Báo cáo Y học: Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain pdf

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

x
, 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 7 °C to a lower temperature, from
50 °C 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 P
i
analogs (V
i
,BeF
x
). 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 4–5 °C 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 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

), beryllium fluoride
(BeF
x
), or aluminum fluoride (AlF
4
)
–
. These complexes are
stable analogs of the S1*ÆATP and S1**ÆADPÆP
i
interme-
diate states of the S1-catalyzed Mg
2+
-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
S1ÆADPÆV
i
and S1ÆADPÆBeF
x
causes 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:
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

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 ÔweakÕ 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 25 kDa, 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 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

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 P
i
analogs (V
i
,BeF
x
), 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 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

trypsin and S1 at 25 °C for 60 min. To prepare Nt-S1, 5 m
M
ATP was added. The concentration of S1 was 3 mgÆmL
)1
in
both cases, and the medium contained 50 m
M
Tris/HCl,
pH 8.0, 30 m
M
KCl, and 5 m
M
MgCl
2
. During the course
Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur. J. Biochem. 269) 5679
of 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 m
M
Hepes, pH 7.3,
containing 1 m
M
MgCl
2
and 0.5 m
M
ADP, 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 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

Æcm
)1
. G-actin was polymerized to F-actin in
G-buffer by the addition of 4 m
M
MgCl
2
.F-Actinwas
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 14 °C [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 P
i
analogs
Trapping of ADP by different phosphate analogs (V
i
,BeF
x
)
was performed by the methods described for the
preparation of stable ternary complexes S1ÆADPÆV
i
and
S1ÆADPÆBeF
x

[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

of the protein. The ATPase activity of S1, t-S1, or Nt-S1
modified by V
i
or BeF
x
in 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 m
M
MgCl
2
,and
0.5 m
M
ADP. S1 solutions contained 30 m
M
Hepes,
pH 7.3, 1 m
M
MgCl
2
,and0.5m
M
ADP. The final concen-
tration of S1, t-S1, or Nt-S1 was 13 l
M
,andF-actin

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

0.5 m
M
ADP. 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
ORIGIN
software (MicroCal Inc.). Transition temperatures
(T
m
) were determined from the maximum of the thermal
transition. Calorimetric enthalpies (DH
cal
) 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.
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

)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
5680 O. P. Nikolaeva et al. (Eur. J. Biochem. 269) Ó FEBS 2002
experiments 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 20 °C 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 (T
m
¼ 42.4 °C) is shifted to a lower temperature by

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

normally sedimenting molecules, and in the value of
calorimetric enthalpy DH
cal
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
25 kDa 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
7 °C 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.Therelativeerrorofthe
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
,0.5m
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

,0.5m
M
ADP. The heating
rate was 1 KÆmin
)1
. The parameters derived from calorimetric data are
showninTable1.
Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur. J. Biochem. 269) 5681
DSC 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
P
i
analogs such as V
i
,BeF
x
,andAlF
4
–
were shown to cause
a significant increase of the thermal stability of the protein,
as judged by the values measured for T
m
and DH
cal
,anda
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-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

x
,thethermaltransitionoft-S1
shiftedto55.1°C and its enthalpy (1400 kJÆmol
)1
)
increased by only 14% in comparison with ADP-containing
t-S1 (Fig. 2A).
The effects of V
i
and BeF
x
on Nt-S1ÆADP (Fig. 2B) were
even more pronounced than in the case of t-S1 and
uncleaved S1. Formation of the complex Nt-S1ÆADPÆV
i
shifted the thermal transition of Nt-S1ÆADP by 10.7 °C,
from 42.4 °Cto53.1°C, and increased its enthalpy by
almost 60%, from 740 to 1170 kJÆmol
)1
. The increase in T
m
was less in the case of the complex Nt-S1ÆADPÆBeF
x
(8.3 °C), although DH
cal
increased 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 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

Nt-S1ÆADPÆBeF
x
meet these criteria (Fig. 2B). These com-
plexes were dialyzed for 48 h at 4 °C against 30 m
M
Hepes,
pH 7.3, containing 1 m
M
MgCl
2
, to remove the free ADP
and V
i
or BeF
x
and were then subjected to calorimetric
measurements performed in the presence of 0.5 m
M
ADP. It
is clear from Fig. 3 that the decomposition of the complexes
Nt-S1ÆADPÆV
i
and Nt-S1ÆADPÆBeF
x
was negligible. The
removal of the reagents caused only some small decrease in
calorimetric enthalpy of the thermal transition, by 16% for
Nt-S1ÆADPÆV
i
(Fig. 3A) and by 25% for Nt-S1ÆADPÆBeF

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

for 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 0.5 m
M
ADP.
Solid line curves were obtained in the presence of 0.5 m
M
ADP and
0.5 m
M
V
i
. Curves shown by dashes-and-dots were obtained in the
presence of 0.5 m
M
ADP, 5 m
M
NaF, and 0.5 m
M
BeCl
2
.Other
conditions were the same as in Fig. 1B.

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

)1
) was digested with trypsin (50 : 1 by mass) in
the presence of 0.5 m
M
ADP and 0.5 m
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
,0.5m
M
ADP, 0.5 m
M
V
i
.
S1 concentration was 1 mgÆmL
)1
. Heating rate was 1 KÆmin
)1

.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

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
,at4 °C for 48 h. After
dialysis ADP was again added to these samples to final concentration
of 0.5 m
M
. Other conditions were the same as in Figs 1B and 2B.
Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur. J. Biochem. 269) 5683
thermal 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 25 kDa 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 T

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

cal
from 740 to 590 kJÆmol
)1
.Asmallpeakat53.5°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 20 °C and rotor speed of
15 000 r.p.m. was equal to 70.0 ± 4.4 S, which is appre-
ciably higher than that of free F-actin (49.0 ± 3.5 S) but
lower than the coefficient of F-actin complexed with t-S1 or
uncleaved S1 under the same conditions (94.5 ± 8.5 S and
117.5 ± 7.5 S, 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 15 m
M

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

sedimenting with the coefficient of about 5 S 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).
DISCUSSION
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 25 kDa/
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 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 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

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 25 kDa fi 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
50kDafi 45 kDa conversion (Fig. 1A). It has been sug-
gested from this correlation that tryptic cleavage within
both 25 and 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 5–7 °Cto
lower temperature. This effect was observed both in the
presence of ATP (Fig. 1B) and in the S1 ternary complex
with ADP and V

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.

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 (T
m
¼ 39 °C)andinthepresenceof
ADP (T
m
¼ 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 T
m
of 45.6 °C in the absence of nucleotides
and 49.1 °C 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 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

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 0.5–1.2 °C 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–
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

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.
5686 O. P. Nikolaeva et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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