Effect of caldesmon on the type of conformation changes in F-actin induced by the binding of myosin 1 subfragment

The effect of caldesmon on the conformational changes of F-actin caused by myosin subfragment 1 (S-1) binding was studied, using the polarized microfluorimetry method. It was demonstrated that the polarized fluorescence of rhodaminil-phalloin specifically bound to F-actin of pure actin filaments as well as of tropomyosin-containing actin filaments changes as a result of binding to S-1. The nature of these changes depends on the presence of caldesmon in the filaments. Caldesmon was supposed to modify the conformational changes in F-actin induced by S-1.     

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin

Previously we provided evidence that myosin subfragment 1 (S1) can bind either one (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results of the kinetics study of binding of S1 to F-actin labeled with fluorescent dye pyrene. A transition from state 1 to state 2 depends on probability that the second actin is free, which is high when molar ratio of S1/actin (R) is less than 0.5, and it decreases dramatically when R>2.0 due to the parking problem. The kinetics data obtained at different molar ratios were well fitted by two binding states model. The sequential binding of myosin head initially with one actin monomer and then with the second actin monomer in F-actin can play a key role in force generation by actin-myosin and their directed movement.     

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

Proteolysis and binding of myosin subfragment 1 to actin

Rates of proteolytic cleavage of myosin subfragment 1 were measured in the absence and presence of different amounts of actin. The rates of tryptic digestion at the 50K/20K junction and papain digestion at the 25K/50K junction of the myosin head were progressively inhibited with increasing substoichiometric molar ratios of actin to myosin subfragment 1. The percentage inhibitions of digestion reactions corresponded precisely to the molar compositions of actin-subfragment 1 solutions and demonstrated that equimolar complexes of these proteins were responsible for the observed changes in the proteolysis of myosin heads.     

51V NMR study of vanadate binding to myosin and its subfragment 1

The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.     

Substructure of skeletal myosin subfragment 1 revealed by thermal denaturation

The stability of myosin subfragment 1 (S1) to thermal denaturation has been followed by limited tryptic proteolysis. Digestions done during the thermal denaturation show that at temperatures at and above 37 degrees C there is a marked increase in the susceptibility of S1 to tryptic degradation, as evidenced by the loss of all bands corresponding to the normally trypsin-resistant fragments of 50, 27, and 21 kDa of the heavy chain and to the light chain. The enhanced digestion of S1 appears to be due to a general unfolding of all segments of S1, although the 50-kDa segment appears to unfold at a lower temperature than the remainder of the S1 structure. Digestions done after 30-min exposure to higher temperatures or after subsequent cooling to 25 degrees C show marked differences in the susceptibility of the S1 to trypsin. This suggests that, on cooling, a substantial portion of the S1, but not the 50-kDa segment, is capable of refolding to a state corresponding closely to that in the native S1. These data indicate that in terms of thermal denaturation the S1 behaves as though it is comprised of two domains--an unstable 50-kDa domain and a more stable domain comprised of the 27- and 21-kDa segments of the heavy chain interacting with the light chain, as proposed recently by Setton and Muhlrad [Setton, A., & Muhlrad, A. (1984) Arch. Biochem. Biophys. 235, 411-417]. The rates of thermal inactivation of the ATPase of S1 are found to correspond closely to the decay rates for the 50-kDa fragment, suggesting that this segment in S1 is closely associated with the ATPase function of the protein.     

Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin

The thermal unfolding of rabbit skeletal heavy meromyosin (HMM), myosin subfragment 1, and subfragment 2 has been studied by differential scanning calorimetry (DSC). Two distinct endotherms are observed in the DSC scan of heavy meromyosin. The first endotherm, with a Tm of 41 degrees C at pH 7.9 in 0.1 M KCl, is assigned to the unfolding of the subfragment 2 domain of HMM based on scans of isolated subfragment 2. The unfolding of the subfragment 2 domain is reversible both in the isolated form and in HMM. The unfolding of subfragment 2 in HMM can be fit as a single two-state transition with a delta Hvh and delta Hcal of 161 kcal/mol, indicating that subfragment 2 exists as a single domain in HMM. The unfolding of subfragment 2 is characterized by an extraordinarily large delta Cp of approximately 30,000 cal/(deg.mol). In the presence of nucleotides, the high-temperature HMM endotherm with a Tm of 48 degrees C shifts to higher temperature, indicating that this peak corresponds to the unfolding of the subfragment 1 domain. This assignment has been confirmed by comparison with isolated subfragment 1. The stabilizing effect of AMPPNP was significantly greater than that of ADP. The vanadate-trapped ADP species was slightly more stable than M.AMPPNP with a Tm at 58 degrees C. The unfolding of subfragment 1, both in the isolated form and in HMM, was irreversible. Only a single endotherm was noted in the DSC scans of the subfragment 1 domain of HMM and in freshly prepared subfragment 1 complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin

The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.     

Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin.

Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross-linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state

Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d > 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.     

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.     

Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method.

1. The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing [gamma-32P]ATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.     

The binding of heat-treated myosin subfragment 1 to actin and substructure considerations

Heat treatment of myosin subfragment 1 at 35 degrees C caused about 95% inactivation of the catalytic function but did not block its binding to actin. Heat-treated subfragment 1 showed specific, strong, and close to stoichiometric binding to actin. MgATP but not MgADP dissociated these complexes. However, in contrast to intact subfragment 1, the heat-treated protein did not polymerize G-actin and was not protected from trypsin by the binding to actin. Tryptic degradation of the 50K fragment abolished, or reduced greatly, the binding of heat-treated subfragment 1 to actin in solution but not on nitrocellulose overlays. These results are discussed in the context of subfragment 1 substructure.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

Photocleavage of myosin subfragment 1 by vanadate

The heavy chain of myosin's subfragment 1 (S1) was cleaved at two distinct sites (termed V1 and V2) after irradiation with UV light in the presence of millimolar concentrations of vanadate and in the absence of nucleotides or divalent metals. The V1 site cleavage appeared to be identical with the previously described active site cleavage at serine-180, which is effected by irradiation of a photomodified form of the S1-MgADP-Vi complex [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6011]. The V2 site was cleaved specifically, without cleavage at the V1 site, first by formation of the light-stable S1-Co2+ADP-Vi complex at the active site [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry 27, 8408-8415] and then by irradiation in the presence of millimolar vanadate. By gel electrophoresis, the V2 site was localized to a region about 20 kDa from the COOH terminus of the S1 heavy chain. From the results of tryptic digestion experiments, the COOH-terminal V2 cleavage peptide appeared to contain lysine-636 in the linker region between the 50- and 20-kDa tryptic peptides of the heavy chain. This site appeared to be the same site cleaved by irradiation of S1 (not complexed with Co2+ADP-Vi) in the presence of millimolar vanadate as previously described [Mocz, G. (1989) Eur. J. Biochem. 179, 373-378]. Cleavage at the V2 site was inhibited by Co2+ but was not significantly affected by the presence of nucleotides or Mg2+ ions. Tris buffer significantly inhibited V2 cleavage. From the results of UV-visible absorption, 51V NMR, and frozen-solution EPR spectral experiments, it was concluded that irradiation with UV light reduced vanadate +5 to the +4 oxidation state, which was then protected from rapid reoxidation by O2 by complexation with the Tris buffer. The relatively stable reduced form or forms of vanadium were not competent to cleave S1 at either the V1 or the V2 site. 51V NMR titration experiments indicated that a tetrameric species of vanadium preferentially bound to S1 and to the S1-MgADP-Vi complex, whereas no binding of either the monomeric or dimeric species could be detected. These results suggest that the vanadate tetramer was responsible for the photocleavage of S1 which occurred at both the V1 and V2 sites in the absence of nucleotides or divalent metals.     

The binding of myosin subfragment 1 to actin can be measured by proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.     

Fluorescence studies of the conformation of pyrene-labeled tropomyosin: effects of F-actin and myosin subfragment 1

The fluorescence of pyrene-TM [rabbit skeletal tropomyosin (TM) labeled at Cys with N-(1-pyrenyl)maleimide] consists of monomer and excimer bands [Betcher-Lange, S., & Lehrer, S.S. (1978) J. Biol. Chem. 253, 3757-3760]; an increase in excimer fluorescence with temperature is due to a shift in equilibrium from a chain-closed state (N) to a chain-open state (X) associated with a helix pretransition [Graceffa, P., & Lehrer, S.S. (1980) J. Biol. Chem. 255, 11296-11300]. In this study, we show that the presence of appreciable excimer fluorescence at temperatures below the N----X pretransition (initial excimer) is due to perturbation of the TM chain-chain interaction by the pyrenes at Cys-190. Fluorescence and ATPase titrations indicated that the label caused a decrease in TM binding to F-actin primarily due to reduced end to end TM interactions on the actin filament. Under conditions where pyrene-TM was bound to F-actin, however, the excimer fluorescence did not increase with temperature, indicating that F-actin stabilizes tropomyosin by inhibiting the N----X transition. The binding of myosin subfragment 1 (S1) to pyrene-TM-F-actin at low ratios to actin caused time-dependent changes in fluorescence. After equilibrium was reached, the initial excimer fluorescence was markedly reduced and remained constant over the pretransition temperature range. Further stabilization of tropomyosin conformation on F-actin is therefore associated with S1 binding. Effects of the binding of S1 to the F-actin-tropomyosin thin filament on the state of tropomyosin were studied by monitoring the monomer fluorescence of pyrene-TM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric studies of the ADP binding to myosin subfragment 1, heavy meromyosin, and to myosin filaments.

A calorimetric titration method was used to study the ADP binding to the chymotryptic subfragments of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1), and to myosin aggregated into filaments at low ionic strength. The binding constant (K) and heat of reaction (deltaH, kiloJoules (moles of ADP bound)-1) were determined. For HMM in 0.5 M KCl, 0.01 M MgCl2, 0.02 M Tris (pH 7.8) at 12 degrees, log K = 5.92 +/- 0.13 and deltaH = -70.9 +/- 3.6 kJ mol-1. These results agree with our previous findings for myosin in 0.5 M KCl at 12 degrees. When the KCl concentration was reduced to 0.1 M, the binding constant did not change significantly (log K = 6.09 +/- 0.06) but the binding was more exothermic (deltaH = -90.1 +/- 3.3 kJ mol-1). Similar results were obtained for myosin filaments in 0.1 M KCl and also for both the isoenzymes of S-1(S-1(A1) and S-1(A2) in 0.1 M KCl. In 0.5 M KCl, the binding curves suggest that about one ADP is bound per active site, but as 0.1 M KCl, the apparent stoichiometry drops from 0.7 to 0.75. The most probable explanation is that there is some site heterogeneity which is more evident at lower ionic strength.     

Differential scanning calorimetric study of the thermal unfolding of myosin rod,light meromyosin,and subfragment 2

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil α-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.