Cross-linking between translationally equivalent sites on the two heads of myosin. Relationship to energy transfer results between the same pair of sites

Mercenaria myosin and scallop pure hybrid myosin possessing Mercenaria regulatory light chains were reacted with various concentrations of 4-4'-dimaleimidylstilbene-2-2'-disulfonic acid (DMSDS). Regulatory light chain homodimers are formed with great efficiency (20-50%). Dimers incorporating essential light chains were not formed upon reaction of DMSDS with Mercenaria myosin but some (less than 5%) essential light chain homodimers were obtained in the case of scallop hybrid myosin, probably occurring through relatively specific intermolecular associations within small myosin aggregates. Results were invariant, irrespective of the presence or absence of calcium and/or ATP. No radioactivity is incorporated into regulatory light chain homodimers upon post-labeling DMSDS-reacted myosin with 14C-labeled N-ethylmaleimide, irrespective of the original labeling ratio of DMSDS to myosin heads. This indicates the absence of free sulfhydryl groups in the regulatory light chain homodimer and suggests, therefore, that DMSDS links the two light chains together between translationally equivalent residues (Cys-50 of the Mercenaria regulatory light chain). These results imply that translationally equivalent sites on the two heads of myosin can come within 18 A of each other, the span of reacted DMSDS. Because energy transfer results between identical pairs of translationally equivalent sites on hybrid myosins indicated a low efficiency of energy transfer between these sites (Chantler, P.D., and Tao, T. (1986) J. Mol. Biol. 192, 87-99), it would appear that even though the two cysteines can come within 18 A of each other, their mean separation is much greater than this distance (greater than 50 A), a result consistent with a considerable flexibility of the two myosin heads with respect to each other.     

Locking regulatory myosin in the off-state with trifluoperazine

Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.     

Proximity of regulatory light chains in scallop myosin

The distance between the regulatory light chains of the two heads of the scallop myosin molecule was estimated with the aid of two photolabile cross-linkers, benzophenone maleimide and p-azidophenacylbromide. These cross-linkers selectively alkylate thiol groups and have a maximum length of about 9 A. One of the two regulatory light chains of scallop myosin was removed by treatment of myofibrils at 10 degrees C with EDTA and replaced with a foreign regulatory light chain carrying a cross-linker. Cross-linking between the scallop and foreign regulatory light chains was effected by photolysis. This was demonstrated by incubating nitrocellulose transfers of sodium dodecyl sulfate/polyacrylamide gels of the photolyzed hybrid myofibrils with specific antibodies against the different light chains, followed by fluorescein isothiocyanate-125I-labeled secondary antibody. Scallop regulatory light chains cross-linked extensively (20 to 50%) with Mercenaria regulatory light chains (cysteine in position approximately 50) in solutions that induce rigor in skinned fibers (no ATP) and in relaxing solutions (ATP but no Ca2+). Neither the regulatory light chains of chicken skeletal myosin (cysteines 129 and 157) nor those of gizzard myosin (cysteine 108) were cross-linked to scallop regulatory light chains in either medium. These results indicate that the N-terminal portions of the myosin regulatory light chains can approach each other within 9 A or less, while the distance between the C-terminal halves exceeds 9 A, and support the view that the N termini of the regulatory light chains point toward the myosin rod. Since the relative distance between the regulatory light chains of the two myosin heads is not altered between rigor and rest, we suggest that motion of the essential light chains is mainly responsible for the observed difference in the relative positions of the regulatory and essential light chains between conditions of rigor and rest.     

Regulatory light-chain Cys-55 sites on the two heads of myosin can come within 2A of each other.

The di-thiol reagent, 5,5'-dithiobis (2-nitrobenzoic acid) is shown to induce disulfide bond formation between Mercenaria regulatory light-chain Cys-55 sites on either head of scallop hybrid myosin. This indicates that these two sites on opposite heads of myosin can come within 2A of each other and this confirms a prediction based on earlier data [Chantler, Tao and Stafford (1991) Biophys. J. 59, 1242-1250]. Results demonstrate that myosin heads in solution show a considerable mutual freedom of movement which can be monitored by probes in the vicinity of regulatory light-chain residue 55. Implications for light-chain movement on the myosin head are discussed.     

Position of Mercenaria regulatory light-chain Cys50 site on the surface of myosin visualized by electron microscopy

Mercenaria regulatory light-chains, specifically labelled at cysteine 50 with N-iodoacetyl-N'-biotinylhexylenediamine, were rebound to regulatory light-chain denuded scallop myosin, and the hybrid myosin formed was decorated with avidin. These hybrid myosins were visualized by rotary-shadowing electron microscopy. Three distinct images of avidin-decorated hybrid myosin molecules were obtained. These comprise singly decorated molecules, where the avidin is bound symmetrically or asymmetrically with respect to the two heads of myosin, in addition to "figures-of-five", where two myosin molecules associate with a centrally placed avidin molecule. Analysis of these images indicates that the Mercenaria regulatory light-chain Cys50 site is located 15 to 35 A from the head-rod junction when the light-chain is bound in situ to myosin. Implications with respect to head topology and probe studies are discussed.     

Electron microscopy of negatively stained scallop myosin molecules. Effect of regulatory light chain removal on head structure

The heads of myosin molecules from the striated adductor muscle of scallop have been studied by electron microscopy after negative staining. In common with vertebrate skeletal muscle myosin visualized by this method, the scallop myosin heads were pear-shaped and often showed pronounced curvature. Staining suggestive of two or, more frequently, three domains could often be observed. Removal of regulatory light chains (R-LCs) resulted in a reduction in the length of the heads of about 2.6 nm, with no significant change in maximum width. In desensitized preparations a majority of heads displayed anticlockwise curvature, whereas intact heads were usually seen curved clockwise. Analysis of the head curvature in both intact and desensitized molecules was consistent with an ability of each head to rotate about its long axis. Desensitization resulted in an increased incidence of heads showing two domains. It seems likely that the reduction in length upon removal of the R-LC is due to the two small domains located in the neck region of the head collapsing into one.     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

Fluorescence intensity and UV absorption changes accompanying dissociation and association of regulatory light chain of scallop adductor myosin

Dissociation and association of regulatory light chains of scallop myosin were found to be accompanied by changes in the fluorescence intensity and in the UV absorption spectrum. The changes in the two optical properties of scallop myosin and the dissociation and association of regulatory light chains were studied as a function of the magnesium and calcium concentrations. The results thus obtained suggested that there are two different types of attachment between regulatory light chains and "desensitized" myosin; one type is a calcium-specific attachment, and the other type of attachment can be mediated by either calcium or magnesium ions. These changes in the optical properties of scallop myosin were distinguishable from those induced by Mg-ATP; for example, with "desensitized" scallop myosin, the former changes were not observed but the latter were.     

Changes in reactivities of scallop adductor myosin with 5,5'-dithiobis-(2-nitrobenzoate) and with 2,4,6-trinitrobenzene sulfonate accompanying dissociation and association of regulatory light chain

1. The reactivities of scallop myosin with 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and with 2,4,6-trinitrobenzene sulfonate (TNBS) were found to be affected by dissociation and association of regulatory light chains (RLC) of myosin. 2. Approximately 4 mol of sulfhydryl groups of "desensitized" myosin (DM) were masked on association of DM with RLC. When these sulfhydryl groups were reacted with DTNB, the modified DM became incapable of associating with RLC, but when the modified DM was treated with 2-mercaptoethanol, the ability to associate with RLC was fully recovered. 3. The DTNB-reactivity of scallop myosin and its RLC content were measured as a function of calcium and magnesium concentrations. The results thus obtained could be explained in terms of our previous suggestion (J. Biochem. 94, 1061 (1983] that there are two different attachments between DM and RLC. 4. The relation between the TNBS-reactivity and the RLC content was not simple but complex. Not the extent, but the rate of trinitrophenylation of scallop myosin was affected by dissociation and association of DM with RLC; thus, the involved TNBS-reactive lysine residues did not seem to be in the regions on DM and RLC that would be physically covered upon DM-RLC association. 5. The amount of the involved lysine residues was estimated to be only 1 mol per mol of myosin. Modification of the specific lysine residues resulted in a partial decrease in the DM-RLC association.     

Regulatory light chains and scallop myosin. Form of light chain removal or reuptake is dependent on the presence of divalent cations

Readdition of regulatory light chains to regulatory light chain denuded scallop myofibrils, in the presence of magnesium, results in a negatively co-operative restoration of calcium sensitivity as a function of regulatory light chain content. The form of the stoichiometry curves obtained in the presence of 10 mM-EDTA, by light chain removal from scallop myofibrils at various temperatures, are parabolic in shape, consistent with a random removal process. However, in the presence of EDTA at low temperatures, regulatory light chains are removed in a biphasic manner, indicating that the binding constants of the light chains for each myosin head are not equivalent under these conditions. It is shown here that as the temperature is raised, light chain removal by EDTA approaches that of a random process. The stoichiometry curves obtained in the presence of 10 mM-EDTA may therefore be seen as a composite of both a biphasic removal process (temperatures below 20 degrees C) and a random removal process (temperatures above 20 degrees C), there being a temperature-dependent switch in the myosin molecule between 17 and 23 degrees C that governs the mode of light chain removal. These results indicate that both myosin heads must contain light chains for calcium sensitivity and are consistent with our earlier proposals for head-head co-operativity within the scallop myosin molecule.     

Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand.

The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.     

Electron microscopy of cross-linked scallop myosin

The N-terminal regions of the regulatory light chains on the two heads of scallop myosin can be cross-linked to one another. Electron microscopy of cross-linked myosin molecules, and of dimers of myosin subfragment-1 produced by digesting them with papain, shows that the site of cross-linking is very close to the head-rod junction.     

Hybrids of Physarum myosin light chains and desensitized scallop myofibrils

The two light chains of Physarum myosin have been purified in a 1:1 ratio with a yield of 0.5-1 mg/100 g of plasmodium and a purity of 40- 70%; the major contaminant is a 42,000-dalton protein. The 17,700 Mr Physarum myosin light chain (PhLC1) binds to scallop myofibrils, providing the regulatory light chains (ScRLC) have been removed. The 16,500 Mr light (PhLC2) does not bind to scallop myofibrils. The calcium control of scallop myosin ATPase is lost by the removal of one of the two ScRLC's and restored equally well by the binding of either PhLC1 or rabbit skeletal myosin light chains. When both ScRLC's are removed, replacement by two plasmodial light chains does not restore calcium control as platelet or scallop light chains do. Purified plasmodial actomyosin does not bind calcium in 10(-6) M free calcium, 1 mM MgCl2. No tropomyosin was isolated from Physarum by standard methods. Because the Physarum myosin light chains can substitute only partially for light chains from myosin linked systems, because calcium does not bind to the actomyosin, and because tropomyosin is apparently absent, the regulation of plasmodial actomyosin by micromolar Ca++ may involve other mechanisms, possibly phosphorylation.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

The light chains of scallop myosin as regulatory subunits

In molluscan muscles contraction is regulated by the interaction of calcium with myosin. The calcium dependence of the aotin-activated ATPase activity of scallop myosin requires the presence of a specific light chain. This light chain is released from myosin by EDTA treatment (EDTA-light chains) and its removal desensitizes the myosin, i.e. abolishes the calcium requirement for the actin-activated ATPase activity, and reduces the amount of calcium the myosin binds; the isolated light chain, however, does not bind calcium and has no ATPase activity. Calcium regulation and calcium binding is restored when the EDTA-light chain is recombined with desensitized myosin preparations. Dissociation of the EDTA-light chain from myosin depends on the concentration of divalent cations; half dissociation is reached at about 10^?5 M-magnesium or 10^?7 M-calcium concentrations. The EDTA-light chain and the residual myosin are fairly stable and the components may be kept separated for a day or so before recombination.Additional light chains containing half cystine residues (SH-light chains) are detached from desensitized myosin by sodium dodecyl sulfate. The EDTA-light chains and the SH-light chains have a similar chain weight of about 18,000 daltons; however, they differ in several amino acid residues and the EDTA-light chains contain no half cystine. The SH-light chains and EDTA-light chains have different tryptic fingerprints. Both light chains can be prepared from washed myofibrils.Densitometry of dodecyl sulfate gel electrophoresis bands and Sephadex chromatography in sodium dodecyl sulfate indicate that there are three moles of light chains in a mole of purified myosin, but only two in myosin treated with EDTA. The ratio of the SH-light chains to EDTA-light chains was found to be two to one in experiments where the total light-chain complements of myosin or myofibril preparations were carboxymethylated. A similar ratio was obtained from the densitometry of urea-acrylamide gel electrophoresis bands. We conclude that a myosin molecule contains two moles of SH-light chain and one mole of EDTA-light chain, and that the removal of a single EDTA-light chain completely desensitizes scallop myosin.Heavy meromyosin and S-1 subfragment can be prepared from scallop myosin. Both of these preparations bind calcium and contain light chains in significant amounts. The heavy meromyosin of scallop is extensively degraded; the S-1 preparation, however, is remarkably intact. Significantly, heavy meromyosin has a calcium-dependent actin-activated ATPase while the S-1 does not require calcium and shows high ATPase activity in its absence. These results suggest that regulation involves a co-operativity between the two globular ends of the myosin.Desensitized scallop myosin and scallop S-1 preparations can be made calcium sensitive when mixed with rabbit actin containing the rabbit regulatory proteins. This result makes it unlikely that specific light chains of myosin are involved in the regulation of the vertebrate system.The fundamental similarity in the contractile regulation of molluscs and vertebrates is that interaction between actin and myosin in both systems requires a critical level of calcium. We propose that the difference in regulation of these systems is that the interaction between myosin and actin is prevented by blocking sites on actin in the case of vertebrate muscles, whereas in the case of molluscan muscles it is the sites on myosin which are blocked in the absence of calcium.     

Effect of regulatory light chain on chymotryptic digestion of scallop adductor myosin

Chymotryptic digestability of scallop myosin was studied by measuring (a) changes in the gel electrophoretic pattern and (b) production of the soluble fraction obtained by centrifugation. Chymotryptic digestion of essential light chain (SH-LC) was strongly inhibited by association of regulatory light chain (R-LC) with myosin. This is in agreement with the observation of Stafford et al. (Biochemistry 18, 5273 (1979]. SH-LC and R-LC were both more resistant to the chymotryptic digestion when R-LCs were associated with myosin in the presence of calcium than when they dissociated from myosin in the presence of EDTA. In contrast, heavy chains of scallop myosin were digested more quickly in the presence of calcium than EDTA. This suggests that association of R-LC induces reversible changes in the heavy chain conformation, which lead to an increase in the chymotryptic digestability of heavy chains. The chymotryptic digestability of scallop myosin increased in two distinct phases as the calcium concentration in the digestion medium was increased, but monophasically as the magnesium concentration was increased. The magnesium increased the digestability by approximately half as much as did calcium. These findings suggest two types of attachment between regulatory light chains and desensitized myosin: one mediated specifically by low concentrations of calcium ions, the second by higher concentrations of either calcium or magnesium.     

Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Myosin light-chain domain rotates upon muscle activation but not ATP hydrolysis.

We have studied the correlation between myosin structure, myosin biochemistry, and muscle force. Two distinct orientations of the myosin light-chain domain were previously resolved using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled regulatory light chains in scallop muscle fibers. In the present study, we measured isometric force during EPR spectral acquisition, in order to define how these two light-chain domain orientations are coupled to force and the myosin ATPase cycle. When muscle fibers are partially activated with increasing amounts of calcium, the distribution between the two light-chain domain orientations shifts toward the one associated with strong actin binding. This shift in distribution is linearly related to the increase in force, suggesting that rotation of the light-chain domain is coupled to strong actin binding. However, when nucleotide analogues are used to trap myosin in the pre- and posthydrolysis states of its ATPase cycle in relaxed muscle, there is no change in the distribution between light-chain domain orientations, showing that the rotation of the light-chain domain is not directly coupled to the ATP hydrolysis step. Instead, it is likely that in relaxed muscle the myosin thick filament stabilizes two light-chain domain orientations that are independent of the nucleotide analogue bound at the active site. We conclude that a large and distinct rotation of the light-chain domain of myosin is responsible for force generation and is coupled to strong actin binding but is not coupled to a specific step in the myosin ATPase reaction.     

Regulatory properties of single-headed fragments of scallop myosin.

Calcium control was studied in single-headed myosin and subfragment-1 (S1) preparations obtained by papain digestion of scallop myosin. Single-headed myosin, containing light chains in stoichiometric amounts, was calcium regulated; in contrast, the actin-activated Mg-ATPase of all S1 species lacked calcium sensitivity. Both regulatory and essential light chains were retained by S1 and single-headed myosin preparations provided divalent cations were present during papain digestion, although a peptide amounting to 10% of the mass was removed from regulatory light chains. The modified regulatory light chain retained its ability to confer calcium binding and restore calcium sensitivity to the ATPase of desensitized myofibrils. Regulatory light chains protected the essential light chains from fragmentation by papain. S1 bound regulatory light chains with a uniformly high affinity and appeared to consist of a single species. The results demonstrate that head to head interactions are not obligatory for calcium control, although they may occur in the intact myosin molecule, and suggest a role for the subfragment-2 region in calcium regulation of myosin.