Structural changes in synthetic myosin minifilaments and their dissociation by adenosine triphosphate and pyrophosphate

Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.     

Effect of pH on the cross-bridge arrangement in synthetic myosin filaments.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.     

Is the SII portion of the cross-bridge in glycerinated rabbit psoas fibers compliant in the rigor state?

To see whether the SII portion of the cross-bridge in rigor fibers is longitudinally compliant, we chemically cross-linked with dimethyl suberimidate the entire rod portion (including the SII portion) of myosin onto the surface of thick filaments in glycerinated rabbit psoas fibers, and studied the effect of the SII fixation on the stiffness of the rigor fibers. The cross-linking of fiber segments with full filament overlap increased the rigor stiffness by approximately 25%. Almost the same absolute amount of the stiffness increase was also observed in rigor fibers with half- or no filament overlap after the cross-linking, and a similar but somewhat larger increment of stiffness was observed in fiber segments cross-linked in relaxing solution. These results indicate that the stiffness increase is not produced by the fixation of the SII portion onto the thick filament surface, but is caused instead by the cross-linking of some parallel elastic elements in muscle, and therefore indicate that the SII portion of the cross-bridge is hardly longitudinally compliant in rigor fibers.     

Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle

The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent cross-linking of single fibers from rabbit psoas increases oscillatory power.

Single fibers from chemically skinned rabbit psoas muscle were treated with 1-ethyl-3-[3-dimethyl-amino)proyl]-carbodiimide (EDC) at 20 degrees C after rigor was induced. A 22-min treatment resulted in 18% covalent cross-linking between myosin heads and the thin filament as determined by stiffness measurements. This treatment also results in covalent cross-linking among rod portions of myosin molecules in the backbone of the thick filament. The fibers thus prepared are stable and do not dissolve in solutions at ionic strengths as high as 1,000 mM. The preparation was subjected to sinusoidal analysis, and the resulting complex modulus data were analyzed in terms of three exponential processes, (A), (B), and (C). Oscillatory work (process B) was much greater in the cross-linked fibers than in untreated ones in activating solutions of physiological ionic strength (200 mM); this difference was attributed to the decline of process (A) with EDC treatment. Consequently, the Nyquist plot of the EDC-treated preparation exhibited an insect-type response. We conclude that, under these conditions, both cross-linked and non-cross-linked myosin heads contribute to the production of oscillatory power. The cross-linked preparations also exhibited oscillatory work in high ionic strength (500-1,000 mM) solutions, indicating that cross-linked myosin heads are capable of utilizing ATP to produce work. We conclude that process (A) does not relate to an elementary step in a cross-bridge cycle, but it may relate to dynamics outside the cross-bridge such as filament sliding or sarcomere rearrangement.     

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.     

A model to account for the elastic element in muscle crossbridges in terms of a bending myosin rod

We advance a structural model to account for the rapid elastic element seen in mechanical transient experiments on vertebrate skeletal muscle (A.F. Huxley & Simmons 1971 Nature, Lond. 233, 533-538). In contrast to other crossbridge models, ours does not envisage a myosin rod made up of two rigid portions connected by a hinge, but rather a gradually bending rod portion connecting the heads to the thick filament shaft. We propose that, in relaxed muscle, the subfragment 2 (S2) portion of the myosin rod is bound to the thick filament shaft by ionic interactions analogous to those between the light meromyosin (LMM) portions of the rod that constitute the body of the shaft. These interactions probably involve the alternating zones of positive and negative charge seen in myosin rod amino acid sequences. As the crossbridge cycle that generates tension begins, we propose that part of S2 detaches from the thick filament shaft and bends to enable the myosin head to attach to actin. When tension develops in the crossbridge, the S2 is straightened and more of it becomes detached from the shaft so that the junction between S2 and the myosin heads moves 3-4 nm axially. As tension declines at the end of the crossbridge stroke, we propose that S2 rebinds to the thick filament shaft and that this provides the restoring force to return the junction of the heads and S2 to its original axial position. Thus this movement would have the characteristics of an elastic element; detailed calculations indicate that it would have properties similar to those observed experimentally. Furthermore, this model can account for the radial attractive force seen in rigor and in contracting muscle, the decrease in stiffness when interfilament spacing is increased in skinned muscle, and the increased rate of proteolysis observed at the S2-LMM junction in contracting muscle.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Movement of myosin fragments in vitro: domains involved in force production

We have used the Nitella-based movement assay to localize the site of force production in myosin. Methods were developed to use nonfilamentous myosin or proteolytic fragments of myosin in place of the thick filaments used in the original assay. In the experiments described here, the tail of myosin or its subfragments is anchored via antibodies to the surface of small particles. Nonfilamentous myosin or its subfragments move along Nitella actin cables at speeds similar to those obtained with filamentous myosin. We generated short HMM, a myosin fragment containing the heads and only 400 A of the tail. Although short HMM lacks the "hinge" region proposed by Harrington to be the site of force generation, and is incapable of forming thick filaments, it moves along actin at speeds above 1 micron/sec. Therefore, neither a thick filament nor the carboxy-terminal 1100 A of the tail is required for movement along actin. The results indicate that force production occurs in or near the myosin heads.     

Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.     

End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin

A 1:1 complex of actin and depactin, an actin-depolymerizing protein isolated from starfish oocytes [Mabuchi, I. (1983) J. Cell Biol. 97, 1612-1621], was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to introduce covalent bonds at their contact site. Locations of cross-linking sites were identified along the depactin sequence by the end-label fingerprinting, which employed site-directed antibodies against the N- and C-termini of depactin as end labels. Mappings with these end labels have revealed that the N-terminal segment of depactin (residues 1-20) contains sites in contact with the N- and C-terminal segments of actin, both of which participate in interaction with depactin [Sutoh, K., & Mabuchi, I. (1986) Biochemistry 25, 6186-6192].     

Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.     

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin-fragmin interactions as revealed by chemical cross-linking

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.     

Evidence for cooperative interactions of myosin heads with thin filament in the force generation of vertebrate skeletal muscle fibers

To examine the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA-and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-phenylenedimaleimide (p-PDM)-treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one, and two of their heads inactivated, and the relationships among these values (expressed relative to the control values) were studied. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, muscle fiber stiffness is determined by the total number of native heads, and there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.     

Packing of myosin molecules in muscle thick filaments

The backbone of the myosin filament is an aggregate of alpha-helical coiled coil myosin rods. Its surface forms a three-stranded helix composed of myosin heads. Currently there is no adequate model to describe the organization of the myosin filament. It is proposed here that, in cross-section the light meromyosin (LMM) of 18 myosin molecules form an outer tube, with nine S2 forming the interior core. At the surface of the thick filament, myosin heads are arranged in three rows, giving the filament a periodicity of 14.3 nm per three myosin molecules. Two of these molecules are organized at an angle of 120 degrees to each other on the same level, while the third is shifted 7.2 nm along the filament axis. This packing gives a striation pattern of 7.2 nm by electron microscopy. An alternative model is also possible, in which the heads of the myosin molecules are uniformly spaced at an interval of 14.3 nm along the filament axis. The packing of individual molecules within the myosin filament is based on a regular pattern of charge on the 28 amino-acid repeat in the rod domain.     

The M.ADP.P(i) state is required for helical order in the thick filaments of skeletal muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.     

The rigor configuration of smooth muscle heavy meromyosin trapped by a zero-length cross-linker

When chicken gizzard heavy meromyosin (HMM) in its rigor complex with actin was reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), HMM cross-linked with actin but also the two heads of the HMM molecule cross-linked to each other [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry 28, 1898-1904, 1905-1912]. By ultracentrifugal fractionation of the EDC-treated acto-HMM in the presence of Mg-ATP, we obtained a preparation enriched for gizzard HMM with cross-linked heads. When HMM molecules in this preparation were rotary-shadowed and observed in an electron microscope, many head pairs were in contact with each other. The amount of HMM with cross-linked heads determined by electron microscopy was equal to that of the cross-linked NH2-terminal 24K tryptic fragments of HMM heavy chains determined by NaDodSO4 gel electrophoresis, indicating that this cross-linking is primarily responsible for the contact observed between two HMM heads. Most pairs of the contacted heads originated in the same HMM molecule, although a few pairs belonged to different HMM molecules. Cross-linking between the two heads of the same HMM molecule appeared to occur within the distal, more globular half of each head. However, the cross-linking sites were located at different positions within the globular portion. The actin-activated Mg-ATPase activity of the HMM sample treated with EDC in the presence of actin increased in a biphasic manner, depending on the concentration of F-actin, with two apparent association constants: 2.9 x 10(4) M-1 and one much less than 1 x 10(4) M-1. Since the apparent association constant obtained with the HMM control was similar to the latter value, the association constant for HMM molecules with cross-linked heads was identified to be the former value. The binding of HMM to actin was thus strengthened at least by a factor of 3 by the cross-linking between two HMM heads. These results suggest that HMM heads are trapped by treatment with EDC in the rigor complex configuration and that this configuration is retained even after the HMM has been released from actin. The EDC reactivity of rabbit skeletal muscle HMM, however, was different from that of chicken gizzard HMM. The treatment of acto-HMM complexes with EDC did not generate cross-linking between two skeletal muscle HMM heads.     

Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone

The site of photocross-linking between Cys-697 (SH2), prelabeled with 4'-[14C]maleimidylbenzophenone, and the 50-kDa segment of myosin S1 on irradiation in the absence of nucleotide has been determined by isolation of the 20-50-kDa adduct and subsequent tryptic proteolysis. Isolation and partial sequencing of the radioactively labeled peptide indicate that the site of cross-linking is Arg-239. This result indicates that, in the absence of nucleotide, Arg-239 resides at about 1.0 nm from SH2 and, on the basis of the recent work of Sutoh and Hiratsuka (Sutoh, K. and Hiratsuka T. (1988) Biochemistry 27, 2964-2969) places Arg-239 at no more than 1.45 nm from either Lys-184 or Lys-189 of the nucleotide-binding "glycine-rich" loop prior to the binding of nucleotide.     

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.