Effect of actin, ATP, phosphates, and pH on vanadate-induced photocleavage of myosin subfragment 1.

Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K+(EDTA)-, Ca2(+)-, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of actin-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the "consensus" ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the binding and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the "consensus" site through a loop. The 74-kDa cleavage site is a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction.     

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].     

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.     

Ligand-induced myosin subfragment 1 global conformational change

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.     

Alcohol-induced biphasic inhibition of myosin subfragment 1 K-EDTA-ATPase

Butanol-induced inhibition of K-EDTA-ATPase of myosin subfragment 1 proceeded by biphasic kinetics, consisting of rapid and slow inactivations. The extent of the rapid inactivation, which was estimated by extrapolating the process of slow inactivation to zero time of the incubation period, was saturated with butanol concentration. Recovery of activity by dilution in the rapid phase indicates that the rapid process is reversible. The slow inactivation was concomitant with a partial denaturation of the 50 kDa domain of S1, which was detected by limited tryptic digestion. Other alcohols (methanol, ethanol, propanol and hexanol) also inhibited the K-EDTA-ATPase in the rapid phase. The Ki decreased with an increase in the number of methylene groups of alcohol. When K-EDTA-ATPase activity in the rapid phase was plotted against viscosity, surface tension or dielectric constant, the curves were different for each of the various alcohol solutions. The rapid inactivation appears to be caused by a binding of the alkyl group to S1, rather than by solvent effects. The kinetics of rapid butanol inhibitions indicate that butanol reduces the maximum activity of ATPase but enhances an apparent affinity of S1 with ATP. These indications suggest that alcohol stabilizes S1.KATP intermediate. The rapid K-EDTA-ATPase inhibition was observed at the same alcohol concentration where S1 Mg-ATPase was activated.     

Intramolecular cross-linking of myosin subfragment 1 with bimane

We previously showed that the fluorescent inter-thiol cross-linker dibromobimane (DBB) [Kosower, N. S., Kosower, E. M., Newton, G. L., & Ranney, H. M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382-3386] cross-links two [50 and 20 kilodaltons (kDa)] of the three major fragments of myosin subfragment 1 (S-1); on intact S-1, DBB quenches tryptophans and inhibits all ATPases [Mornet, D., Ue, K., & Morales, M. F. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1658-1662]. Here we characterize the modification chemically: DBB cross-links Cys-522 (50 kDa) with Cys-707 (20 kDa), thereby sealing a large preexisting heavy-chain loop containing important functionalities. Cross-linking rate is insensitive to nucleotides, but apparently sterically, either monobromobimane or DBB reduces Ca2+-ATPase to low, nonzero levels.     

Incorporation of 6-carboxyfluorescein into myosin subfragment 1

We describe for the first time the introduction of a label into the "50K" domain of myosin subfragment 1 (S-1), and we investigate the properties of this fluorescent modification in relation to the ATPase and actin-binding activities, both residing in the myosin head. The labeling consists of a major incorporation of 6-carboxyfluorescein into the "50K" domain of S-1. Using different conditions for tryptic digestion that allowed a fragmentation of the "50K" domain with a loss of 5 kilodaltons (kDa) leading to a final product of 45 kDa, we have shown that the fluorescent dye remains in the 45-kDa final product. By studying cross-linking as a function of time, we have demonstrated that the "50K" domain and the 45-kDa fluorescent peptide are equally cross-linkable to actin. We have also investigated the K+EDTA-, Ca2+-, Mg2+-, and actin-activated ATPase activities of this modified S-1 and after purification observed no enzymatic changes.     

Interactions of myosin subfragment 1 isozymes with G-actin

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.     

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.     

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)     

The interdomain motions in myosin subfragment 1.

The interdomain motions in myosin subfragment 1 (S1) were studied by steady-state and time-resolved fluorescence of tryptophan residues and N-(iodoacetyl)-N'-(5-sulfo-1-naphtyl)ethylenediamine (AEDANS) attached to Cys178 of alkali light chain 1 (A1) exchanged into S1. The efficiency of fluorescence resonance energy transfer (FRET) from tryptophan residues of motor domain to AEDANS at A1 decreased dramatically after addition of ATP to S1A1-AEDANS. The efficiency of FRET calculated from the crystal structure of chicken S1 corresponded to the experimental one measured in the presence of ATP. The results showed that AEDANS at Cys178 of A1 became more mobile and distant from the motor domain of S1 upon ATP binding. These findings led to the suggestion that a release of the products of ATP hydrolysis and power stroke might be associated with movement of light chain-binding domain towards the N-terminal domain of S1.     

Nucleotide-induced states of myosin subfragment 1 cross-linked to actin

Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.     

Structure of myosin subfragment 1 from low-angle X-ray scattering

The X-ray scattering pattern produced by a solution of myosin subfragment 1 has been measured to a resolution (Bragg spacing) of 2 nm. We find that for subfragment 1 (S1) prepared by limited papain digestion in the presence of ethylenediaminetetraacetate the radius of gyration is 3.28 +/- 0.06 nm, the volume is 151 +/- 6 nm3, the surface area is 330 +/- 15 nm2, and the length of the maximum chord is 12.0 +/- 1.0 nm. The theoretical scattering patterns from several objects of uniform electron density have been calculated and compared with the observed scattering produced by S1. The recent three-dimensional electron micrograph reconstruction of S1-decorated actin by J. Seymour and E. O'Brien (private communication) generated the calculated pattern that best fit the observed scattering. This fit strongly suggests that this reconstruction resembles subfragment 1. The good correspondence between an S1 structure derived when S1 is attached to actin and a study of free S1 in solution strongly suggests that binding to actin does not grossly distort the shape of S1. This is consistent with the notion that S1 changes its orientation on actin, rather than its shape, in order to generate the contractile force in muscle.     

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.     

Structural characterization of beta-cardiac myosin subfragment 1 in solution

beta-cardiac myosin subfragment 1 (betaS1) tertiary structure and dynamics were characterized with proteolytic digestion, nucleotide analogue trapping kinetics, and intrinsic fluorescence changes accompanying nucleotide binding. Proteolysis of betaS1 produces the 25, 50, and 20 kDa fragments and a new cut within the 50-kDa fragment at Arg369. F-actin inhibits cleavage of the 50-kDa fragment and fails to inhibit cleavage at the 50/20 kDa junction, suggesting betaS1 presents an actoS1 conformation fundamentally different from skeletal S1. Time-dependent changes in Mg(2+)-ATPase accompanying proteolysis identifies cleavage points that lie within the energy transduction pathway. The nucleotide analogue trapping kinetics reveal the presence of a reversible weakly actin attached state. Comparison of nucleotide analogue induced betaS1 structures with the transient structures occurring during ATPase indicates analogue induced and transient structures are in a one-to-one correspondence. Tryptophan fluorescence enhancement accompanies the binding or trapping of nucleotide or nucleotide analogues. Isolation of Trp508 fluorescence shows it is an ATP-sensitive tryptophan and that its vicinity changes conformation sequentially with the transient intermediates accompanying ATPase. These studies elucidate energy transduction and suggest how mutations of betaS1 implicated in disease might undermine function, stability, or efficiency.     

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.     

Effect of nucleotide structure on cardiac myosin subfragment 1 transient kinetics

Transient kinetic data of the hydrolysis of several nucleotides (TTP, CTP, UTP, GTP) by cardiac myosin subfragment 1 (S1) were analyzed to obtain values for the equilibrium constant for nucleotide binding and rate constants for the S1-nucleotide isomerization and the subsequent nucleotide hydrolysis as well as the magnitudes of the relative fluorescence enhancements of the myosin that occur upon isomerization and hydrolysis. These data are compared with data from a previous study with ATP. Nucleotide binding is found to be relatively insensitive to nucleotide ring structure, being affected most by the group at position C6. Isomerization and hydrolysis are more sensitive to nucleotide structure, being inhibited by the presence of a bulky group at position C2. Kinetic parameters decrease as follows: for binding, GTP greater than UTP approximately TTP greater than ATP greater than CTP; for isomerization, ATP greater than UTP approximately TTP approximately CTP greater than GTP; for hydrolysis, ATP greater than TTP greater than CTP approximately UTP greater than GTP. Fluorescence enhancements appear to be most dependent upon the relative values of the individual rate constants.     

Vanadate-mediated photocleavage of rabbit skeletal myosin

The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with near-ultraviolet light in the presence of 0.1-1.0 mM vanadate. The sigmoidal dependence upon vanadate concentration, with half-maximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2-terminus; these sites are cleaved equally well in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half-times for cleavage of the 23-kDa and 75-kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2-3-fold by the presence of greater than 10 microM MgATP. The third photocleavage site is located about 5-10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half-time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate-mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2+, Mg2+ and actin-activated Mg2+ ATPases becoming elevated, whereas the K+/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding.