Eur J Biochem 269, 5678–5688 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03279.x Differential scanning calorimetric study of myosin subfragment 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 A N Belozersky Institute of Physico-Chemical Biology, Moscow State University; and 2A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia The thermal unfolding of myosin subfragment (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 S1ỈADPỈVi or S1ỈADPỈBeFx, 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 thermal transition by about °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 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 4–5 °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 prevents 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 stabilization 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 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 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 Ômolecular 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 Pi analogs, such as orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4)– These complexes are stable analogs of the S1*ỈATP and S1**ỈADPỈPi intermediate 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 S1ỈADPỈVi and S1ỈADPỈBeFx 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: 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) Keywords: myosin subfragment 1; thermal unfolding; differential scanning calorimetry Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur J Biochem 269) 5679 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 Pi 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 discoideum 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 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-phenylenedimaleimide 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 actininduced 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 conformational changes due to interaction with F-actin and nucleotides 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 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 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 Pi analogs (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 S1ỈADPỈVi and S1ỈADPỈBeFx 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 concentration of S1 was determined by measuring A280 using an absorption coefficient of 0.75 mgỈmL)1Ỉcm)1 The preparation 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 : 50 (w/w) ratio of trypsin and S1 at 25 °C for 60 To prepare Nt-S1, mM ATP was added The concentration of S1 was mgỈmL)1 in both cases, and the medium contained 50 mM Tris/HCl, pH 8.0, 30 mM KCl, and mM MgCl2 During the course Ó FEBS 2002 5680 O P Nikolaeva et al (Eur J Biochem 269) of digestion, aliquots were taken and analysed by SDS/ PAGE [31] Digestion was terminated by adding soybean trypsin inhibitor at a 1.5 : (w/w) ratio to trypsin The proteins were dialyzed against 30 mM Hepes, pH 7.3, containing mM MgCl2 and 0.5 mM 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 Pi Actin was prepared from rabbit skeletal-muscle acetone powder [33] Monomeric G-actin was stored in low-strength buffer composed of mM Tris/HCl, pH 8.0, 0.2 mM ATP, 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 mgỈmL)1Ỉcm)1 G-actin was polymerized to F-actin in G-buffer by the addition of 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 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 Pi analogs Trapping of ADP by different phosphate analogs (Vi, BeFx) was performed by the methods described for the preparation of stable ternary complexes S1ỈADPỈVi and S1ỈADPỈBeFx [5,7,15] To obtain these complexes, t-S1 or Nt-S1 (1 mgỈmL)1) were incubated with 0.5 mM Vi or BeFx for 30 at 20 °C in a medium containing 30 mM Hepes, pH 7.3, mM MgCl2, and 0.5 mM ADP Beryllium fluoride complexes were obtained by addition of 0.5 mM BeCl2 in the presence of mM 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 Vi or BeFx 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, mM MgCl2, and 0.5 mM ADP S1 solutions contained 30 mM Hepes, pH 7.3, mM MgCl2, and 0.5 mM ADP The final concentration 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 photoelectric 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 Sedimentation 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 differential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described previously [13–20] Prior to measurements, all S1 samples were dialyzed against 30 mM Hepes, pH 7.3, containing mM MgCl2 and 0.5 mM ADP All experiments were performed at a scanning rate of KỈmin)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 irreversibility 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 (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 RESULTS Calorimetric characterization of the S1 species modified by tryptic cleavage Figure 1A shows electrophoretic pattern of the S1 preparations 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 transformation 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 transformation 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 lmolỈPiỈmin)1Ỉmg)1), the activity of Nt-S1 was about 50–60% of that for t-S1 and uncleaved S1 The trypsincleaved S1 preparations, t-S1 and Nt-S1, were also examined for their homogeneity by sedimentation velocity Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur J Biochem 269) 5681 Table Calorimetric parameters obtained from the DSC data for S1, t-S1, and Nt-S1 The absolute error of the given values of transition temperature (Tm) did not exceed ± 0.2 °C The relative error of the given values of calorimetric enthalpy, DHcal, did not exceed ± 7% Experimental conditions for measurements were 30 mM Hepes, pH 7.3, mM MgCl2, 0.5 mM ADP Molecular mass of 115 kDa was used for calculation of DHcal for all proteins studied Protein experiments performed under the same conditions, at the same protein concentration (1 mgỈmL)1), and even in the same rotor Both these S1 species demonstrated sharp sedimentation boundaries, and sedimentation coefficients DHcal (kJỈmol)1) S1 t-S1 Nt-S1 Fig Electrophoretic patterns (A), and DSC scans (B) of S1 (1), t-S1 (2), and Nt-S1 (3) Protein concentrations were mgỈmL)1 Conditions: 30 mM Hepes, pH 7.3, mM MgCl2, 0.5 mM ADP The heating rate was KỈmin)1 The parameters derived from calorimetric data are shown in Table Tm (°C) 50.0 49.4 42.4 1320 1230 740 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 thermostable than t-S1 and S1 The transition temperature for Nt-S1 (Tm ¼ 42.4 °C) is shifted to a lower temperature by °C, in comparison with that of t-S1 (Tm ¼ 49.4 °C) The main calorimetric parameters extracted from these data (Tm, DHcal) are summarized in Table The value of calorimetric enthalpy DHcal determined for Nt-S1 (740 kJỈmol)1) is about 55–60% of that for S1 and t-S1 (Table 1) This great difference in the DHcal value 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 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 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 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 °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 Ó FEBS 2002 5682 O P Nikolaeva et al (Eur J Biochem 269) Fig Temperature dependence of the excess molar heat capacity for t-S1 (A) and Nt-S1 (B) in the presence of ADP and Vi or BeFx Curves shown by dashed lines were obtained in the presence of 0.5 mM ADP Solid line curves were obtained in the presence of 0.5 mM ADP and 0.5 mM Vi Curves shown by dashes-and-dots were obtained in the presence of 0.5 mM ADP, mM NaF, and 0.5 mM BeCl2 Other conditions were the same as in Fig 1B 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 Pi analogs 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 Tm and DHcal, and a considerable increase in the cooperativity of the thermal transition [13,15,17,20] Figure shows the effects of the formation of the ternary complexes of t-S1 and Nt-S1 with ADP-Vi and 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 Pi analogs were very similar to those observed with uncleaved S1 [13,15] Formation of the complex t-S1ỈADPỈVi increased Tm by 7.7 °C, from 49.4 to 57.1 °C, and caused a pronounced increase of DHcal by 18%, from 1230 to 1450 kJỈmol)1 In the case of the complex t-S1ỈADPỈBeFx, the thermal transition of t-S1 shifted to 55.1 °C and its enthalpy (1400 kJỈmol)1) increased by only 14% in comparison with ADP-containing t-S1 (Fig 2A) The effects of Vi and BeFx on Nt-S1ỈADP (Fig 2B) were even more pronounced than in the case of t-S1 and uncleaved S1 Formation of the complex Nt-S1ỈADPỈVi shifted the thermal transition of Nt-S1ỈADP by 10.7 °C, from 42.4 °C to 53.1 °C, and increased its enthalpy by almost 60%, from 740 to 1170 kJỈmol)1 The increase in Tm was less in the case of the complex Nt-S1ỈADPỈBeFx (8.3 °C), although DHcal increased in this case by more than 60%, to 1240 kJỈmol)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-S1ỈADPỈVi and Nt-S1ỈADPỈBeFx 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 S1ỈADPỈVi and S1ỈADPỈBeFx 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 distinguishable from the peaks of nucleotide-free S1 or S1ỈADP on the thermogram [15,16,36] The complexes Nt-S1ỈADPỈVi and Nt-S1ỈADPỈBeFx meet these criteria (Fig 2B) These complexes were dialyzed for 48 h at °C against 30 mM Hepes, pH 7.3, containing mM MgCl2, to remove the free ADP and Vi or BeFx and were then subjected to calorimetric measurements performed in the presence of 0.5 mM ADP It is clear from Fig that the decomposition of the complexes Nt-S1ỈADPỈVi and Nt-S1ỈADPỈBeFx was negligible The removal of the reagents caused only some small decrease in calorimetric enthalpy of the thermal transition, by 16% for Nt-S1ỈADPỈVi (Fig 3A) and by 25% for Nt-S1ỈADPỈBeFx (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 Pi analogs are as stable as the complexes obtained with control uncleaved S1 as they not significantly decompose a few days after removal of excess reagents Tryptic cleavage of S1 in the S1ỈADPỈVi and S1ỈADPỈBeFx 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 S1ỈADPỈVi [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 50 kDa fi 45 kDa, is responsible for destabilizing the S1 molecule S1 was digested in the presence of 0.5 mM ADP and 0.5 mM Vi for 60 and aliquots were taken at several times Digestion was stopped by addition of soybean trypsin inhibitor, and then the samples were subjected to DSC Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur J Biochem 269) 5683 Fig Temperature dependence of the excess molar heat capacity for Nt-S1 in the ternary complexes with ADP and Vi (A) or BeFx (B) before and after removal of excess reagents Curves shown by dashed lines were obtained for Nt-S1 in the presence of 0.5 mM ADP and 0.5 mM Vi (A) or 0.5 mM ADP, mM NaF and 0.5 mM BeCl2 (B) Solid line curves were obtained after removal of excess ADP and Vi or BeFx from the complexes Nt-S1ỈADPỈVi and Nt-S1ỈADPỈBeFx by dialysis against 30 mM Hepes, pH 7.3, containing mM MgCl2, at °C for 48 h After dialysis ADP was again added to these samples to final concentration of 0.5 mM Other conditions were the same as in Figs 1B and 2B 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 S1ỈADPỈVi complex turns into transition with maximum at 53.1 °C which corresponds to Nt-S1 in the ternary complex with ADP and Vi (Fig 2B) The disappearance 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 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 Fig Electrophoretic patterns (A) and DSC scans (B) of the S1 samples obtained in the course of tryptic digestion of S1 in the S1ỈADPỈVi complex S1 (2 mgỈmL)1) was digested with trypsin (50 : by mass) in the presence of 0.5 mM ADP and 0.5 mM Vi for different time intervals, 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 Conditions: 30 mM Hepes, pH 7.3, mM MgCl2, 0.5 mM ADP, 0.5 mM Vi S1 concentration was mgỈmL)1 Heating rate was KỈmin)1 The vertical bar corresponds to 100 kJỈmol)1ỈK)1 the same time, the 50 kDa fi 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 S1ỈADPỈBeFx complex (data not shown) Overall, these data support the above suggestion that the changes in the 5684 O P Nikolaeva et al (Eur J Biochem 269) Ó FEBS 2002 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 Tm 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 shows the excess heat capacity curves for actinbound 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 °C to 54.1 °C (Fig 6A), and by increasing the DHcal value for t-S1 from 1230 to 1340 kJỈmol)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) Fig 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 Fig The experimental DSC curves of F-actin stabilized by phalloidin (A) and its complexes with t-S1 (B) or Nt-S1 (C) Conditions: 26 lM F-actin, 50 lM phalloidin, 13 lM t-S1 or Nt-S1 in 15 mM Hepes, pH 7.3, mM MgCl2, 0.5 mM ADP, and twice-diluted G-buffer Heating rate KỈmin)1 The vertical bar corresponds to 10 lW 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 Tm to lower temperature, from 42.5 to 41.1 °C, and a decrease in DHcal from 740 to 590 kJỈmol)1 A small peak at 53.5 °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 : 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 appreciably 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 Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur J Biochem 269) 5685 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 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, welldefined 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 nucleotideand 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 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 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 has no appreciable influence on the actin-induced changes in the thermal unfolding of S1 (Fig 6A) S1 cleaved at loop (t-S1) probably retains quite a number of positively charged lysyl residues for electrostatic 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 investigate 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 50 kDa fi 45 kDa conversion (Fig 1A) It has been suggested from this correlation that tryptic cleavage within both 25 and 50 kDa segments causes dramatic destabilization 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 °C 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 35 °C 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 °C 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 Ó FEBS 2002 5686 O P Nikolaeva et al (Eur J Biochem 269) 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 (Tm ¼ 39 °C) and in the presence of ADP (Tm 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 Tm 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 Pi 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 [20] or with deleted loop [41] These fragments demonstrated 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 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 DSCrevealed 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 with the negatively charged N-terminal part of actin [38–40] Thus, the interaction of loop 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 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 preventing the global conformational changes in the myosin head induced by actin binding to loop 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 communication pathway between the motor domain and the regulatory domain, and it may play a crucial role in the transmission of actin-induced conformational changes from loop 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 Ó FEBS 2002 Thermal unfolding of tryptically cleaved myosin S1 (Eur J Biochem 269) 5687 REFERENCES Rayment, I & Holden, H.M (1994) Myosin subfragment-1: structure and function of a molecular motor Curr Opin Struct Biol 3, 944–952 Spudich, J.A (1994) How molecular motors work Nature 372, 515–518 Privalov, P.L & Potekhin, S.A (1984) Scanning microcalorimetry in studying temperature-induced changes in proteins Methods Enzymol 131, 4–51 Shnyrov, V.L., Sanchez-Ruiz, J.M., Boiko, B.N., Zhadan, G.G & Permyakov, E.A (1997) Applications of scanning microcalorimetry in biophysics and biochemistry Thermochim Acta 302, 165–180 Goodno, C.C (1982) Myosin active-site trapping with vanadate ion Methods Enzymol 85, 116–123 Phan, B & Reisler, E (1992) Inhibition of myosin ATPase by beryllium fluoride Biochemistry 31, 4787–4793 Werber, M.M., Peyser, Y.M & Muhlrad, A (1992) Characteră ization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes Biochemistry 31, 7190–7197 Highsmith, S & Eden, D (1990) Ligand-induced myosin subfragment global conformational change Biochemistry 29, 4087–4093 Gopal, D & Burke, M (1996) Myosin subfragment hydrophobicity changes associated with nucleotide-induced conformations Biochemistry 35, 506–512 10 Smith, C.A & Rayment, I (1996) X-ray structure of the magnesium (II)ỈADPỈvanadate 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 & Muhlrad, A (2001) ă Eect of ionic strength on the conformation of myosin subfragment 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 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 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 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 actomyosin chemomechanical transduction by non-nucleoside triphosphate 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 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 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 unfolding of p-phenylenedimaleimide-modified myosin subfragment induced by its ÔweakÕ binding to F-actin FEBS Lett 489, 144–148 Ponomarev, M.A., Furch, M., Levitsky, D.I & Manstein, D.J (2000) Charge changes in loop affect the thermal unfolding of the myosin motor domain bound to F-actin Biochemistry 39, 4527–4532 Applegate, D & Reisler, E (1984) Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment Biochemistry 23, 4779–4784 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 ´ Pinter, K., Lu, R.C & Szilagyi, L (1986) Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides FEBS Lett 200, 221–225 Bonet, A., Mornet, D., Audemard, E., Derancourt, J., Bertrand, R & Kassab, R (1987) Comparative structure of the proteasesensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins J Biol Chem 262, 16524–16530 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 Hozumi, T (1983) Structure and function of myosin subfragment as studied by tryptic digestion Biochemistry 22, 799–804 Blotnick, E & Muhlrad, A (1992) Eect of actin on the tryptic ă digestion of myosin subfragment in the weakly attached state Eur J Biochem 210, 873–879 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 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 Weeds, A.G & Taylor, R.S (1975) Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin Nature 257, 54–56 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 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 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 Le Bihan, T & Cicquaud, C (1991) Stabilization of actin by phalloidin: a differential scanning calorimetric study Biochem Biophys Res Commun 181, 542–547 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 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 with ADP and vanadate or beryllium fluoride J Muscle Res Cell Motil 17, 475–485 ´ Ajtai, K., Szilagyi, L & Biro, E.N.A (1982) Study of the structure of HMMỈvanadate complex FEBS Lett 141, 74–77 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 5688 O P Nikolaeva et al (Eur J Biochem 269) 39 Cheung, P & Reisler, E (1992) Synthetic peptide of the sequence 632–642 on myosin subfragment 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 affect the thermal unfolding of myosin head fragments while complexed to F-actin J Muscle Res Cell Motil 20, 72 Ó FEBS 2002 42 Cope, M.J.T.V., Whisstock, J., Rayment, I & Kendrick-Jones, J (1996) Conservation within the myosin motor domain: implications for structure and function Structure 4, 969–987 43 Reizes, O., Barylko, B., Li, C., Sudnof, T.C & Albanesi, J.P ă (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-phenylenedimaleimidemodified 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  ... removal of excess reagents Tryptic cleavage of S1 in the S1ỈADPỈVi and S1ỈADPỈBeFx complexes The N-terminal tryptic cleavage of the S1 heavy chain can be achieved not only in the presence of ATP,... 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. .. 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