Differential scanning calorimetric study of myosin subfragment 1
with tryptic cleavage at the N-terminal region of the heavy chain
Olga P. Nikolaeva
1
, Victor N. Orlov
1
, Andrey A. Bobkov
2,
* and Dmitrii I. Levitsky
1,2
1
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University; and
2
A. 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 (V
i
) or beryllium fluoride (BeF
x
), and
in the presence of F-actin. In the presence of ATP and in the
complexes S1ÆADPÆV
i
or S1ÆADPÆBeF

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 Ômole-
cular motorÕ 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 P
i
analogs, such as orthovanadate (V
i

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:
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)
Eur. J. Biochem. 269, 5678–5688 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03279.x
ATPase reaction. It has also been concluded from DSC
experiments on recombinant fragments of the head of
Dictyostelium discoideum myosin II that the changes in the
thermal unfolding, that are due to formation of stable
ternary complexes with ADP and P
i
analogs, 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-
eum myosin head fragment corresponding to the globular
motor portion of the head [20]. It has been shown that

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 25 kDa-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
attachedstate(inthepresenceofADP)[26]andweakly
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 ÔweakÕ binding of

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 S1ÆADPÆV
i
and S1ÆADPÆBeF
x
and to undergo structural changes due to formation of these
complexes.
MATERIALS AND METHODS
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 A
280
using an
absorption coefficient of 0.75 mgÆmL
)1
Æcm
)1
.Theprepar-
ation of the trypsin-modified derivatives t-S1 and Nt-S1 was
performed according to Mornet et al.[22].Thet-S1was
obtained by tryptic digestion using a 1 : 50 (w/w) ratio of

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 P
i
.
Actin was prepared from rabbit skeletal-muscle acetone
powder [33]. Monomeric G-actin was stored in low-strength
buffer composed of 2 m
M
Tris/HCl, pH 8.0, 0.2 m
M
ATP,
0.2 m
M
CaCl
2
,0.5m
M
2-mercaptoethanol, and 0.01%
NaN
3
(G-buffer). Actin concentration was determined
by measuring A
290
using absorption coefficient of
0.63 mgÆmL
)1

[5,7,15]. To obtain these complexes, t-S1 or
Nt-S1 (1 mgÆmL
)1
)wereincubatedwith0.5m
M
V
i
or BeF
x
for 30 min at 20 °C in a medium containing 30 m
M
Hepes,
pH 7.3, 1 m
M
MgCl
2
,and0.5 m
M
ADP. Beryllium fluoride
complexes were obtained by addition of 0.5 m
M
BeCl
2
in
the presence of 5 m
M
NaF. The formation of the complexes
was controlled by measuring the K
+
-EDTA ATPase activity

concentration was 26 l
M
.
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, sedimentation 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 m
M
Hepes, pH 7.3, containing 1 m
M
MgCl
2
and

of instrumental background, concentration normalization,
and chemical baseline correction, they can be used for the
description of the irreversible thermal denaturation of S1.
RESULTS
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 (95 kDa) was cleaved with
trypsin into three large fragments (25 kDa, 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 25 kDa
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 25 kDa 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 lmolÆP
i
Æmin
)1
Æmg

7 °C, in comparison with that of t-S1 (T
m
¼ 49.4 °C).
The main calorimetric parameters extracted from these
data (T
m
, DH
cal
) are summarized in Table 1. The value
of calorimetric enthalpy DH
cal
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 DH
cal
value cannot
be explained by possible contributions of DC
p
(i.e. the
difference in heat capacity, C
p
,betweenthenativeand
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 45 kDa segment (Fig. 1A, lane 3) and the decrease by
about 50% in the ATPase activity, in the amount of

pH 7.3, 1 m
M
MgCl
2
,0.5m
M
ADP. Molecular mass of 115 kDa was
used for calculation of DH
cal
for all proteins studied.
Protein T
m
(°C) DH
cal
(kJÆmol
)1
)
S1 50.0 1320
t-S1 49.4 1230
Nt-S1 42.4 740
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

ADP-V
i
and ADP-BeF
x
, 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 BeF
x
is less
pronounced than the effect of V
i
(Fig. 2A,B). In the case
of t-S1 (Fig. 2A), the effects of P
i
analogs were very similar
to those observed with uncleaved S1 [13,15]. Formation of
the complex t-S1ÆADPÆV
i
increased T
m
by 7.7 °C, from 49.4
to 57.1 °C, and caused a pronounced increase of DH
cal
by
18%, from 1230 to 1450 kJÆmol
)1
. In the case of the
complex t-S1ÆADPÆBeF

S1 and t-S1, to undergo global structural changes due to
formation of ternary complexes with ADP and P
i
analogs. Formation of the complexes Nt-S1ÆADPÆV
i
and
Nt-S1ÆADPÆBeF
x
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 S1ÆADPÆV
i
and S1ÆADPÆBeF
x
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-S1ÆADPÆV
i
and

x
(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 P
i
analogs 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 S1ÆADPÆV
i
and S1ÆADPÆBeF
x
complexes
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 S1ÆADPÆV
i
[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, 25 kDa fi 22 kDa or
50kDafi 45 kDa, is responsible for destabilizing the S1
molecule. S1 was digested in the presence of 0.5 m
M
ADP
and 0.5 m
M
V
i

5682 O. P. Nikolaeva et al. (Eur. J. Biochem. 269) Ó FEBS 2002
analysis and to SDS/PAGE (Fig. 4). Figure 4B shows that
in the course of tryptic digestion the initial transition with
maximum at 58 °C characteristic for control uncleaved S1
in the S1ÆADPÆV
i
complex turns into transition with
maximum at 53.1 °C which corresponds to Nt-S1 in the
ternary complex with ADP and V
i
(Fig. 2B). The disap-
pearance of the transition at 58 °C on Fig. 4B and its
conversion into transition at 53.1 °C correlates well with
disappearance of the band of 25 kDa fragment on the
electrophoretogram (Fig. 4A). After 40 min of incubation
with trypsin, when the 25 kDa band had almost completely
disappeared (Fig. 4A), only the thermal transition at
53.1 °C was observed on the thermogram (Fig. 4B). At
thesametime,the50kDafi 45 kDa 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
S1ÆADPÆBeF
x
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

.The
vertical bar corresponds to 100 kJÆmol
)1
ÆK
)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 0.5 m
M
ADP and 0.5 m
M
V
i
(A) or 0.5 m
M
ADP, 5 m
M
NaF and 0.5 m
M
BeCl
2
(B). Solid line
curves were obtained after removal of excess ADP and V
i

m
for F-actin from 62 to
82 °C, 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
4.7 °C, from 49.4 °Cto54.1 °C (Fig. 6A), and by increasing
the DH
cal
value 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 T
m
to
lower temperature, from 42.5 to 41.1 °C, and a decrease in
DH

Hepes,
pH 7.3, 2 m
M
MgCl
2
,0.5m
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.
5684 O. P. Nikolaeva et al. (Eur. J. Biochem. 269) Ó FEBS 2002
was 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

part of actin, and this electrostatic interaction is mainly
responsible for the ÔweakÕ 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. discoideum myosin 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 25 kDa 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

i
(Fig. 4B). These results are in good
agreement with literature data showing that in the course of
incubation at 35 °CtheK
+
-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 5 °C decrease of T
m
), 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
Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur. J. Biochem. 269) 5685
two 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.

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 P
i
analogs 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 10 °C, 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

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–
115 form 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.
ACKNOWLEDGMENTS
We thank Mr P. V. Kalmykov and Mrs N. N. Magretova for their help

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

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

Nature 364, 171–174.
Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur. J. Biochem. 269) 5687
39. Cheung, P. & Reisler, E. (1992) Synthetic peptide of the
sequence 632–642 on myosin subfragment 1 inhibits actomyosin
ATPase activity. Biochem. Biophys. Res. Commun. 189,
1143–1149.
40. Chaussepied, P. & Morales, M.F. (1988) Modifying preselected
sites on proteins: the stretch of residues 633–642 of the myosin
heavy chain is part of the actin-binding site. Proc. Natl Acad. Sci.
USA 85, 7471–7475.
41. Ponomarev, M., Furch, M., Knetsch, M., Manstein, D. &
Levitsky, D. (1999) Changes in loop 2 affect the thermal unfolding
of myosin head fragments while complexed to F-actin. J. Muscle
Res. Cell Motil. 20,72.
42. Cope, M.J.T.V., Whisstock, J., Rayment, I. & Kendrick-Jones, J.
(1996) Conservation within the myosin motor domain: implica-
tions for structure and function. Structure 4, 969–987.
43. Reizes, O., Barylko, B., Li, C., Su
¨
dnof, T.C. & Albanesi, J.P.
(1994) Domain structure of a mammalian myosin Ib. Proc. Natl
Acad. Sci. USA 91, 6349–6353.
44. Greene, L.E., Chalovich, J.M. & Eisenberg, E. (1986) Effect of
nucleotide on the binding of N,N¢-p-phenylenedimaleimide-
modified S-1 to unregulated and regulated actin. Biochemistry 25,
704–709.
45. Milligan, R.A. (1996) Protein–protein interactions in the rigor
actomyosin complex. Proc. Natl Acad. Sci. USA 93, 21–26.
5688 O. P. Nikolaeva et al. (Eur. J. Biochem. 269) Ó FEBS 2002