectral
properties, and distinct conformations can be assigned to
these states. Our primary aim was to use the spin label EPR
technique to measure changes in label orientation and/or
rotational rate that results from binding of AdoPP[CH
2

]P
to myosin in muscle fibers, and to find some correlation with
the myosin head structure. The AdoPP[CH
2
]P state can be
stabilized for long enough to conduct measurements by
Correspondence to J. Belagyi, Central Research Laboratory, Univer-
sity of Pe
´
cs School of Medicine, 12 Szigeti Str., H-7643 Pe
´
cs, Hungary.
Fax: + 36 72 536254, Tel.: + 36 72 536255,
E-mail:
Abbreviations:AdoPP[CH
2
]P, adenosine 5¢-[b,c-imido]triphosphate;
ST EPR, saturation transfer electron paramagnetic resonance
spectroscopy; MSL, 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl;
TCSL, 4-isothiocyanato-2,2,6,6-tetramethylpiperidino-oxyl; TNBS,
2,4,6-trinitrobenzenesulfonate.
(Received 7 August 2001, revised 4 March 2002,
accepted 7 March 2002)
Eur. J. Biochem. 269, 2168–2177 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02872.x
saturation transfer (ST) EPR. ST EPR is widely applied to
biological problems [9,10,12,21]. Motion of the label is slow
in biological samples, and the spectral intensity is not too
high, therefore this problem motivated us to find a way to
eliminate the critical setting of the out-of-phase null at
unfavorable signal-to-noise ratio.

MATERIALS AND METHODS
Materials
KCl, MgCl
2
, EGTA, histidine/HCl, glycerol, ADP, ATP,
AdoPP[CH
2
]P, N-ethylmaleimide, 4-maleimido-2,2,6,
6-tetramethylpiperidino-oxyl (MSL) and 4-isothiocyanato-
2,2,6,6-tetramethanepiperidino-oxyl (TCSL), phosphoenol-
pyruvic acid, pyruvate kinase, and lactate dehydrogenase
were obtained from Sigma (Germany). 2,4,6-Trinitroben-
zenesulfonate (TNBS) was obtained from Fluka (Germany).
Fiber preparation
Glycerol-extracted muscle fiber bundles were prepared from
rabbit psoas muscle. Small strips of muscle fibers were
stored after osmotic shock in buffer containing 50% (v/v)
glycerol, 80 m
M
KCl, 5 m
M
MgCl
2
,1m
M
EGTA and
25 m
M
Tris/HCl, pH 7.0, at )18 °C for up to 1 month.
Fiber bundles from glycerinated muscle were washed for

60 min in rigor buffer (80 m
M
potassium propionate, 5 m
M
MgCl
2
,1m
M
EGTA, 25 m
M
Tris/HCl buffer, pH 7.0),
which removed glycerol, and then transferred to fresh
buffer. This state models the rigor state of the muscle. We
added MgADP to the rigor solution at a final concentration
of 5 m
M
to simulate the strongly binding state of myosin to
actin, which may correspond to the AM–ADP state. In
experiments involving MgADP, the activity of adenylate
kinase was inhibited by the addition of 50 l
M
diadenosine
pentaphosphate. The other analogue of intermediates in the
ATPase pathway was formed by AdoPP[CH
2
]P,which
stoichiometrically binds at the active site of myosin to form
a stable complex. The muscle fibers were stored in solution
containing 80 m
M

potassium propionate, 5 m
M
MgCl
2
,
1m
M
EGTA, 5 or 16 m
M
AdoPP[CH
2
]P and 25 m
M
Tris/
HCl, pH 7.0, for 15 min at 0 °C,andthenspectrawere
recorded at ambient temperature (20–22 °C). Only freshly
opened bottles of AdoPP[CH
2
]P were used to avoid
degradation of the compound [31].
Preparation of MSL–hemoglobin
Freshly prepared human hemoglobin was spin-labeled with
MSL for 24 h; the molar ratio of label and protein was one
to one. The labeled site was the b-93 cysteine residue. The
spin-labeled protein was dialyzed against 0.1
M
phosphate
buffer at pH 7.0 for 48 h at 4 °C and lyophilized. Then
5 mg protein was dissolved in 1 mL buffer (final concen-
tration) to obtain samples. Different amounts of glycerol up

to 70% (v/v) and sucrose up to 50% (w/v) were added to
increase the viscosity.
Spin labeling of muscle fibers
Spin labeling of fibers was performed in labeling medium
(rigor solution plus 2 m
M
pyrophosphate at pH 6.5) with
% 2 mol TCSL to 1 mol myosin for 20 min at 0 °C. Before
spin labeling, the fibers were incubated in low-ionic strength
buffer [1 m
M
EGTA, 5 m
M
MgCl
2
,1m
M
5,5¢-dithiobis(2-
nitrobenzoic acid) and 20 m
M
Mops, pH 7.5] for 1 h to
achieve selective labeling of the reactive thiols [32]. After
spin labeling, the fiber bundles were washed in rigor buffer
plus 5 m
M
dithiothreitol for 30 min at 0 °C, pH 7.0, to
remove the unreacted labels and restore the preblocked thiol
groups. The ratio of the attached spin labels to moles of
myosin varied between 0.22 and 0.41 (mean value 0.33) as
calculated from the double integral of the EPR spectra after

comparison with the double integral of an MSL solution of
10 l
M
using the same sample cell and spectrometer
parameters.
EPR measurements
Conventional and ST EPR spectra were recorded with an
ESP 300E (Bruker) spectrometer. First harmonic, in-phase,
absorption spectra were obtained by using 20 mW micro-
wave power and 100 kHz field modulation with amplitude
of 0.1 or 0.2 mT. Second harmonic, 90° out-of-phase,
absorption spectra were recorded with 63 mW and 50 kHz
field modulation of 0.5 mT amplitude detecting the signals
at 100 kHz out-of-phase. The 63 mW microwave power
corresponds to an average microwave field amplitude of
0.025 mT in the central region of the standard tissue cell of
Zeiss (Carl Zeiss), and the values were obtained by using the
standard protocol of Fajer & Marsh [33]. In this region of
the tissue cell, small segments of the muscle fibers (6–7 mm
long) were mounted parallel to each other. The spectra were
recorded in two positions at a temperature of 22 ± 1 °C,
where the longer axis of the fibers was oriented parallel and
perpendicular to the laboratory field. The spectra were
normalized to the same number of unpaired electrons by
calculating the double integral of the derived spectra. We
assumed that the spectra from TCSL-fibers in different
states would be composed of a linear combination of
spectra; normalized EPR spectra were manipulated by
digital subtraction.
To obtain the precise phase setting for the ST EPR

technique, a different procedure was applied [34,35]. The
idea originates from B. H. Robinson (Department of
Chemistry, Nashville University, Nashville, KS, USA).
Assuming that at low microwave power the variance of the
EPR signal would be minimum over the whole field scan at
the out-of-phase setting, the correct phase angle can be
calculated from two high-power spectra differing in phase
angle by exactly 90°.
Using the least-squares assumption at the out-of-phase
setting for an EPR spectrum, we obtain
S ¼
X
n
i
½spðiÞÀm
2
¼ min
where sp(i) is the signal intensity of the digitized spectrum,
and m is the mean value of the EPR signal. The summation
variable i ranges from 1 to n,wheren denotes the number of
digitized data. In general the spectrum at an arbitrary phase
setting is the linear combination of the in-phase and out-of-
phase components
spðiÞ¼AðiÞ cos 0 þ BðiÞ sin 0
Ó FEBS 2002 Effect of AdoPP[CH
2
]P on myosin head domain movements (Eur. J. Biochem. 269) 2169
where J is the phase angle, and A(i)andB(i)arethe
amplitudes of the in-phase and out-of-phase components.
Differentiating S with respect to variables m and J,and

setting the derivatives equal to zero results in
m ¼
spðiÞ and tan 20 ¼
2V
AB
V
B
À V
A
where V
A
, V
B
are the variances of the components A(i)and
B(i), and V
AB
is the common variance. To apply this
procedure to a given sample and to obtain the in-phase and
out-of-phase spectra, two independent data sets (two
spectra) are required for the same sample, for which the
phase angles differ by exactly 90°:
sp1ðiÞ¼AðiÞ cos 0 þ BðiÞ sin 0
sp2ðiÞ¼ÀAðiÞ sin 0 þ BðiÞ cos 0
where sp1(i) is taken at an arbitrary phase angle J,and
sp2(i) is recorded at a phase angle J +90°.Linear
transformation of the two spectra through the calculated
phase angle J allows the second harmonic in-phase and out-
of-phase EPR spectra to be estimated. In practice, the
digitized data of sp1(i)andsp2(i) are used to obtain the
variances and the phase angle. The linear transformation

through the calculated phase angle gives the required out-
of-phase spectrum. The upper spectra in Fig. 1 are two
second-harmonic high-power EPR spectra for MSL-hae-
moglobin at phase angles a and a +90°, and the lower
spectrum is the out-of-phase spectrum calculated from the
new phase angle. In control experiments, the 0-degree was
obtained by adjusting the phase angle by the method of
Squier & Thomas [36]. In this paper we use the expression
method of variance to distinguish this procedure from the
conventionally used low-power phase setting (null method).
The procedure was tested on MSL-haemoglobin in
different regions of the rotational correlation times. The
viscosity of the samples was increased by the addition of
sucrose or glycerol. The temperature of the samples was
varied in the range 20 °Cto)30 °C by using an ER412 VT
temperature regulator from Bruker. In other cases, skeletal
muscle F-actin and glycerinated muscle fibers labeled with
N-maleimide or isothiocyanate spin labels were measured.
ATPase activity
ATPase activity was determined using a pyruvate kinase/
lactate dehydrogenase-coupled optical test [29]. The assay
medium for Mg
2+
-ATPase consisted of 100 m
M
KCl,
20 m
M
Mops, 1 m
M

MgCl
2
,0.5m
M
EGTA, 0.15 m
M
NADH, 1 m
M
phosphoenolpyruvate, 20 UÆmL
)1
pyru-
vate kinase, 40 UÆmL
)1
lactate dehydrogenase, and
0.5 m
M
ATP, pH 7.0. For Ca
2+
,Mg
2+
-ATPase, the assay
medium also contained 1 m
M
CaCl
2
. At 340 nm, the
absorption change was measured with a Perkin–Elmer
spectrophotometer interfaced to a computer. The molar
absorption coefficient (e
340

)ofNADHwas
6.22 · 10
3
mol
)1
Æcm
)1
. In the experiments, the Mg
2+
-
ATPase and Ca
2+
,Mg
2+
-ATPase activities of thin fiber
bundles over 10-min intervals were determined. Fiber
bundles of 8–10 mg wet weight were slightly stretched on
a rectangular support made of platinum. The support was
diagonally fitted into a standard quartz cuvette, which
was filled in with the solutions. The solution was
continuously mixed with a small magnetic bar. The
decrease in absorbance resulted in a straight line, and the
slope of the straight line was used to estimate ATPase
activity. We assumed that 50% of the dried muscle weight
was myosin. The Mg
2+
-ATPase activity was
4.131 ± 0.718 lmol P
i
Æ(mg myosin)

)1
Æmin
)1
(n ¼ 4) for
control fibers and 4.024 ± 0.742 lmol P
i
Æ(mg myo-
sin)
)1
Æmin
)1
(n ¼ 4) for TCSL-fibers. The ATPase
activity of active fiber bundles was 5.565 ± 0.816 lmol
P
i
Æ(mg myosin)
)1
Æmin
)1
(n ¼ 4) for control fibers, and
3.199 ± 0.457 lmol P
i
Æ(mg myosin)
)1
Æmin
)1
(n ¼ 4) was
calculated for TCSL-fibers. The K
+
/EDTA ATPase

activity of myosin extracted from muscle fibers was
measured by determining the rate of release of P
i
as
described previously [26]. The mean ATPase activity of
myosin was reduced to % 60% of the control after spin
labeling with TCSL.
Fig. 1. Component spectra of MSL-hemoglobin (A) and ST EPR
spectrum of MSL-hemoglobin calculated from the two high-power
component spectra (B). The phase angles of the spectra in (A) differ
by 90°.
2170 N. Hartvig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Rotational dynamics of spin-labeled hemoglobin
Figures 2 and 3 show the comparison of the ratio of the
diagnostic peaks L¢¢/L and the EPR spectra. The MSL-
haemoglobin samples were subsequently measured with the
variance method and the conventional null technique [36].
Appropriate fitting provides evidence that the variance
method is useful, especially when the rotational motion of
large proteins with low concentration of spin labels should
be detected. At low concentration of spin labels where high
receiver gain is required to obtain a spectrum of good
quality, it is difficult to follow the widely used low-power
(£ 1 mW) phase setting method because of the unfavorable
signal-to-noise ratio.
In a series (n ¼ 9) of spin-labeled muscle fibers, the
phase setting adjusted by the null method (u ¼ 0°)was
changed by exactly 40°. The spectra were then recorded, and
the phase angle was calculated by the variance technique on

each sample. The result of the measurements was
u ¼ 42.38 ± 2.09 (mean ± SD). Good agreement was
obtained with the integral method [37,38] (Fig. 4). On the
basis of the comparison, we suggest that the variance
method is almost equivalent to the other methods.
Orientation of probe molecules
Myosin in fibers was spin-labeled with an isothiocyanate-
basedspinlabel,whichisbelievedtobindtothefast
reacting thiol sites in the catalytic domain of myosin. The
labels in the fibers were immobilized on the microsecond
time scale: the effective rotational correlation time was 60 ls
in the absence of nucleotides calculated from ST EPR
spectra (Fig. 5C). Spectroscopic probes provided direct
information about the orientation of the myosin heads; in
rigor, they had only one mode of binding [22,23]. The MSL
probes showed a narrow distribution with respect to the
longer axis of the fibers, with a mean angle of 82° and an
angular spread of 6°. With TCSL probes, the EPR spectra
also reported a high dependence of orientation [26], but with
different mean angle and angular spread (J ¼ 75°,
r ¼ 16°, Fig. 4) compared with MSL-fibers. Blocking
the reactive thiol site, Cys707, with 0.1 m
M
N-ethylmale-
imide before spin labeling, but after incubation with
5,5¢-dithiobis(2-nitrobenzoic acid), greatly reduced the spec-
tral intensity of the TCSL-fibers. Using the same procedure
of TCSL labeling without and with N-ethylmaleimide, the
molar ratio of the bound spin label to mol of protein was
about 25 : 1. After N-ethylmaleimide pretreatment, the

spectrum showed a fraction of weakly immobilized labels,
and the orientation-dependence of TCSL-fibers was greatly
reduced. Pretreating the fibers with TNBS before
Fig. 2. Representative ST EPR spectra recorded for MSL-hemoglobin.
Lyophilized MSL-hemoglobin was dissolved in 0.1
M
phosphate buf-
fer, pH 7.0, and either glycerol or sucrose plus glycerol was added to
the samples to increase viscosity. The final concentration of hemo-
globin was 5 mgÆmL
)1
. Upper curve: null method; lower curve: vari-
ance method. (A) MSL-hemoglobin in 70% (v/v) glycerol at 0 °C;
(B) MSL-hemoglobin in 70% (v/v) glycerol plus 30% (w/v) sucrose at
0 °C; (C) MSL-hemoglobin in 70% (v/v) glycerol plus 50% (w/v)
sucrose at )30 °C; (D) lyophilized MSL-hemoglobin.
Fig. 3. Comparison of the spectroscopic methods using the low-field
diagnostic peaks (L¢¢/L) on different spin-labeled samples. For the
variance method the phase angle was calculated from two high-power
out-of-phase EPR spectra. The regression coefficient of the fitted
straight line is shown.
Ó FEBS 2002 Effect of AdoPP[CH
2
]P on myosin head domain movements (Eur. J. Biochem. 269) 2171
spin-labeling did not significantly affect the orientation-
dependence of the EPR spectra compared with that of fibers
pretreated with 5,5¢-dithiobis(2-nitrobenzoic acid). On the
other hand, trinitrophenylation of the reactive lysine residue
before TCSL treatment did not significantly modify the
orientation-dependence of the labels derived from the

spectra.
Spectrum simulation based on our earlier work [26]
showed that, although the TCSL probe molecules were
probably attached to the same sites as the MSL probe
molecules, they exhibited different orientational distribu-
tion. To determine the orientation of the probe molecule
within the myosin head, the electron microscopic data of the
actomyosin complex [39,40], the crystal structure of sub-
fragment 1 [4], and the procedure developed by Fajer [23]
were taken into account. The average angle of attachment
of the myosin head to the actin filament was % 40° at
practically all stages of the hydrolytic cycle of ATP [39]; only
a few degrees of difference were detected in the tilt angle. It
suggests that, in the presence of ADP, the TCSL label
reflects an internal rearrangement of the structure in the
environment of the labeled site. Accepting the reference
system for the head of myosin defined by Fajer [23], the
x axis of the reference frame lies in the plane of the head
projection and inclined at 40° with respect to the long axis of
the fibres, while the y-axis is perpendicular to this plane.
Then to a rough approximation, the tilt angle of the
principal z-axis of the TCSL label is about 30°,using
the data derived from our model-independent approach for
the calculation of orientational distribution. A significant
change in the tilt angle of the principal z axis after addition
of ADP or AdoPP[CH
2
]P would mean reorientation of the
segment, i.e. the broken helix containing the two reactive
thiols, that holds the label. The uncertainty arising from the

incorrect orientation of the fibre segments with respect to
Fig. 5. EPR spectra of TCSL-fibers in rigor and the ADP state. Spectra
A and B were recorded in parallel orientation, and spectrum C was
obtained in perpendicular orientation to the fibers. The fibers were
kept in buffer solution (see Materials and methods) during spectrum
accumulation. MgADP was added at a concentration of 5 m
M
,and
the fibers were kept for 10 min in buffer before EPR measurement. The
change in the hyperfine splitting (2A¢
zz
) shows the different static
orders of spin labels in rigor and the ADP state. (A) Rigor state; (B)
ADP state; (C) rigor state; (D) ST EPR spectrum of rigor fibers.
ST EPR spectra were recorded for fibers oriented perpendicular to the
long axis of the fibers. The field scan was 10 mT.
Fig. 4. Comparison of the two methods by calculating the first integrals
of the corresponding two spectra. The regression coefficient of the fitted
straight line is shown.
2172 N. Hartvig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
the magnetic field and from the assumption of an axially
symmetrical EPR parameter A in the model-independent
approach of the calculation has an impact on the values for
probe orientation.
Effect of nucleotides
The addition of MgADP resulted in a change in the mean
angle of the distribution of the spin labels. It decreased from
75° to 56°, and the angular spread increased by 4°,butthe
orientation order remained (Fig. 5). In contrast,
AdoPP[CH

2
]P produced orientation disorder of the myosin
heads, and a random population of spin labels was
superimposed on the ADP-like spectrum, which demon-
strates conformational/motional changes and dissociation
of the myosin heads (Fig. 6). In agreement with previous
data, the fraction of the ordered population was estimated
to be % 50% of the total concentration [41]. However, there
was a large difference between the spectra of MSL-fibers
and TCSL-fibers. One component of the spectra of MSL-
fibers reflected exactly the same orientation as in rigor; the
myosin heads apparently remained bound to actin. The
second component was characteristic of randomly oriented
myosin heads (Fig. 7). In the case of the TCSL-fibers, the
component of the spectrum with a high degree of order was
the same as in the ADP state; the second component
represented the disordered heads. The ratio of the double
integrals of the component spectra was about 50 : 50. It is
Fig. 7. Conventional EPR spectra of MSL-fibers in the AdoPP[CH
2
]P
state (A) and in rigor (B). The longer axis of the fibers was oriented
parallel to the laboratory magnetic field. Bottom: residual spectrum
after digital subtraction (spectrum A ) B).
Fig. 6. Conventional EPR spectrum of TCSL-fibers in the
AdoPP[CH
2
]P state. The longer axis of the fibers was oriented parallel
to the laboratory magnetic field, except for spectrum B. Spectrum B
was recorded on AdoPP[CH

2
]P-fibers in perpendicular orientation.
Digital subtraction of the ADP spectrum (C) from the AdoPP[CH
2
]P
spectrum (A) resulted in a spectrum (D) that was characteristic of
randomly oriented spin labels. Bottom: the residual spectrum gener-
ated from the spectrum of the ADP–V
I
state of the fibers (not shown)
and spectrum D. The field scan was 10 mT.
Ó FEBS 2002 Effect of AdoPP[CH
2
]P on myosin head domain movements (Eur. J. Biochem. 269) 2173
known that the addition of MgATP plus orthovanadate to
rigor solution produced large changes in muscle fibers, and
only one spectral component could be detected, which was
characteristic of a random population of spin labels [42].
Digital subtraction of the ATP–V
i
spectrum from the
spectrum of TCSL-fibers in the AdoPP[CH
2
]P state resul-
ted in a spectrum similar to that of TCSL-fibers in the ADP
state (Fig. 6).
Comparison of EPR spectra obtained for homogenized
fibers in rigor and the AdoPP[CH
2
]P state showed a

significant decrease in the hyperfine splitting constant 2A¢
zz
,
which is evidence of the increased rotational mobility.
The hyperfine splitting in the AdoPP[CH
2
]P state was
6.667 ± 0.03 mT (n ¼ 4), whereas a value of 6.780 ±
0.02 mT (n ¼ 6) was estimated in rigor fibers. The
apparent rotational correlation time (s
r
¼ 0.14 ls) calcu-
lated with the Goldman equation [43] corresponds to the
detached myosin heads, rotating rapidly, or the binding of
AdoPP[CH
2
]P produces segmental mobility in the environ-
ment of the labeled sites.
The comparison of spectra recorded in perpendicular
orientation of fibers with respect to the laboratory magnetic
field in rigor and the AdoPP[CH
2
]P state (see Figs 5 and 6)
showed no appreciable difference. It provides evidence that
lineshapes of ST EPR spectra are not affected by the
orientation order of probe molecules. The ST EPR spectra
of TCSL-fibers in the presence of AdoPP[CH
2
]P showed
changes in the rotational mobility (Fig. 8). The ratio of the

low-field diagnostic peaks L¢¢/L changed from 0.800 ±
0.064 (n ¼ 5) to 0.675 ± 0.094 (n ¼ 3).
DISCUSSION
Characterization of the labeled sites
Earlier experiments showed that the maleimide spin labels
were located on the reactive cysteine residue (Cys707), and,
as a consequence of the modification, the ATPase activities
of myosin changed [20,44,45]. TCSL may react with the
most reactive lysine residue (Lys84) of myosin: blocking it
with TNBS enhanced the Mg
2+
-ATPase activity of myosin
by a factor 20 [46]. Our experiments on the Mg
2+
-ATPase
activity of TCSL-fibers did not show an increase in the
activity in contrast with the findings with untreated fibers.
The presence of low concentrations of MgPP
i
in some way
protected the reactive lysine residues from modification
[47,48]. The experimental results together with the similar
dependence of the probe orientation in MSL-fibers and
TCSL-fibers suggested that the paramagnetic labels were
probably bound to the same sites. This is supported by the
similar value of the ratio of the attached spin labels and that
of the decrease in K
+
/EDTA ATPase activity. The
difference in the mean angle of label orientation and the

larger angular spread can be explained by the different
chemical structure and different attaching linkage of the two
labels.
Recent results of the effects of Cys707 labeling on myosin
showed that, after modification, the myosin heads had
limited ability to propel the actin filaments in the in vitro
motility assay [19,20,28]. However, the spin-labeling of
myosin heads in muscle fibers did not significantly affect the
shortening and force generation [11,26]. The lower
Ca
2+
,Mg
2+
-ATPase activity suggests that spin labeling
blocked actin activation by preventing the accelerated
release of hydrolysis product from the myosin heads.
Rotational dynamics of labels in the presence
of Ado
PP
[CH
2
]
P
In comparison with rigor fibers, the EPR spectra exhibited
significant changes at H || k orientation. Thomas and
coworkers came to the conclusion that approximately half
of the cross-bridges had a different state [41]. This fraction
showed dynamic disorder, the heads being dissociated from
actin, whereas the other population had the same spectral
feature as in rigor. Our results on AdoPP[CH

2
]P-fibers led
us to a similar conclusion, but the second fraction with a
high degree of order exhibited a state similar to the ADP
state; the state of these heads differed from that of rigor.
AdoPP[CH
2
]P, similarly to ADP, may induce a change in
the orientation of the protein segment that holds the label
by means of rotation, resulting in another stable con-
formational state. It seems that the nucleotide-induced
Fig. 8. ST EPR spectra of spin-labeled fibers. The spectra of TCSL-
fibers and MSL-fibers were taken in perpendicular orientations.
MgAdoPP[CH
2
]P was added at a concentration of 16 m
M
,andthe
incubation of fibers lasted for 10 min before EPR measurement. The
decrease in the ratio of the L¢¢/L diagnostic peaks in the AdoPP[CH
2
]P
states shows the increased rotational mobility of spin labels attached to
theheadregionofmyosin.Thefieldscanwas10mT.
2174 N. Hartvig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
conformational change is independent of whether ADP or
AdoPP[CH
2
]P binds to the myosin head. However, ADP
forms a tight AM–ADP complex, therefore it should favor

two-headed binding. The binding of AdoPP[CH
2
]P is
almost complete (85% saturation) at a concentration of
0.5 m
M
[49,50]. This supports the view that, in the
AdoPP[CH
2
]P state, the myosin heads may have two
structural states even in the ordered array of cross-bridges
obtained from X-ray analysis [51]. Electron micrographs
and X-ray diffraction patterns of insect flight muscle fibers
provided evidence that the cross-bridges in the weakly
binding state were highly ordered at a uniform 90° angle to
the filament axis, but the spin-labeled nucleotide reported
disorder. The low-angle X-ray diffraction patterns were
modified by AdoPP[CH
2
]P in insect flight muscle; the ratio
of the two inner equatorial peaks was lowered when the
concentration of AdoPP[CH
2
]P was increased [49,52].
Similarly, glycerol-extracted muscle fibers from rabbit psoas
muscle gave low-angle X-ray diffraction patterns that
differed from the diffraction diagrams of either relaxed or
rigor muscle fibers [53].
The results obtained for muscle proteins at lower
concentrations of MgAdoPP[CH

2
]P have recently been
criticised [31]. Using X-ray diffraction, the authors conclu-
ded that, at saturating concentrations of MgAdoPP[CH
2
]P
(20 m
M
in the absence of calcium), at high ionic strength
and low temperature the cross-bridges bound weakly to
actin. This conclusion seems to agree with the EPR results.
Barnett & Thomas [42] proposed that a single chemical state
of a nucleotide could give rise to more than one conforma-
tion of myosin, and a change in the chemical state would
alter the equilibrium between these states. Our EPR
measurements were performed at two concentrations of
MgAdoPP[CH
2
]P (5 and 16 m
M
), but significant differen-
ces between the EPR spectra were not observed. The spin
labels attached to proteins reflect local conformations and
dynamic changes in the microsecond or shorter term
regimes. Therefore, the appearance of two populations
detected by EPR in the presence of MgAdoPP[CH
2
]P does
not contradict the results of the X-ray diffraction technique.
Earlier measurements based on exchangeability of bound

nucleotide and
31
P-NMR observations also showed two
states of the myosin heads in the absence of nucleotides and
ADP or AdoPP[CH
2
]P states, which implies the rapid
interconversion of different conformational states [54,55].
Moreover, the equilibrium, i.e. nearly equal populations at
25 °C between the states, was temperature-dependent: at the
lower temperature (4 °C) only one form was detected.
Our results are also not in contradiction with earlier EPR
results using MSL-fibers. MSL probes reported a more rigid
attachment (L¢¢/L ¼ 1.2) to myosin than did TCSL probes
(L¢¢/L ¼ 0.83). Therefore, the former may indicate the
orientation of the entire head, whereas TCSL may be
sensitive to smaller changes in the internal structure. The
addition of AdoPP[CH
2
]P induced increased rotational
mobility of both spin labels, which implied alteration of the
binding state of myosin to actin. However, it seems that
TCSL probes report local change as well, as deduced from
the comparison of the rigor and ADP spectra. Precise
measurements made on muscle fibers with the use of
15
N
and deuterized maleimide spin labels and sophisticated
spectral analysis gave evidence that, even in the case of
maleimide spin label, small conformational changes were

produced on myosin following nucleotide binding to myosin
[18,56].
Our data on AdoPP[CH
2
]P binding suggest that the
structural states of the myosin head in rigor, the ADP state
and the AdoPP[CH
2
]P state differ significantly from each
other. The differences in the EPR spectra in different states
indicate significant alterations in the internal structure of the
myosin head region and the properties of binding to actin.
Accordingly, our experiments support the view that the
rotational motion of myosin reflects functionally relevant
conformational changes during ATP hydrolysis by myosin.
The TCSL probe attached to the small SH1–SH2 helix may
be a sensitive detector that reports the coupling between the
motor and regulatory domains of myosin.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Research
Foundation (OTKA T 030248, CO 123) and Ministry of Education
(FKFP 0387/2000).
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Ó FEBS 2002 Effect of AdoPP[CH
2
]P on myosin head domain movements (Eur. J. Biochem. 269) 2177