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.     

Reversible inactivation of myosin subfragment 1 activity by mechanical immobilization.

The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, epsilonADP binds aggregated S1 with reduced affinity, and the bound epsilonADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1 x MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.     

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids

The formation of hybrid myosin and subfragment 1 species by incubation of these proteins with free alkali light chains at physiological ionic and temperature conditions is described. Exchange of bound alkali light chain on myosin by free alkali light chains under these conditions is readily demonstrated from the subunit composition of the isolated myosin. Therefore, the light chain exchange previously described for the one-headed subfragment 1 [Sivaramakrishnan, M., & Burke, M (1981) J. Biol. Chem. 256, 2607--2610] also occurs in the two-headed myosin molecule. It is found than the isozyme to hybrid transformation is dependent on both the temperature and the ionic strength of the incubation mixture but is relatively independent of pH in the range 6.5--8.0. A comparison of the SF1(A1) leads to SF1(A2)h system with the SF1(A2) leads to SF1(A1)h system indicates that more hybrid is formed in the latter case. With the assumption that hybrid formation reflects the degree of reversible dissociation exhibited by the isozyme, under the particular experimental condition employed, the data signify that the subunit interactions in the two isozymes are not identical and that the heavy chain--A1 interactions are significantly more stable that the heavy chain--A2 ones. An examination of the ATPase properties of the thermal hybrids in the presence and absence of actin indicates close similarities to their corresponding "native" isozymic counterparts.     

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.     

Isolating and localizing ATP-sensitive tryptophan emission in skeletal myosin subfragment 1

The fluorescence intensity difference between rabbit skeletal myosin subfragment 1 (S1) and nucleotide-bound or trapped S1 isolates ATP-sensitive tryptophans (ASTs) emission from the total tryptophan signal. Neutral (acrylamide) quenching of the ASTs is sensitive to the binding or trapping of nucleotide to the active site of S1. Anion (I(-)) quenching of the ASTs, sensitive to charge separation in the tryptophan micro environment, is negligible. These findings suggest the ASTs sense conformational change during ATPase from negatively charged surroundings. Specific chemical modifications of S1 identified the location of the ASTs. Trp131 was quenched by chemical modification, and its emission was isolated by taking the intensity difference between unmodified and modified S1. Trp131 fluorescence intensity and quenching constant do not distinguish among the bound or trapped nucleotides, suggesting that the vicinity of Trp131 does not change conformation during the ATPase cycle and eliminating Trp131 as an AST. Trp510 fluorescence was quenched by 5'-iodoacetamidofluorescein (5'IAF) modification of the reactive thiol (SH1) of S1. The tryptophan emission enhancement increment due to active site trapping decreases linearly with SH1 modification and extrapolates to 0 for 100% modification. These data identify Trp510 as the primary AST in skeletal S1 in agreement with observations from Dictyostelium (Batra and Manstein (1999) Biol. Chem. 380, 1017-1023) and smooth muscle S1 (Yengo et al. (2000) Biophys. J. 78, 242A). With Trp510 identified as the sole AST, fluorescence difference spectroscopy provides a novel means to monitor the concentration of myosin transient intermediates in ATP hydrolysis.     

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.     

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.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Binding between maleimidobenzoyl-G-actin and myosin subfragment 1.

It is well known that the structural interactions between S-1 moieties of myosin molecules ("cross bridges") and actin molecules in polymerized ("F") form are thought to underlie muscle contraction. It is surmised that such interactions are unitary (actin:S-1 = 1:1), but actual demonstration thereof is handicapped by intrinsic properties of the proteins. Recently, it has been reported that chemically modified [with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric ("G") form and makes a stable unitary complex with S-1 but does not activate the S-1 ATPase [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. However, we recently showed that when MBS-G-actin and S-1 are covalently cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), ATPase activity is restored [Hozumi, T. (1991) Biochem. Int. 23, 835-843]. Here we investigated the interface between MBS-G-actin and S-1 using the techniques of tryptic digestion and EDC-cross-linking. MBS-G-actin specifically protected the N-terminal region of S-1 heavy chain against tryptic cleavage at the 25 kDa/50 kDa junction, which is different from the effect that a protomer within F-actin has on the protection of the 25 kDa/50 kDa junction. In addition, the cross-linking pattern between MBS-G-actin and S-1 was different from that between F-actin and S-1. When MBS-G actin was cross-linked to trypsin-treated S-1, no cross-linked product was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Evidence for heavy chain-alkali light chain association-dissociation equilibrium

Modification of the free alkali light chains of myosin by iodoacetylation results in a much lower extent of exchange into myosin subfragment 1 by the thermal hybridization procedure (Burke, M., and Sivaramakrishnan, M. (1981) Biochemistry 20, 5908-5913). As reported by others (Wagner, P. D., and Stone, D. B. (1983) J. Biol. Chem. 258, 8876-8882), free alkali light chains modified by iodoacetate at their single sulfhydryl residue exhibit minimal exchange into intact myosin. However, when unmodified alkali light chain is used to probe for exchange, close to the theoretical limit of exchange is observed for subfragment 1, and significant levels of exchange are found for myosin. It appears that modification of the free alkali light chain alters the structure of the protein, and this causes either a marked reduction in its affinity for the heavy chain or in its ability to enter the light chain binding site. This conclusion is supported by tryptic digestions done on the unmodified and modified free light chains where it is found that the latter is degraded at a much faster rate, indicating a more open structure for the modified protein. The observation that alkali light chain exchanges into myosin when unmodified alkali light chains are used indicates that the presence of the associated 5,5'-dithiobis-(2-nitrobenzoic acid) light chains does not preclude the reversible dissociation of this subunit from myosin under ionic and temperature conditions approaching the physiological state.     

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.     

The interaction of skeletal myosin subfragment 1 with the polyanion, heparin

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross-linking techniques and limited digestion studies. 1. The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly-(L-glutamic acid) causes a specific structural change in the 50-kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45-kDa species by a proteolytic cleavage restricted to its COOH-terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50-kDa----45-kDa conversion, suggesting that the S1-nucleotide interactions exhibit some resemblances to the polyanion-S1 binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45-kDa and 20-kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains. 2. Moreover, the binding of heparin to S1 resulted in the self-association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross-linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg-nucleotides. The interaction of cationic sites on S1 and possibly on the 50-kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F-actin abolished S1 aggregation.     

Effects of tryptic digestion on myosin subfragment 1 and its actin-activated adenosinetriphosphatase

Myosin subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol ("SH1"). Short exposure to trypsin cuts the S-1 heavy chain into three still-associated fragments (20K, 50K, and 27K) [Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J., & Sreter, F.A. (1978) Arch. Biochem. Biophys. 190, 793-799] which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (at 4 degrees C, pH 7, in 0.15 M KC1 and 5 mM MgC12, +/- 1 mM ADP). These results are thus in agreement with turbidity measurements on similar systems as reported by Mornet et al. [Mornet, D., Pantel, P., Audemard, E., & Kassab, R. (1979) Biochem. Biophys. Res. Commun. 89, 925-932]. The excited-state lifetime of the fluorescent label on cut S-1 is indistinguishable from that on normal S-1 (+/- ADP, +/- F-actin). F-Actin activation of MgATPase of cut S-1 is lower than that for normal S-1 at moderate concentrations of F-actin, as reported by Mornet et al. (1979). But as the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an eight-species steady-state kinetics scheme involving actin binding to free S-1, S-1 . ATP, S-1. ADP X P, and S-1 . ADP, actin affinity for the species S-1 . ADP X P was found to be 13.4 times greater for uncut S-1 than for cut S-1 [at 24 degrees C, pH 7.0, in 3 mM KC1, 1 mM ATP, 1 mM MgCl2, and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].     

Subunit interactions in myosin subfragment 1. Subunit scrambling is independent of catalytic center involvement

Evidence is presented that, under conditions of 4.7 M NH4Cl and 10 mM Mg-ATP where no subunit dissociation can be detected by transport methods, a dynamic equilibrium exists in subfragment 1 between the associated and dissociated subunits. This is readily discerned by the formation of hybrid subfragment 1 species when a subfragment 1 isozyme is incubated with excess free light chains of the alternate isozyme. A similar process occurs with p-N,N'-phenylenedimaleimide (pPDM)-modified subfragment 1 containing [14C]Mg-ADP, but in this case, although extensive amounts of hybrid are formed, no loss of the trapped nucleotide is observed. Subunit scrambling without loss of the trapped nucleotide is apparent from incubating pPDM-SF1(A2)-[14C]Mg-ADP with unmodified SF1(A1) under similar conditions since the mixture subsequently contains SF1(A1), SF1(A2)h, pPDM-SF1(A1)h-[14C]Mg-ADP and pPDM-SF1(A2)-[14C]Mg-ADP. These data show that the nucleotide trapped in the presumptive active site does not escape during the dissociation-reassociation cycle, and suggest that the ATPase site resides solely on the heavy chain.     

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states.

Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch 2 element [Geeves, M.A. & Holmes, K.C. (1999) Annu. Rev. Biochem.68, 687-728]. The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide. In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment 1. Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes identified three fluorescence resonance energy transfer pairs. The effect of ADP, ADP.BeFx, ADP.AlF4- and ADP.Vi on the conformation of the motor domain was shown by applying temperature-dependent fluorescence resonance energy transfer methods. The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin. The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation. The converter region of subfragment 1 became more rigid during the open-to-closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide-binding pocket to the light-chain-binding domain.     

o-Iodosobenzoic oxidation and cleavage of myosin subfragment 1.

1. o-Iodosobenzoic acid (IOB) caused the formation of a disulfide bridge between SH1 and SH2 groups of myosin SF1 rendering inactive its ATPase activity. 2. IOB at high concentrations provoked fragmentation of SF1 at its tryptophan residues. 3. The main fragmentation point was located at 15 K from the amino terminus of the myosin heavy chain. 4. Actin was not fragmented by IOB. It protected SF1 tryptophans from IOB attack. 5. These results suggest a possible use of IOB as a reagent to study protein tryptophan under nondenaturing conditions.     

Cross-linking of the 25- and 20-kilodalton fragments of skeletal myosin subfragment 1 by a bifunctional ATP analogue

The bifunctional photoreactive ATP analogue azidonitrobenzoyl-8-azido-ATP (ANB-8-N3-ATP) was synthesized. This ATP analogue carriers photoreactive azido groups at the eighth position of the adenine ring and at the 3' position of ribose. Photolysis of this analogue in the presence of skeletal muscle alpha-chymotryptic subfragment 1 (S-1) resulted in a new 120-kDa band, while photolysis in the presence of the tryptic S-1 produced a new 45-kDa band. The 45-kDa peptide was shown to be combined with the 25-kDa N-terminal and 20-kDa C-terminal fragments since it was labeled with a monoclonal antibody specific for the N-terminal 25-kDa segment of the S-1 heavy chain, and it was also found to retain the fluorescence of (iodoacetamido)fluorescein attached specifically to the SH-1 thiol of the C-terminal 20-kDa segment. These results indicate that the 25- and 20-kDa peptides are in close contact with the ATPase active site.     

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.