Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain Olga P.. However, S1 cleaved by trypsin in the N-termin
Trang 1Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain
Olga P Nikolaeva1, Victor N Orlov1, Andrey A Bobkov2,* and Dmitrii I Levitsky1,2
1
A N Belozersky Institute of Physico-Chemical Biology, Moscow State University; and2A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia
The thermal unfolding of myosin subfragment 1 (S1) cleaved
by trypsin was studied by differential scanning calorimetry
In the absence of nucleotides, trypsin splits the S1 heavy
chain into three fragments (25, 50, and 20 kDa) This
cleavage has no appreciable influence on the thermal
unfolding of S1 examined in the presence of ADP, in the
ternary complexes of S1 with ADP and phosphate analogs,
such as orthovanadate (Vi) or beryllium fluoride (BeFx), and
in the presence of F-actin In the presence of ATP and in the
complexes S1ÆADPÆVi or S1ÆADPÆBeFx, trypsin produces
two additional cleavages in the S1 heavy chain: a faster
cleavage in the N-terminal region between Arg23 and Ile24,
and a slower cleavage at the 50 kDa fragment It has been
shown that the N-terminal cleavage strongly decreases the
thermal stability of S1 by shifting the maximum of its
ther-mal transition by about 7C to a lower temperature, from
50C to 42.4 C, whereas the cleavage at both these sites
causes dramatic destabilization of the S1 molecule leading to
total loss of its thermal transition Our results show that S1
with ATP-induced N-terminal cleavage is able, like
uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx) These changes are reflected in a pronounced increase of S1 thermal stability However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4–5C shift of the S1 thermal transition to higher temperature Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it pre-vents structural changes that occur when S1 is bound to F-actin The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabili-zation of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites
Keywords: myosin subfragment 1; thermal unfolding; differential scanning calorimetry
Cyclic association–dissociation of actin and myosin coupled
with ATP hydrolysis by myosin ATPase is the most
essential process of muscle contraction The globular head
of myosin, called subfragment 1 or S1, where both the
nucleotide- and actin-binding sites of the molecule are
located, is responsible for the generation of force during
contraction The function of the myosin head as a
mole-cular motor is explained by significant conformational
changes, which occur in the head during ATPase reaction
and alter the character of actin–myosin interaction [1,2]
Thus the description of nucleotide- and actin-induced
structural changes in the myosin head is essential for the understanding of the motor mechanism
Among a variety of methods employed, the method of differential scanning calorimetry (DSC) is especially useful for probing global structural changes that occur in the myosin head due to interaction with nucleotides and F-actin DSC is the most effective and commonly employed method to study the thermal unfolding of proteins [3,4] This method has been used successfully for studying structural changes, which occur in the myosin head due to formation of stable ternary complexes with ADP and Pi analogs, such as orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4)– These complexes are stable analogs of the S1*ÆATP and S1**ÆADPÆPi interme-diate states of the S1-catalyzed Mg2+-ATPase reaction [5–7] and, therefore, they are often used for probing the conformational changes occurring in the myosin head in the course of the ATPase reaction [8–12] It has been shown
by using DSC that the formation of the ternary complexes S1ÆADPÆViand S1ÆADPÆBeFxcauses a global change of S1 conformation, which is expressed in a pronounced increase
of S1 thermal stability and in a significant change of S1 domain structure [13,14] The use of various naturally occurring nucleoside diphosphates [15] and their synthetic non-nucleoside analogs [16] allowed us to conclude that these changes revealed by DSC adequately reflect those changes which occur in the S1 molecule in the course of the
Correspondence to D I Levitsky, A N Bach Institute of
Biochemistry, Russian Academy of Sciences, Leninsky prospect 33,
Moscow 119071, Russia.
Fax: +7 095 9542732, Tel.: +7 095 9521384,
E-mail: levitsky@inbi.ras.ru
Abbreviations: DSC, differential scanning calorimetry; S1, myosin
subfragment 1; t-S1, S1 with heavy chain cleaved by trypsin into
the fragments 25, 50, and 20 kDa; Nt-S1, t-S1 with additional
N-terminal tryptic cleavage between Arg23 and Ile24.
*Present address: The Burnham Institute, 10901 N Torrey Pines
Road, La Jolla, CA 92037, USA.
(Received 31 May 2002, revised 10 August 2002,
accepted 24 September 2002)
Trang 2ATPase reaction It has also been concluded from DSC
experiments on recombinant fragments of the head of
Dictyostelium discoideummyosin II that the changes in the
thermal unfolding, that are due to formation of stable
ternary complexes with ADP and Pianalogs, occur mainly
in the globular motor portion of the head [17] Moreover,
DSC was also successfully used for probing the structural
changes that occur in the myosin head due to its strong
binding to F-actin in the presence of ADP It was shown
that the binding of skeletal S1 to F-actin significantly
increased the thermal stability of S1 [18,19] A very similar
effect was observed by DSC with recombinant D
discoid-eummyosin head fragment corresponding to the globular
motor portion of the head [20] It has been shown that
charge changes in the actin-binding surface loop 2 of myosin
strongly affect the thermal unfolding of the myosin motor
domain bound to F-actin [20] It has also been concluded
from DSC experiments with S1 modified by
p-phenylene-dimaleimide that actin-induced structural changes occur not
only upon strong binding but also on weak binding of the
head to F-actin [19] Therefore it can be suggested that
actin-induced structural changes play an important role in
the motor function of the myosin head
Hence the use of the DSC method represents a powerful
experimental approach for probing nucleotide- and
actin-induced structural changes in the myosin head The main
goal of these studies was to understand the mechanism of
these changes, i.e the mechanism of transmission of
structural changes from the nucleotide- and actin-binding
sites to the entire motor portion of the head For this
purpose, specially modified S1 preparations were studied by
DSC to reveal their ability to undergo global
conforma-tional changes due to interaction with F-actin and
nucleo-tides The most interesting modifications were those which
did not directly affect the actin- and nucleotide-binding
sites, but impaired the spread of conformational changes
from these sites to the entire motor domain of the myosin
head In this respect, a cleavage at the N-terminal region of
myosin heavy chain was of particular interest as it is induced
by nucleotides and prevented by actin, although this region
does not seem to directly involved in actin binding
It is well known that in the absence of nucleotides, trypsin
and many other proteases cleave the heavy chain of rabbit
skeletal S1 into three fragments of 25kDa, 50 kDa, and
20 kDa (aligned from the N-terminus in this order) [21] that
remain tightly associated under nondenaturing conditions
This cleavage occurs at two flexible surface loops: the first
loop, termed loop 1, is located near the active site of myosin
ATPase at the 25kDa-50 kDa junction, while loop 2,
connecting 50 kDa and 20 kDa segments, is part of the
actin-binding interface ATP and ADP open a new site for
tryptic cleavage in the N-terminal region of the heavy chain
of rabbit skeletal S1 between Arg23 and Ile24 [22,23]
Similar nucleotide-induced cleavage at the N-terminal
region has also been demonstrated for different S1 species
(rabbit or chicken skeletal S1, smooth muscle S1 from
chicken gizzard) with many other proteases, such as
subtilisin, thermolysin, and chymotrypsin [21,24,25] It is
therefore quite possible that the 3D structure of this region
and the spatial relationship to the nucleotide-binding site are
similar among all S1 species It has been suggested from
secondary structure predictions that this region is a random
coil held between the two a-helices [25] Nucleotide-induced
conformational changes in the myosin head probably expose this N-terminal region to proteases On the other hand, actin was found to suppress the nucleotide-induced tryptic cleavage at the N-terminal region of S1 in both strongly attached state (in the presence of ADP) [26] and weakly attached state (in the presence of ATP analogs) [27] As the N-terminal region is located spatially far from actin-binding sites in the 3D structure of S1 [28], these effects of actin can
be explained by long-range conformational changes induced
by the attachment of actin to its binding sites, primarily to loop 2 which is mainly responsible for the weak binding of S1 to F-actin Therefore we can expect that the actin-induced conformational changes in the S1 molecule should also be affected by the N-terminal tryptic cleavage
Very little is known about the properties of S1 modified by the N-terminal cleavage This cleavage was found to accelerate the inactivation of the S1 ATPase upon mild heat treatment with the loss of the ability of nucleotides to protect the S1 against thermal denaturation [23] There is some discrepancy in the literature about the effect of the N-terminal cleavage on the ATPase activity of S1: some authors have shown significant inhibition [26] and others have observed no changes in the activity [23] When nucleotides were not removed from S1 after the N-terminal tryptic cleavage, the cleaved S1 was shown to retain ATPase activity and actin binding similar to that of uncleaved S1 [29]
In the present study, we applied the DSC approaches described above to examine the effects of N-terminal tryptic cleavage on the thermal unfolding of S1 The main goal of this research was to investigate how this cleavage affects the ability of S1 to undergo global nucleotide-induced and actin-induced conformational changes For this purpose we studied the thermal unfolding of S1 cleaved by trypsin in the N-terminal region (Nt-S1) in the presence of ADP, in the ternary complexes with ADP and Pianalogs (Vi, BeFx), and
in the presence of F-actin For comparison, the thermal unfolding of S1 cleaved by trypsin in the absence of nucleotides into the fragments 25, 50, and 20 kDa (t-S1) was also studied by DSC under the same conditions Our results show that the N-terminal tryptic cleavage of the S1 heavy chain dramatically decreases the thermal stability of S1 and completely prevents the actin-induced conformational changes in the S1 molecule On the other hand, we show that this cleavage does not significantly affect the ability of S1 to form stable complexes S1ÆADPÆViand S1ÆADPÆBeFx and to undergo structural changes due to formation of these complexes
M A T E R I A L S A N D M E T H O D S
Proteins S1 from rabbit skeletal myosin was prepared by digestion of myosin filaments with a-chymotrypsin [30] The concentra-tion of S1 was determined by measuring A280 using an absorption coefficient of 0.75mgÆmL)1Æcm)1 The prepar-ation of the trypsin-modified derivatives t-S1 and Nt-S1 was performed according to Mornet et al [22] The t-S1 was obtained by tryptic digestion using a 1 : 50 (w/w) ratio of trypsin and S1 at 25C for 60 min To prepare Nt-S1, 5mM ATP was added The concentration of S1 was 3 mgÆmL)1in both cases, and the medium contained 50 mM Tris/HCl,
pH 8.0, 30 mMKCl, and 5mMMgCl During the course
Trang 3of digestion, aliquots were taken and analysed by SDS/
PAGE [31] Digestion was terminated by adding soybean
trypsin inhibitor at a 1.5: 1 (w/w) ratio to trypsin The
proteins were dialyzed against 30 mM Hepes, pH 7.3,
containing 1 mM MgCl2and 0.5mMADP, stored in the
same buffer, and used for experiments during three days
The concentrations of t-S1 and Nt-S1 were measured by the
Bradford protein assay [32] using undigested S1 as standard
K+-EDTA-ATPase activities of S1, t-S1, and Nt-S1 were
determined by measuring the released Pi
Actin was prepared from rabbit skeletal-muscle acetone
powder [33] Monomeric G-actin was stored in low-strength
buffer composed of 2 mMTris/HCl, pH 8.0, 0.2 mMATP,
0.2 mM CaCl2, 0.5 mM 2-mercaptoethanol, and 0.01%
NaN3 (G-buffer) Actin concentration was determined
by measuring A290 using absorption coefficient of
0.63 mgÆmL)1Æcm)1 G-actin was polymerized to F-actin in
G-buffer by the addition of 4 mM MgCl2 F-Actin was
stabilized by the addition of a twofold molar excess of
phalloidin (Sigma) to obtain a better separation of the
thermal transitions of actin-bound S1 and F-actin on DSC
thermograms Specific binding of this cyclic heptapeptide to
F-actin was shown to increase the temperature of the thermal
denaturation of F-actin by 14C [34] A similar effect of
phalloidin was observed in our DSC experiments [35]
Preparation of the complexes of t-S1 and Nt-S1
with ADP and Pianalogs
Trapping of ADP by different phosphate analogs (Vi, BeFx)
was performed by the methods described for the
preparation of stable ternary complexes S1ÆADPÆVi and
S1ÆADPÆBeFx[5,7,15] To obtain these complexes, t-S1 or
Nt-S1 (1 mgÆmL)1) were incubated with 0.5 mMVior BeFx
for 30 min at 20C in a medium containing 30 mMHepes,
pH 7.3, 1 mMMgCl2, and 0.5 mMADP Beryllium fluoride
complexes were obtained by addition of 0.5mM BeCl2in
the presence of 5mMNaF The formation of the complexes
was controlled by measuring the K+-EDTA ATPase activity
of the protein The ATPase activity of S1, t-S1, or Nt-S1
modified by Vior BeFxin the presence of ADP did not exceed
3–5% of the activity of unmodified protein preparation
Actin binding assay
Complexes of S1, t-S1, or Nt-S1 with F-actin were formed
by mixing equal volumes of F-actin and S1 solutions
F-actin solutions contained G-buffer, 3 mM MgCl2, and
0.5mM ADP S1 solutions contained 30 mM Hepes,
pH 7.3, 1 mMMgCl2, and 0.5 mMADP The final
concen-tration of S1, t-S1, or Nt-S1 was 13 lM, and F-actin
concentration was 26 lM
The binding of S1, t-S1, or Nt-S1 to phalloidin-stabilized
F-actin was determined by a cosedimentation assay The
complexes of F-actin with t-S1, Nt-S1, or uncleaved S1 were
examined by sedimentation velocity experiments in a
Beckman model E analytical ultracentrifuge with a
photo-electric scanning system at rotor speed from 12 000 to
24 000 r.p.m All the experiments were performed in a
standard four-hole rotor An-F Ti After precipitation of
the acto-S1 complexes by the low-speed centrifugation, the
samples were subjected to high-speed centrifugation at
rotor speed from 48 000 to 60 000 r.p.m in order to reveal
any S1 molecules remained in the supernatant Sedimenta-tion properties (homogeneity, sedimentaSedimenta-tion coefficients) of S1 and its derivatives in the absence of F-actin were also examined in these experiments
Differential scanning calorimetry (DSC) DSC experiments were performed on a DASM-4M differ-ential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described previously [13–20] Prior to measurements, all S1 samples were dialyzed against 30 mMHepes, pH 7.3, containing 1 mMMgCl2and 0.5mMADP All experiments were performed at a scanning rate of 1 KÆmin)1(the rate which was used in all our previous DSC experiments with S1 and other fragments of the myosin head [13–20]) The reversibility of the thermal transitions was verified by checking the reproducibility of the calorimetric trace in a second heating of the sample immediately after cooling from the first scan The thermal denaturation of all protein samples studied was fully irreversible This irreversi-bility can be explained by protein aggregation which was shown to occur after heating of S1 and its complexes with nucleotides [13] The calorimetric traces were corrected for the instrumental background and for possible aggregation artifacts by subtracting the scans obtained from the second heating of the samples The temperature dependence of the excess heat capacity was farther analysed and plotted using ORIGINsoftware (MicroCal Inc.) Transition temperatures (Tm) were determined from the maximum of the thermal transition Calorimetric enthalpies (DHcal) were calculated from the area under the excess heat capacity curves Because these parameters can be obtained directly from experimental calorimetric traces after simple treatment such as subtraction
of instrumental background, concentration normalization, and chemical baseline correction, they can be used for the description of the irreversible thermal denaturation of S1
R E S U L T S
Calorimetric characterization of the S1 species modified by tryptic cleavage
Figure 1A shows electrophoretic pattern of the S1 prepa-rations obtained after limited tryptic digestion of S1 in the absence and in the presence of ATP In the absence of nucleotides the S1 heavy chain (95kDa) was cleaved with trypsin into three large fragments (25kDa, 50 kDa, and
20 kDa) The presence of ATP during tryptic digestion induces two additional cleavages in the S1 heavy chain leading to a faster conversion of the N-terminal 25kDa segment into the product of 22 kDa and a slower transfor-mation of the 50 kDa segment into the 45 kDa product [22,26] In our preparation of S1 treated with trypsin in the presence of ATP (Nt-S1) the 25kDa fi 22 kDa transfor-mation was almost complete, while only about half of the 50 kDa segment was converted into the 45 kDa segment (Fig 1A) While t-S1 (i.e S1 cleaved with trypsin
in the absence of nucleotides) demonstrated the same
K+-EDTA ATPase activity as uncleaved S1 did (about
3 lmolÆPiÆmin)1Æmg)1), the activity of Nt-S1 was about 50–60% of that for t-S1 and uncleaved S1 The trypsin-cleaved S1 preparations, t-S1 and Nt-S1, were also exam-ined for their homogeneity by sedimentation velocity
Trang 4experiments performed under the same conditions, at the
same protein concentration (1 mgÆmL)1), and even in the
same rotor Both these S1 species demonstrated sharp
sedimentation boundaries, and sedimentation coefficients
measured at 20C and rotor speed of 60 000 r.p.m were equal to 5.4 ± 0.1 S for both t-S1 and Nt-S1 However, an amplitude of the boundary (i.e the optical density at
280 nm of the normally sedimenting protein) for Nt-S1 was about half of that observed with t-S1 These results allow us
to suggest that about 50% of the molecules in the Nt-S1 preparation undergo to unfolding with full loss of the ATPase activity
Figure 1B shows calorimetric traces of the thermally induced unfolding of Nt-S1, t-S1, and uncleaved S1 All the samples contained ADP, which was shown to protect Nt-S1 from rapid inactivation upon storage [29] Under these conditions, the DSC curve of t-S1 is similar to that
of uncleaved S1, whereas Nt-S1 is clearly less thermo-stable than t-S1 and S1 The transition temperature for Nt-S1 (Tm¼ 42.4 C) is shifted to a lower temperature by
7C, in comparison with that of t-S1 (Tm¼ 49.4 C) The main calorimetric parameters extracted from these data (Tm, DHcal) are summarized in Table 1 The value
of calorimetric enthalpy DHcal determined for Nt-S1 (740 kJÆmol)1) is about 55–60% of that for S1 and t-S1 (Table 1) This great difference in the DHcalvalue cannot
be explained by possible contributions of DCp (i.e the difference in heat capacity, Cp, between the native and denatured states of the protein) which were similar for S1, t-S1, and Nt-S1
It should be noted that a good correlation exists between the conversion of about 50% of the 50 kDa segment into the 45kDa segment (Fig 1A, lane 3) and the decrease by about 50% in the ATPase activity, in the amount of normally sedimenting molecules, and in the value of calorimetric enthalpy DHcal of Nt-S1 (Table 1) This correlation suggests that the cleavage in the 50 kDa segment causes a full denaturation of S1, and that only part of the Nt-S1 molecules, which has the uncleaved 50 kDa segment,
is responsible for ATPase activity, sedimentation properties, and cooperative thermal transition of Nt-S1 This means that N-terminal cleavage leading to conversion of the 25kDa segment into 22 kDa segment itself does not significantly alter the ATPase activity and calorimetric enthalpy of S1, but it dramatically decreases the thermal stability of S1 by shifting the thermal transition of S1 by
7C to a lower temperature
The ternary complexes of t-S1 and Nt-S1 with ADP and phosphate analogs
Previous DSC studies performed with skeletal S1 and with
D discoideum myosin head fragments have shown that
Table 1 Calorimetric parameters obtained from the DSC data for S1, t-S1, and Nt-S1 The absolute error of the given values of transition temperature (T m ) did not exceed ± 0.2 C The relative error of the given values of calorimetric enthalpy, DH cal , did not exceed ± 7% Experimental conditions for measurements were 30 m M Hepes,
pH 7.3, 1 m M MgCl 2 , 0.5 m M ADP Molecular mass of 115kDa was used for calculation of DH cal for all proteins studied.
Protein T m (C) DH cal (kJÆmol)1)
Fig 1 Electrophoretic patterns (A), and DSC scans (B) of S1 (1), t-S1
(2), and Nt-S1 (3) Protein concentrations were 1 mgÆmL)1
Condi-tions: 30 m M Hepes, pH 7.3, 1 m M MgCl 2 , 0.5 m M ADP The heating
rate was 1 KÆmin)1 The parameters derived from calorimetric data are
shown in Table 1.
Trang 5DSC is useful for probing global conformational changes in
the myosin motor caused by ligand binding Formation of
stable ternary complexes of the myosin head with ADP and
Pianalogs such as Vi, BeFx, and AlF4 were shown to cause
a significant increase of the thermal stability of the protein,
as judged by the values measured for Tmand DHcal, and a
considerable increase in the cooperativity of the thermal
transition [13,15,17,20] Figure 2 shows the effects of the
formation of the ternary complexes of t-S1 and Nt-S1 with
ADP-Viand ADP-BeFx, in comparison with the proteins
containing ADP alone (control curves shown by dashed
lines) For both proteins, the formation of the ternary
complexes causes a significant shift of the thermal transition
to higher temperature and the effect of BeFx is less
pronounced than the effect of Vi(Fig 2A,B) In the case
of t-S1 (Fig 2A), the effects of Pianalogs were very similar
to those observed with uncleaved S1 [13,15] Formation of
the complex t-S1ÆADPÆViincreased Tmby 7.7C, from 49.4
to 57.1C, and caused a pronounced increase of DHcalby
18%, from 1230 to 1450 kJÆmol)1 In the case of the
complex t-S1ÆADPÆBeF, the thermal transition of t-S1
shifted to 5 5 1C and its enthalpy (1400 kJÆmol)1) increased by only 14% in comparison with ADP-containing t-S1 (Fig 2A)
The effects of Viand BeFxon Nt-S1ÆADP (Fig 2B) were even more pronounced than in the case of t-S1 and uncleaved S1 Formation of the complex Nt-S1ÆADPÆVi shifted the thermal transition of Nt-S1ÆADP by 10.7C, from 42.4C to 5 3.1 C, and increased its enthalpy by almost 60%, from 740 to 1170 kJÆmol)1 The increase in Tm was less in the case of the complex Nt-S1ÆADPÆBeFx (8.3C), although DHcalincreased in this case by more than 60%, to 1240 kJÆmol)1
Thus, the DSC experiments show that Nt-S1 is able, like S1 and t-S1, to undergo global structural changes due to formation of ternary complexes with ADP and Pi analogs Formation of the complexes Nt-S1ÆADPÆVi and Nt-S1ÆADPÆBeFx has a strong stabilizing effect on Nt-S1, leading to significant increase of the thermal stability of the protein This effect observed with Nt-S1 is even more pronounced than in the case of t-S1 and uncleaved S1 The DSC method can also be used to examine the relative stability of the S1ÆADPÆVi and S1ÆADPÆBeFx complexes obtained with modified S1 or with various nucleoside diphosphates [15,16,36] The complexes decompose slowly after removal of excess reagents, and this process is linked to the disappearance of calorimetric peak attributed to the complex and the corresponding appearance of the peak assigned to nucleotide-free S1 This approach can be used only for the characterization of the stability of those ternary complexes whose calorimetric peaks are clearly distinguish-able from the peaks of nucleotide-free S1 or S1ÆADP on the thermogram [15,16,36] The complexes Nt-S1ÆADPÆViand Nt-S1ÆADPÆBeFxmeet these criteria (Fig 2B) These com-plexes were dialyzed for 48 h at 4C against 30 mMHepes,
pH 7.3, containing 1 mMMgCl2, to remove the free ADP and Vi or BeFx and were then subjected to calorimetric measurements performed in the presence of 0.5mMADP It
is clear from Fig 3 that the decomposition of the complexes Nt-S1ÆADPÆVi and Nt-S1ÆADPÆBeFx was negligible The removal of the reagents caused only some small decrease in calorimetric enthalpy of the thermal transition, by 16% for Nt-S1ÆADPÆVi(Fig 3A) and by 25% for Nt-S1ÆADPÆBeFx (Fig 3B) These results are very similar to those obtained earlier with uncleaved S1 [15,16,36] Thus, the ternary complexes of Nt-S1 with ADP and Pianalogs are as stable
as the complexes obtained with control uncleaved S1 as they
do not significantly decompose a few days after removal of excess reagents
Tryptic cleavage of S1 in the S1ÆADPÆVi and S1ÆADPÆBeFxcomplexes
The N-terminal tryptic cleavage of the S1 heavy chain can
be achieved not only in the presence of ATP, but also in the ternary complex S1ÆADPÆVi [37] This approach is very convenient to investigate the changes in the thermal unfolding of S1 in the course of tryptic digestion and to determine which transformation, 25kDafi 22 kDa or
5 0 kDafi 45kDa, is responsible for destabilizing the S1 molecule S1 was digested in the presence of 0.5mMADP and 0.5mMVifor 60 min and aliquots were taken at several times Digestion was stopped by addition of soybean trypsin inhibitor, and then the samples were subjected to DSC
Fig 2 Temperature dependence of the excess molar heat capacity for
t-S1 (A) and Nt-S1 (B) in the presence of ADP and V i or BeF x Curves
shown by dashed lines were obtained in the presence of 0.5m M ADP.
Solid line curves were obtained in the presence of 0.5m M ADP and
0.5m M V i Curves shown by dashes-and-dots were obtained in the
presence of 0.5m M ADP, 5m M NaF, and 0.5m M BeCl 2 Other
conditions were the same as in Fig 1B.
Trang 6analysis and to SDS/PAGE (Fig 4) Figure 4B shows that
in the course of tryptic digestion the initial transition with
maximum at 58C characteristic for control uncleaved S1
in the S1ÆADPÆVi complex turns into transition with
maximum at 53.1C which corresponds to Nt-S1 in the
ternary complex with ADP and Vi (Fig 2B) The
disap-pearance of the transition at 58C on Fig 4B and its
conversion into transition at 53.1C correlates well with
disappearance of the band of 25kDa fragment on the
electrophoretogram (Fig 4A) After 40 min of incubation
with trypsin, when the 25kDa band had almost completely
disappeared (Fig 4A), only the thermal transition at
53.1C was observed on the thermogram (Fig 4B) At
the same time, the 5 0 kDafi 45kDa transformation was also observed, but this conversion did not exceed 50%, even after prolonged proteolysis (Fig 4A) Very similar results were obtained with tryptic digestion of S1 in the S1ÆADPÆBeFx complex (data not shown) Overall, these data support the above suggestion that the changes in the
Fig 4 Electrophoretic patterns (A) and DSC scans (B) of the S1 samples obtained in the course of tryptic digestion of S1 in the S1ÆADPÆV i
complex S1 (2 mgÆmL)1) was digested with trypsin (50 : 1 by mass) in the presence of 0.5m M ADP and 0.5m M V i for different time inter-vals, and the digestion was terminated by the addition of soybean trypsin inhibitor (1.5: 1, by mass, to trypsin) (A) Lane 1, undigested S1; lanes 2–7, S1 digested for 5, 10, 20, 30, 40 and 60 min, respectively (B) Time intervals of digestion are indicated for each curve Condi-tions: 30 m M Hepes, pH 7.3, 1 m M MgCl 2 , 0.5 m M ADP, 0.5m M V i S1 concentration was 1 mgÆmL)1 Heating rate was 1 KÆmin)1 The vertical bar corresponds to 100 kJÆmol)1ÆK)1.
Fig 3 Temperature dependence of the excess molar heat capacity for
Nt-S1 in the ternary complexes with ADP and V i (A) or BeF x (B) before
and after removal of excess reagents Curves shown by dashed lines
were obtained for Nt-S1 in the presence of 0.5m M ADP and 0.5m M
V i (A) or 0.5m M ADP, 5m M NaF and 0.5m M BeCl 2 (B) Solid line
curves were obtained after removal of excess ADP and V i or BeF x from
the complexes Nt-S1ÆADPÆV i and Nt-S1ÆADPÆBeF x by dialysis against
30 m M Hepes, pH 7.3, containing 1 m M MgCl 2 , at 4 C for 48 h After
dialysis ADP was again added to these samples to final concentration
of 0.5m M Other conditions were the same as in Figs 1B and 2B.
Trang 7thermal unfolding of S1 that are expressed in a significant
shift of the S1 thermal transition to lower temperature, are
due to the splitting in the 25kDa segment
Binding of t-S1 and Nt-S1 to F-actin
It has been suggested that DSC studies of acto-S1 offer a
new and promising approach to investigate the changes that
occur in the S1 molecule due to its interaction with F-actin
[18,19] In the present work, we applied this approach to
study the interaction of t-S1 and Nt-S1 with F-actin The
addition of phalloidin shifted the Tmfor F-actin from 62 to
82C, thus providing a very good separation between the
calorimetric peaks of actin-bound t-S1 or Nt-S1 and F-actin
(Fig 5) This separation allowed us to carry out the
treatment and detailed analysis of the thermal transitions
for actin-bound t-S1 and Nt-S1
Figure 6 shows the excess heat capacity curves for
actin-bound t-S1 (Fig 6A) and Nt-S1 (Fig 6B), in comparison
with the curves obtained in the absence of F-actin under the
same conditions Strong binding of t-S1 to F-actin in the
presence of ADP increases the thermal stability of t-S1
substantially by shifting whole the thermal transition by
4.7C, from 49.4 C to 5 4.1 C (Fig 6A), and by increasing
the DHcalvalue for t-S1 from 1230 to 1340 kJÆmol)1 This
effect is very similar to that observed under the same
conditions with control uncleaved S1 [19] On the other
hand, Nt-S1 does not demonstrate any actin-induced shift
of its thermal transition to a higher temperature (Fig 6B)
Moreover, interaction with F-actin even decreases, through slightly, the thermal stability of Nt-S1 This actin-induced destabilization of Nt-S1 is reflected in a small shift of Tmto lower temperature, from 42.5to 41.1C, and a decrease in
DHcalfrom 740 to 590 kJÆmol)1 A small peak at 5 3.5 C observed on the thermogram of Nt-S1 in the presence of F-actin (Fig 6B) can be assigned to the thermal unfolding
of actin-bound t-S1, as some small admixture of t-S1, of about 5–7%, is usually present in the Nt-S1 preparation due
to incomplete N-terminal cleavage
Formation of the complex of F-actin with Nt-S1 was verified by sedimentation velocity experiments performed under the same conditions as for DSC experiments, i.e in the same medium and at the same molar ratio, Nt-S1/ F-actin equal to 1 : 2 The sedimentation coefficient of this complex measured at 20C and rotor speed of
15000 r.p.m was equal to 70.0 ± 4.4 S, which is appre-ciably higher than that of free F-actin (49.0 ± 3.5S) but lower than the coefficient of F-actin complexed with t-S1 or uncleaved S1 under the same conditions (94.5± 8.5S and 117.5± 7.5S, respectively) However, only about 50–60%
of Nt-S1 molecules retain the folded tertiary structure, as it
Fig 5 The experimental DSC curves of F-actin stabilized by phalloidin
(A) and its complexes with t-S1 (B) or Nt-S1 (C) Conditions: 26 l M
F-actin, 50 l M phalloidin, 13 l M t-S1 or Nt-S1 in 15m M Hepes,
pH 7.3, 2 m M MgCl 2 , 0.5 m M ADP, and twice-diluted G-buffer.
Heating rate 1 KÆmin)1 The vertical bar corresponds to 10 lW.
Fig 6 Temperature dependence of the excess molar heat capacity for t-S1 (A) and Nt-S1 (B) in the absence (dashed line curves) and in the presence (solid line curves) of F-actin The temperature region above
65 C, corresponding to the region of thermally induced denaturation
of phalloidin-stabilized F-actin, is not shown Conditions were the same as in Fig 5.
Trang 8was shown above, and only these molecules are probably
able to bind to F-actin At the same time, the sedimentation
coefficient of the acto-S1 complex is strongly dependent on
the S1/F-actin molar ratio When we increased the molar
concentration of Nt-S1 by 1.5–2 times, the sedimentation
coefficient for F-actin complexed with Nt-S1 became very
similar to that obtained with control, uncleaved S1 After
precipitation of the acto-S1 complexes by low-speed
centrifugation the samples were subjected to high-speed
centrifugation at a rotor speed of 48 000 r.p.m., in order to
reveal S1 molecules unbound to F-actin and retained in the
supernatant We observed no boundaries of the protein
sedimenting with the coefficient of about 5S in these
experiments These results indicated that Nt-S1, like S1 and
t-S1, was completely bound to F-actin under the conditions
used for the DSC experiments
In agreement with earlier published data [29], these results
mean that Nt-S1 is able, like S1 and t-S1, to bind to F-actin
in the presence of ADP However, it is unable, unlike S1 and
t-S1, to undergo actin-induced structural changes expressed
in a significant shift of the thermal transition to higher
temperature (Fig 6)
D I S C U S S I O N
In this study, we used DSC to analyze the effects of the
tryptic cleavage of the S1 heavy chain on S1 structure and
its changes induced by nucleotides and actin For this
purpose, we compared the thermal unfolding of S1 species
with the heavy chain cleaved by trypsin at specific,
well-defined sites
Thermal unfolding of S1 cleaved by trypsin
in the absence of nucleotides
First, the effects of the tryptic cleavage at the 25kDa/
50 kDa and 50 kDa/20 kDa junctions of the S1 heavy
chain were studied The results show that the cleavage at
these sites has no appreciable effect on the thermal
unfolding of S1 (Fig 1) Moreover, this cleavage does not
significantly affect the ability of S1 to undergo
nucleotide-and actin-induced structural changes (Fig 2A nucleotide-and 6A) The
50 kDa/20 kDa junction corresponds to so-called loop 2, a
lysine-rich surface segment of the myosin motor domain
which forms part of the actin-binding site Loop 2 is known
to interact directly with the negatively charged N-terminal
part of actin, and this electrostatic interaction is mainly
responsible for the weak binding of the myosin head to
F-actin [38–40] Previous DSC studies showed that charge
changes in loop 2 strongly affected the thermal unfolding of
the myosin motor domain bound to F-actin [20] For
example, introduction of additional negative charges into
the loop caused a significant decrease in the actin-induced
shift to higher temperature of the thermal transition of
D discoideummyosin motor domain [20], and deletion of
the loop led to complete disappearance of this actin-induced
shift [41] On the other hand, the results presented here show
that tryptic cleavage at loop 2 has no appreciable influence
on the actin-induced changes in the thermal unfolding of S1
(Fig 6A) S1 cleaved at loop 2 (t-S1) probably retains quite
a number of positively charged lysyl residues for
electro-static interaction with the negatively charged residues in the
N-terminal part of actin, and therefore in the presence of
F-actin it demonstrates changes in the thermal unfolding very similar to those observed with uncleaved S1 [19]
The cleavage within 50 kDa segment causes full destabilization of S1
The presence of nucleotides during tryptic digestion induces two additional cleavages in the heavy chain of S1: the cleavage between Arg23 and Ile24 in the N-terminal region leading to conversion of the 25kDa segment into the product of 22 kDa and the cleavage in the C-terminal part
of 50 kDa segment converting it into the 45 kDa product [22,23,26] (Fig 1A and 4A) Therefore, in order to investi-gate the effects of the N-terminal cleavage, their separation from possible effects of the cleavage in 50 kDa segment was required
The N-terminal cleavage is known to occur much faster than the cleavage at the 50 kDa segment [22,23,26] As a result, we obtained S1 preparation with almost complete conversion 25kDafi 22 kDa, whereas less than half of the
50 kDa segment was converted into the 45 kDa product (Fig 1A) This S1 preparation (Nt-S1) demonstrated the
K+-EDTA ATPase activity, the amount of normally sedimenting protein, and the calorimetric enthalpy of about 50–60% of those observed with uncleaved S1 and t-S1 The decrease in these parameters correlated well with the
5 0 kDafi 45kDa conversion (Fig 1A) It has been sug-gested from this correlation that tryptic cleavage within both 25and 50 kDa segments causes dramatic destabiliza-tion of the S1 molecule leading to full loss of its tertiary structure and native properties Therefore, when we used the Nt-S1 for DSC experiments, we observed the thermal unfolding of only those S1 molecules, which were cleaved between Arg23 and Ile24 in the N-terminal region, but not within the 50 kDa segment
N-terminal cleavage dramatically decreases the thermal stability of S1
The results of this work show that tryptic cleavage between Arg23 and Ile24 itself dramatically decreases the thermal stability of S1 by shifting its thermal transition by 5–7C to lower temperature This effect was observed both in the presence of ATP (Fig 1B) and in the S1 ternary complex with ADP and Vi (Fig 4B) These results are in good agreement with literature data showing that in the course of incubation at 35C the K+-EDTA ATPase of Nt-S1 inactivated much faster than those of S1 and t-S1 [23]
It seems possible that the N-terminal region of the S1 heavy chain is very important for stabilization of the entire motor part of S1 The cleavage in this region does cause a significant destabilization of the protein In this context, it is noteworthy that a very similar destabilization, i.e a dramatic decrease of the protein thermal stability (more than 5C decrease of Tm), has been observed for isolated motor domain of the D discoideum myosin head devoid of seven C-terminal residues, the residues 755–761 [17] These residues of D discoideum myosin II correspond to residues 776–782 in the junction between the motor domain and regulatory domain of skeletal S1, and they are located near the N-terminal cleavage site in the atomic structure of S1 [28] Comparison of these data suggests that this junction, which also serves as a communication pathway between the
Trang 9two domains, is of crucial importance for the structural
integrity of the myosin head An important role of the
N-terminal region of the myosin head in the communication
between the motor domain and regulatory domain can also
be proposed
In the crystal structures of the class II myosins, the
N-terminal region forms an independently folding domain
[11,28] It should be noted that in the other myosin classes,
this region is either truncated or absent [42] (e.g the entire
N-terminal region of more than 70 residues is missing in
myosins of class I [43]) As the N-terminal region is
proposed to be very important for stabilization of the entire
motor domain of myosin II, the DSC studies on the thermal
unfolding of myosin I devoid of this region are of particular
interest It has been shown recently that isolated motor
domain of myosin I (MyoIE700) expressed in D discoideum
demonstrates a very low thermal stability both in the
absence of nucleotides (Tm¼ 39 C) and in the presence of
ADP (Tm¼ 43.3 C) (D Levitsky, unpublished results) In
this respect, it is similar to Nt-S1 (Table 1), but it is much
less thermostable than the isolated motor domain of
D discoideum myosin II which unfolds, under the same
conditions, with Tmof 45.6C in the absence of nucleotides
and 49.1C in the presence of ADP [17,20] These results
are in favor of the above suggestion that the N-terminal
region of myosin II is very important for structural
stabilization of the entire motor domain of the myosin head
The N-terminal cleavage prevents actin-induced
structural changes in S1
An intriguing result of the present work is that the
N-terminal cleavage of the S1 heavy chain completely
prevents the changes in the thermal unfolding of S1, i.e a
significant increase in the protein thermal stability, that
occur when S1 is strongly bound to F-actin in the presence
of ADP (Fig 6) This effect cannot be explained only by
destabilization of the entire S1 molecule caused by the
N-terminal tryptic cleavage The results presented here show
that Nt-S1 is able, like S1, to form stable ternary complexes
with ADP and Pianalogs and to undergo global structural
changes due to formation of these complexes (Figs 2B and
3) The recombinant fragment M754 of D discoideum
myosin II, i.e the isolated motor domain devoid of seven
C-terminal residues, showed, like Nt-S1, a very low thermal
stability [17]; however, M754 was able, unlike Nt-S1, to
undergo actin-induced structural changes expressed in a
significant increase of its thermal stability Furthermore, in
the presence of F-actin, another myosin fragment with very
low thermal stability, MyoIE700 (i.e the isolated motor
domain of D discoideum myosin I), showed a very
pronounced shift, more than 10C, of its thermal transition
to a higher temperature (D Levitsky, unpublished results)
Thus, low thermal stability itself can not be the only reason
for inability of the Nt-S1 to undergo structural changes
induced by its binding to F-actin
A very similar effect, i.e the absence of the actin-induced
structural changes, was observed earlier by DSC only in the
case of D discoideum myosin head fragments with many
additional negatively charged residues inserted into loop 2
[20] or with deleted loop 2 [41] These fragments
demon-strated ability to undergo nucleotide-induced structural
changes and to bind to F-actin, but their thermal transitions
shifted by only 0.5–1.2C to a higher temperature in the presence of F-actin [20,41] These effects can be explained easily as loop 2 is part of the actin-binding site and, therefore, alterations in this loop affect the actin–myosin interaction and those structural changes which occur in the myosin head due to this interaction On the other hand, we observed actin-induced structural changes, i.e the DSC-revealed shift of the S1 thermal transition to higher temperature, even in the case of weak binding to F-actin
of S1 with two reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), cross-linked by p-phenylenedimaleimide [19] Such type modified S1 (pPDM-S1) is known to bind to F-actin weakly even in the absence of nucleotides [44], and this weak binding is realized mainly through electrostatic interaction of loop 2 with the negatively charged N-terminal part of actin [38–40] Thus, the interaction of loop 2 with actin seems to be mainly responsible for actin-induced structural changes in the myosin head that are reflected in a pronounced shift of the thermal transition to higher temperature
The effect of the N-terminal cleavage, i.e the absence of the shift to higher temperature of the thermal transition of actin-bound Nt-S1 (Fig 6B), cannot be explained by direct interaction between N-terminal region and loop 2 in S1 as these sites are spatially located rather far from each other in the atomic structure of S1 [28] It seems more likely that a long-distance communication pathway exists between these sites In favor of this suggestion are literature data showing that F-actin suppresses the N-terminal tryptic cleavage of S1 both in the strongly attached state [26] and in the weakly attached state [27] The cleavage between Arg23 and Ile24 probably disrupts this communication pathway, thus pre-venting the global conformational changes in the myosin head induced by actin binding to loop 2
Examination of the S1 structure has suggested that there are contacts between the essential light chain in the regulatory domain and some parts of the heavy chain in the motor domain [45] Essential light-chain residues 103– 115form a helix and lie in close proximity to a helix-loop motif near the N-terminus of the heavy chain (residues 21– 31) This contact may serve as an additional communica-tion pathway between the motor domain and the regula-tory domain, and it may play a crucial role in the transmission of actin-induced conformational changes from loop 2 to the regulatory domain through the motor domain The cleavage between Arg23 and Ile24 in the N-terminal region of the heavy chain may interrupt this transmission by the break of the contact between the motor domain and the light chain
In conclusion, the DSC approach makes it possible to reveal a crucial importance of the N-terminal region of myosin heavy chain for structural stabilization of the myosin head and for conformational changes in the head induced by actin binding
A C K N O W L E D G M E N T S
We thank Mr P V Kalmykov and Mrs N N Magretova for their help
in performing experiments on analytical centrifugation and in analysis
of sedimentation properties of the proteins This work was supported in part by grants 00-04-48167 and 00-15-97787 to D.I.L from the Russian Fund for Basic Research (RFBR) and by INTAS-RFBR joint grant IR-97–577 to D.I.L.
Trang 10R E F E R E N C E S
1 Rayment, I & Holden, H.M (1994) Myosin subfragment-1:
structure and function of a molecular motor Curr Opin Struct.
Biol 3, 944–952.
2 Spudich, J.A (1994) How molecular motors work Nature 372,
515–518.
3 Privalov, P.L & Potekhin, S.A (1984) Scanning microcalorimetry
in studying temperature-induced changes in proteins Methods
Enzymol 131, 4–51.
4 Shnyrov, V.L., Sanchez-Ruiz, J.M., Boiko, B.N., Zhadan, G.G &
Permyakov, E.A (1997) Applications of scanning
micro-calorimetry in biophysics and biochemistry Thermochim Acta
302, 165–180.
5 Goodno, C.C (1982) Myosin active-site trapping with vanadate
ion Methods Enzymol 85, 116–123.
6 Phan, B & Reisler, E (1992) Inhibition of myosin ATPase by
beryllium fluoride Biochemistry 31, 4787–4793.
7 Werber, M.M., Peyser, Y.M & Mu¨hlrad, A (1992)
Character-ization of stable beryllium fluoride, aluminum fluoride, and
vanadate containing myosin subfragment 1-nucleotide complexes.
Biochemistry 31, 7190–7197.
8 Highsmith, S & Eden, D (1990) Ligand-induced myosin
subfragment 1 global conformational change Biochemistry 29,
4087–4093.
9 Gopal, D & Burke, M (1996) Myosin subfragment 1
hydrophobicity changes associated with nucleotide-induced
conformations Biochemistry 35, 506–512.
10 Smith, C.A & Rayment, I (1996) X-ray structure of the
magne-sium (II)ÆADPÆvanadate complex of the Dictyostelium discoideum
myosin motor domain to 1.9 A˚ resolution Biochemistry 35, 5404–
5417.
11 Dominguez, R., Freyzon, Y., Trybus, K.M & Cohen, C (1998)
Crystal structure of a vertebrate smooth muscle myosin motor
domain and its complex with the essential light chain: visualization
of the pre-power stroke state Cell 94, 559–571.
12 Peyser, Y.M., Ajtai, K., Burghardt, T.P & Mu¨hlrad, A (2001)
Effect of ionic strength on the conformation of myosin
subfrag-ment 1-nucleotide complexes Biophys J 81, 1101–1114.
13 Levitsky, D.I., Shnyrov, V.L., Khvorov, N.V., Bukatina, A.E.,
Vedenkina, N.S., Permyakov, E.A., Nikolaeva, O.P & Poglazov,
B.F (1992) Effects of nucleotide binding on thermal transitions
and domain structure of myosin subfragment 1 Eur J Biochem.
209, 829–835.
14 Bobkov, A.A., Khvorov, N.V., Golitsina, N.L & Levitsky, D.I.
(1993) Calorimetric characterization of the stable complex of
myosin subfragment 1 with ADP and beryllium fluoride FEBS
Lett 332, 64–66.
15 Bobkov, A.A & Levitsky, D.I (1995) Differential scanning
calorimetric study of the complexes of myosin subfragment 1 with
nucleoside diphosphates and vanadate or beryllium fluoride.
Biochemistry 34, 9708–9713.
16 Gopal, D., Bobkov, A.A., Schwonek, J.P., Sanders, C.R., Ikebe,
M., Levitsky, D.I & Burke, M (1995) Structural basis for
acto-myosin chemomechanical transduction by non-nucleoside
tri-phosphate analogues Biochemistry 34, 12178–12184.
17 Levitsky, D.I., Ponomarev, M.A., Geeves, M.A., Shnyrov, V.L &
Manstein, D.J (1998) Differential scanning calorimetric study
of the thermal unfolding of the motor domain fragments of
Dictyostelium discoideum myosin II Eur J Biochem 251,
275–280.
18 Nikolaeva, O.P., Orlov, V.N., Dedova, I.V., Drachev, V.A &
Levitsky, D.I (1996) Interaction of myosin subfragment 1 with
F-actin studied by differential scanning calorimetry Biochem.
Mol Biol Int 40, 653–661.
19 Kaspieva, O.V., Nikolaeva, O.P., Orlov, V.N., Ponomarev, M.A.,
Drachev, V.A & Levitsky, D.I (2001) Changes in the thermal
unfolding of p-phenylenedimaleimide-modified myosin subfrag-ment 1 induced by its weak binding to F-actin FEBS Lett 489, 144–148.
20 Ponomarev, M.A., Furch, M., Levitsky, D.I & Manstein, D.J (2000) Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin Biochemistry 39, 4527–4532.
21 Applegate, D & Reisler, E (1984) Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1 Biochemistry 23, 4779–4784.
22 Mornet, D., Pantel, P., Audemard, E., Derancourt, J & Kassab,
R (1985) Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle.
J Mol Biol 183, 479–489.
23 Pinter, K., Lu, R.C & Szila´gyi, L (1986) Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides FEBS Lett 200, 221–225.
24 Bonet, A., Mornet, D., Audemard, E., Derancourt, J., Bertrand,
R & Kassab, R (1987) Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins J Biol Chem 262, 16524–16530.
25 Yamamoto, K (1989) ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites
on the primary structure J Mol Biol 209, 703–709.
26 Hozumi, T (1983) Structure and function of myosin subfragment
1 as studied by tryptic digestion Biochemistry 22, 799–804.
27 Blotnick, E & Mu¨hlrad, A (1992) Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state Eur J Biochem 210, 873–879.
28 Rayment, I., Rypniewski, W.R., Schmidt-Base, K., Smith, R., Tomchick, D.R., Benning, M.M., Winkelmann, D.A., Wesenberg, G & Holden, H.M (1993) Three-dimensional structure of myosin subfragment 1: a molecular motor Science
261, 50–58.
29 Bobkov, A.A., Chen, T., Nikolaeva, O.P., Levitsky, D.I & Reisler, E (1995) N-terminal cleavage in myosin heavy chain affects the properties of myosin head Biophys J 68, A162.
30 Weeds, A.G & Taylor, R.S (1975) Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin Nature 257, 54–56.
31 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.
32 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
33 Spudich, J.A & Watt, S (1971) The regulation of rabbit skeletal muscle contraction I Biochemical studies of the interaction of the tropomyosin–troponin complex with actin and the proteolytic fragments of myosin J Biol Chem 246, 4866–4871.
34 Le Bihan, T & Cicquaud, C (1991) Stabilization of actin by phalloidin: a differential scanning calorimetric study Biochem Biophys Res Commun 181, 542–547.
35 Levitsky, D.I., Rostkova, E.V., Orlov, V.N., Nikolaeva, O.P., Moiseeva, L.N., Teplova, M.V & Gusev, N.B (2000) Complexes
of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry Eur J Biochem 267, 1869–1877.
36 Golitsina, N.L., Bobkov, A.A., Dedova, I.V., Pavlov, D.A., Nikolaeva, O.P., Orlov, V.N & Levitsky, D.I (1996) Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride.
J Muscle Res Cell Motil 17, 475–485.
37 Ajtai, K., Szila´gyi, L & Biro, E.N.A (1982) Study of the structure
of HMMÆvanadate complex FEBS Lett 141, 74–77.
38 Schroder, 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 364, 171–174.