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

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[CH2]P states, indica-

Effect of adenosine 5¢-[b,c-imido]triphosphate on myosin

head domain movements

Saturation transfer EPR measurements without low-power phase setting

No´ra Hartvig1, De´nes Lo˜rinczy2, Nelli Farkas1and Joseph Belagyi1

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[CH2]P) states, indica-ting changes in the internal dynamics and domain orienta-tion of myosin In the AdoPP[CH2]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-[CH2]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–Pi[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]:

AMþ ATP $ A þ MATP $ A þ MATP $

AMADPPi$ AMADP þ Pi$ AM þ ADP þ Pi

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[CH2]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[CH2]P

to myosin in muscle fibers, and to find some correlation with the myosin head structure The AdoPP[CH2]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: belagyi@apacs.pote.hu

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)

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

M A T E R I A L S A N D M E T H O D S

Materials

KCl, MgCl2, EGTA, histidine/HCl, glycerol, ADP, ATP,

AdoPP[CH2]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 mM KCl, 5 mM MgCl2, 1 mM EGTA and

25 mM 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 mMpotassium propionate, 5 mM

MgCl2, 1 mM EGTA, 25 mM 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 mMto 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 lMdiadenosine

pentaphosphate The other analogue of intermediates in the

ATPase pathway was formed by AdoPP[CH2]P, which

stoichiometrically binds at the active site of myosin to form

a stable complex The muscle fibers were stored in solution

containing 80 mM potassium propionate, 5 mM MgCl2,

1 mMEGTA, 5 or 16 mMAdoPP[CH2]P and 25 mMTris/

HCl, pH 7.0, for 15 min at 0C, and then spectra were

recorded at ambient temperature (20–22C) Only freshly

opened bottles of AdoPP[CH2]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 0.1M phosphate

buffer at pH 7.0 for 48 h at 4C 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 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 mMEGTA, 5 mMMgCl2, 1 mM 5,5¢-dithiobis(2-nitrobenzoic acid) and 20 mM 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 mM dithiothreitol for 30 min at 0C, 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 lM 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 ± 1C, 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 Xn i

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, where n 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

spðiÞ ¼ AðiÞ cos 0 þ BðiÞ sin 0

where J is the phase angle, and A(i) and B(i) are the

amplitudes of the in-phase and out-of-phase components

Differentiating S with respect to variables m andJ, and

setting the derivatives equal to zero results in

m ¼ spðiÞ and tan 20 ¼ 2VAB

where VA, VBare the variances of the components A(i) and

B(i), and VAB 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:

sp1ðiÞ ¼ AðiÞ cos 0 þ BðiÞ sin 0

sp2

where sp1(i) is taken at an arbitrary phase angleJ, and

sp2(i) is recorded at a phase angle J + 90 Linear

transformation of the two spectra through the calculated

phase angleJ allows the second harmonic in-phase and

out-of-phase EPR spectra to be estimated In practice, the

digitized data of sp1(i) and sp2(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 20C to)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 Mg2+-ATPase consisted of 100 mM KCl,

20 mM Mops, 1 mM MgCl2, 0.5 mM EGTA, 0.15 mM

NADH, 1 mM phosphoenolpyruvate, 20 UÆmL)1

pyru-vate kinase, 40 UÆmL)1 lactate dehydrogenase, and

0.5 mMATP, pH 7.0 For Ca2+,Mg2+-ATPase, the assay

medium also contained 1 mM CaCl2 At 340 nm, the

absorption change was measured with a Perkin–Elmer

spectrophotometer interfaced to a computer The molar

absorption coefficient (e340) of NADH was

6.22· 103mol)1Æcm)1 In the experiments, the Mg2+

-ATPase and Ca2+,Mg2+-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 Mg2+-ATPase activity was 4.131 ± 0.718 lmol PiÆ(mg myosin))1Æmin)1 (n ¼ 4) for control fibers and 4.024 ± 0.742 lmol PiÆ(mg myo-sin))1Æmin)1 (n ¼ 4) for TCSL-fibers The ATPase activity of active fiber bundles was 5.565 ± 0.816 lmol

PiÆ(mg myosin))1Æmin)1 (n ¼ 4) for control fibers, and 3.199 ± 0.457 lmol PiÆ(mg myosin))1Æmin)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 Pi 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 STEPR spectrum of MSL-hemoglobin calculated from the two high-power component spectra (B) The phase angles of the spectra in (A) differ

by 90.

R E S U L T S

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-based spin label, which is believed to bind to the fast 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 mM 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 STEPR 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.

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

xaxis 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[CH2]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.

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, but the orientation order remained (Fig 5) In contrast, AdoPP[CH2]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.

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–Vi spectrum from the

spectrum of TCSL-fibers in the AdoPP[CH2]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[CH2]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[CH2]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 (sr ¼ 0.14 ls)

calcu-lated with the Goldman equation [43] corresponds to the

detached myosin heads, rotating rapidly, or the binding of

AdoPP[CH2]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[CH2]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[CH2]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)

D I S C U S S I O N

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 Mg2+-ATPase activity of myosin

by a factor 20 [46] Our experiments on the Mg2+-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 MgPPiin 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

Ca2+,Mg2+-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 AdoPP[CH2]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[CH2]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[CH2]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 STEPR spectra of spin-labeled fibers The spectra of TCSL-fibers and MSL-TCSL-fibers were taken in perpendicular orientations MgAdoPP[CH 2 ]P was added at a concentration of 16 m M , and the 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 the head region of myosin The fi eld scan was 10 mT.

conformational change is independent of whether ADP or

AdoPP[CH2]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[CH2]P is

almost complete (85% saturation) at a concentration of

0.5 mM [49,50] This supports the view that, in the

AdoPP[CH2]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[CH2]P in insect flight muscle; the ratio

of the two inner equatorial peaks was lowered when the

concentration of AdoPP[CH2]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[CH2]P have recently been

criticised [31] Using X-ray diffraction, the authors

conclu-ded that, at saturating concentrations of MgAdoPP[CH2]P

(20 mMin 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[CH2]P (5 and 16 mM), 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[CH2]P does

not contradict the results of the X-ray diffraction technique

Earlier measurements based on exchangeability of bound

nucleotide and 31P-NMR observations also showed two

states of the myosin heads in the absence of nucleotides and

ADP or AdoPP[CH2]P states, which implies the rapid

interconversion of different conformational states [54,55]

Moreover, the equilibrium, i.e nearly equal populations at

25C between the states, was temperature-dependent: at the

lower temperature (4C) 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[CH2]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 of15N

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[CH2]P binding suggest that the structural states of the myosin head in rigor, the ADP state and the AdoPP[CH2]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

A C K N O W L E D G E M E N T S

This work was supported by grants from the National Research Foundation (OTKA T 030248, CO 123) and Ministry of Education (FKFP 0387/2000).

R E F E R E N C E S

1 Huxley, H.E (1969) The mechanism of muscle contraction Science 164, 1356–1365.

2 Huxley, H.E (1979) Time resolved x-ray diffraction studies on muscle In Cross-Bridge Mechanism in Muscle Contraction (Sugi,

H & Pollack, G.H., ed.), pp 391–401 University of Tokyo Press, Tokyo.

3 Rayment, I., Holden, H.M., Whittaker, M., Yohn, C.B., Lorenz, M., Holmes, K.C & Milligan, R.A (1993) Structure of the actin-myosin complex and its implications for muscle contraction Science 261, 58–65.

4 Smith, C.A & Rayment, I (1996) X-ray structure of the magne-sium (II) ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A˚ resolution Biochemistry 35, 5404– 5417.

5 Holmes, K.C (1998) A molecular model for muscle contraction Acta Crystallogr A 54, 789–797.

6 Ajtai, K., Peyser, M., Park, S., Burghardt, T.P & Muhlrad, A (1999) Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during consecutive steps of ATP hydrolysis Biochemistry 38, 6428–6440.

7 Bagshaw, C.R & Trentham, D.R (1974) The characterization of myosin–product complexes and of product release step during magnesium ion-dependent adenosine triphosphatase reaction Biochem J 141, 331–349.

8 Geeves, M.A & Holmes, K.C (1999) Structural mechanisms of muscle contraction Annu Rev Biochem 68, 687–728.

9 Roopnarine, O., Szent-Gyo¨rgyi, A.G & Thomas, D.D (1998) Microsecond rotational dynamics of spin-labeled myosin reg-ulatory light chain induced by relaxation and contraction of scallop muscle Biochemistry 37, 14428–14436.

10 Fajer, P.G., Fajer, E.A., Schoenberg, M & Thomas, D.D (1991) Orientational disorder and motion of weakly attached cross-bridges Biophys J 60, 642–649.

11 Cooke, R (1986) The mechanism of muscle contraction CRC Crit Rev Biochem 21, 53–118.

12 Hambly, B., Franks, K & Cooke, R (1992) Paramagnetic spin probes attached to a light chain on the myosin head are highly disordered in active muscle fibres Biophys J 63, 1306–1313.

13 Reedy, M.C., Reedy, M.K & Tregear, R.T (1988) Two attached non-rigor crossbridge forms in insect flight muscle J Mol Biol.

204, 357–383.

14 Fisher, A.J., Smith, C.A., Thoden, J., Smith, R., Sutoh, K., Holden, H.M & Rayment, I (1995) Structural studies of myosin–

nucleotide complexes: a revised model for the molecular basis of

muscle contraction Biophys J 68, 19s–28s.

15 Ling, N., Shrimpton, C., Sleep, J., Kendrick-Jones, J & Irving, M.

(1996) Fluorescent probes on orientation of myosin regulatory

light chains in relaxed, rigor and contracting muscle Biophys J.

70, 1836–1846.

16 Holmes, K.C (1998) A powerful stroke Nat Struct Biol 5, 940–

942.

17 Houdusse, A., Kalabokis, V.N., Himmel, D., Szent-Gyo¨rgyi,

A.G & Cohen, C (1999) Atomic structure of scallop myosin

subfragment S1 complexed with MgADP: a novel conformation

of the myosin head Cell 97, 459–470.

18 Burghardt, T.P., Garamszegi, S.P., Park, S & Ajtai, K (1998)

Tertiary structural changes in the cleft containing the ATP

sensi-tive tryptophan and reacsensi-tive thiol are consistent with pivoting the

myosin heavy chain at Gly699 Biochemistry 37, 8035–8047.

19 Bobkov, A.A., Bobkova, E.A., Homsher, E & Reisler, E (1997)

Activation of regulated actin by SH1-modified myosin

subfrag-ment 1 Biochemistry 36, 7733–7738.

20 Bobkova, E.A., Bobkov, A.A., Levitsky, D.I & Reisler, E (1999)

Effects of SH1 and SH2 modifications on myosin Similarities and

differences Biophys J 76, 1001–1007.

21 Thomas, D.D., Ishiwata, S., Seidel, J.C & Gergely, J (1980)

Submillisecond rotational dynamics of spin-labeled myosin heads

in myofibrils Biophys J 32, 873–889.

22 Thomas, D.D & Cooke, R (1980) Orientation of spin-labeled

myosin heads in glycerinated muscle fibers Biophys J 32, 891–

906.

23 Fajer, P.G (1994) Determination of spin-label orientation within

the myosin head Proc Natl Acad Sci USA 91, 937–941.

24 Zhao, L., Gollub, J & Cooke, R (1996) Orientation of

para-magnetic probes attached to gizzard regulatory light chain bound

to myosin heads in rabbit skeletal muscle Biochemistry 35, 10158–

10165.

25 Baker, J.E., Brust-Mascher, I., Ramachandran, S., LaConte,

L.E.W & Thomas, D.D (1998) A large and distinct rotation of

the myosin light chain domain occurs upon muscle contraction.

Proc Natl Acad Sci USA 95, 2944–2949.

26 Belagyi, J., Frey, I & Po´to´, L (1994) ADP-induced changes in

ordering of spin-labelled myosin heads in muscle fibres Eur J.

Biochem 224, 215–222.

27 Mulhern, S.A & Eisenberg, E (1978) Interaction of spin-labeled

and N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid

SH1-blocked heavy meromyosin and myosin with actin and

ade-nosine triphosphate Biochemistry 17, 4419–4425.

28 Marriott, G & Heidecker, M (1996) Light-directed generation of

the actin-activated ATPase Activity of caged heavy meromyosin.

Biochemistry 35, 3170–3174.

29 Trentham, D.R., Bardsley, R.G., Eccleston, J.P & Weeds, A.G.

(1972) Elementary processes of the magnesium ion-dependent

adenosine triphosphatase activity of heavy meromyosin Biochem.

J 126, 635–644.

30 Ma´lna´si-Csizmadia, A., Wooley, R.J & Bagshaw, C.R (2000)

Resolution of conformational states of Dictyostelium myosin II

motor domain using tryptophan (W501) mutants: implications for

the open-closed transition identified by crystallography

Bio-chemistry 39, 16135–16146.

31 Frisbie, S.M., Xu, S., Chalovich, J.M & YuL.C (1998)

Char-acterization of cross-bridges in the presence of saturating

con-centrations of MgAMP-PNP in rabbit permeabilized psoas

muscle Biophys J 74, 3072–3082.

32 Zhao, L., Naber, N & Cooke, R (1995) Muscle cross-bridges

bound to actin are disordered in the presence of 2,3-butanedione

monoxime Biophys J 68, 1980–1990.

33 Fajer, P.G & Marsh, D (1982) Microwave and modulation field

inhomogeneities and effect of cavity Q in saturation transfer EPR

spectra Dependence of sample size J Magn Reson 49, 212–224.

34 Shimoyama, Y & Watari, H (1986) Analysis of saturation transfer electron paramagnetic resonance spectra in term of amplitude and phase J Chem Phys 84, 3688–3693.

35 Po´to´, L., Frey, I & Belagyi, J (1993) ST-EPR without phase setting at low power A calculation method for weak spectra Abstr 11th International Biophysics Congress, Budapest.

36 Squier, T.C & Thomas, D.D (1986) Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics Biophys J 49, 921–929.

37 Evans, C.A (1981) Use of integral of saturation transfer electron paramagnetic spectra to determine molecular rotational correla-tion time Slowly tumbling spin labels in the presence of rapidly tumbling spin labels J Magn Reson 44, 109–116.

38 Horva´th, L.I & Marsh, D (1983) Analysis of multicomponent saturation transfer EPR spectra using the integral method: application to membrane systems J Magn Reson 54, 363–373.

39 Pollard, T.D., Bhandari, B., Maupin, P., Wachsstock, D., Weeds, A.G & Zot, H.G (1993) Direct visualization by electron micro-scopy of the weakly bound intermediates in the actomyosin ade-nosine triphosphatase cycle Biophys J 64, 454–471.

40 Schro¨der, R.R., Manstein, D.J., Jahn, W., Holden, H., Rayment, I., Holmes, K.C & Spudich, J.A (1993) Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1 Nature (London) 364, 171–174.

41 Fajer, P.G., Fajer, E.A., Brunsvold, N.J & Thomas, D.D (1988) Effect of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle crossbridges Biophys J 53, 513–524.

42 Barnett, V.A & Thomas, D.D (1987) Resolution of conforma-tional states of spin-labeled myosin during steady-state ATP hydrolysis Biochemistry 26, 314–323.

43 Goldman, S.A., Bruno, G.V & Freed, J.H (1972) Estimating slow-motional rotational correlation times for nitroxides by elec-tron spin resonance J Phys Chem 76, 1858–1860.

44 Sekine, T & Kielley, W.W (1964) The enzymic properties of N-ethylmaleimide modified myosin Biochim Biophys Acta 81, 336–345.

45 Crowder, M.S & Cooke, R (1984) The effect of myosin sulfhydryl modification on the mechanics of fibre contraction J Mus Res Cell Motil 5, 131–146.

46 Fabian, F & Muhlrad, A (1968) Effect of trinitrophenylation on myosin ATPase Biochim Biophys Acta 162, 596–603.

47 Miyanishi, T., Inoue, A & Tonomura, Y (1979) Differential modification of specific lysine residues in the two kinds of sub-fragment-1 of myosin with 2,4,6-trinitrobenzenesulfonate J Bio-chem 85, 747–753.

48 Hozumi, T & Muhlrad, A (1981) Reactive lysyl of myosin sub-fragment 1: location on the 27K sub-fragment and labeling properties Biochemistry 20, 2945–2950.

49 Goody, R.S., Barrington-Leigh, J., Mannherz, H.G., Tregear, R.T & Rosenbaum, G (1976) X-ray titration of binding of b,c-imido-ATP to myosin in insect flight muscle Nature 262, 613–615.

50 Marston, S.B., Rodger, C.D & Tregear, R.T (1976) Changes in muscle crossbridges when AMPPNP binds to myosin J Mol Biol 104, 263–276.

51 Reedy, M.K., Lucaveche, C., Naber, N & Cooke, R (1992) Insect crossbridges, relaxed by spin labeled nucleotide, show well-ordered 90 state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder J Mol Biol 227, 678–697.

52 Tregear, R.T., Wakabayashi, K., Tanaka, H., Iwamoto, H., Ready, M.C., Ready, M.K., Sugi, H & Amemiya, Y (1990) X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol J Mol Biol 214, 129–141.

53 Lymn, R.W (1975) Low-angle x-ray diagrams from skeletal

muscle: the effect of AMP.PNP, a non-hydrolyzed analogue of

ATP J Mol Biol 99, 567–582.

54 Mihashi, K., Ooi, A & Hiratsuka, T (1990) Evidence for the

existence of two equilibrium conformations of the ternary complex

of myosin subfragment-1, ADP, and orthovanadate J Biochem.

107, 464–469.

55 Shriver, J.W & Sykes, B.D (1981) Phosphorus-31 nuclear mag-netic resonance evidence for two conformations of myosin sub-fragment-1–nucleotide complexes Biochemistry 20, 2004–2012.

56 Fajer, P.G., Fajer, E.A., Matta, J.M & Thomas, D.D (1990) Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high resolution study with deuterated spin labels Biochemistry 29, 5865–5871.