Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy.

Inter- and intradomain flexibility of the myosin head was measured using phosphorescence anisotropy of selectively labeled parts of the molecule. Whole myosin and the myosin head, subfragment-1 (S1), were labeled with eosin-5-iodoacetamide on the catalytic domain (Cys 707) and on two sites on the regulatory domain (Cys 177 on the essential light chain and Cys 154 on the regulatory light chain). Phosphorescence anisotropy was measured in soluble S1 and myosin, with and without F-actin, as well as in synthetic myosin filaments. The anisotropy of the former were too low to observe differences in the domain mobilities, including when bound to actin. However, this was not the case in the myosin filament. The final anisotropy of the probe on the catalytic domain was 0.051, which increased for probes bound to the essential and regulatory light chains to 0.085 and 0.089, respectively. These differences can be expressed in terms of a "wobble in a cone" model, suggesting various amplitudes. The catalytic domain was least restricted, with a 51 +/- 5 degrees half-cone angle, whereas the essential and regulatory light chain amplitude was less than 29 degrees. These data demonstrate the presence of a point of flexibility between the catalytic and regulatory domains. The presence of the "hinge" between the catalytic and regulatory domains, with a rigid regulatory domain, is consistent with both the "swinging lever arm" and "Brownian ratchet" models of force generation. However, in the former case there is a postulated requirement for the hinge to stiffen to transmit the generated torque associated by nucleotide hydrolysis and actin binding.     

The regulatory domain of the myosin head behaves as a rigid lever.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Inhibition of the ATP-dependent interaction of actin and myosin by the catalytic domain of the myosin light chain kinase of smooth muscle: possible involvement in smooth muscle relaxation

Myosin light chain kinase (MLCK) phosphorylates the light chain of smooth muscle myosin enabling its interaction with actin. This interaction initiates smooth muscle contraction. MLCK has another role that is not attributable to its phosphorylating activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a glass surface coated with phosphorylated myosin. To analyze the inhibitory effect of MLCK, the catalytic domain of MLCK was obtained with or without the regulatory sequence adjacent to the C-terminal of the domain, and the inhibitory effect of the domain was examined by the movement of actin filaments. All the domains work so as to inhibit actin filament movement whether or not the regulatory sequence is included. When the domain includes the regulatory sequence, calmodulin in the presence of calcium abolishes the inhibition. Since the phosphorylation reaction is not involved in regulating the movement by MLCK, and a catalytic fragment that shows no kinase activity also inhibits movement, the kinase activity is not related to inhibition. Higher concentrations of MLCK inhibit the binding of actin filaments to myosin-coated surfaces as well as their movement. We discuss the dual roles of the domain, the phosphorylation of myosin that allows myosin to cross-bridge with actin and a novel function that breaks cross-bridging.     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod phosphorylation in catch muscle, a peptide corresponding to the specific N-terminal region of the protein was synthesized in phosphorylated and unphosphorylated form. We report, for the first time, that unphosphorylated full-length myorod and its unphosphorylated N-terminal synthetic peptide are able to interact with rabbit F-actin and thin filaments from molluscan catch muscle. The binding between thin filaments and the peptide was Ca²⁺-dependent. In addition, we found that phosphorylated N-terminal peptide of myorod has higher affinity for myosin compared to the unphosphorylated peptide. Together, these observations suggest the direct involvement of the N-terminal domain of myorod in the regulation of molluscan catch muscle.     

Domain characterization of rabbit skeletal muscle myosin light chain kinase.

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.     

Segmental flexibility and head-head interaction in scallop myosin. A study using saturation transfer electron paramagnetic resonance spectroscopy

Saturation transfer electron paramagnetic resonance spectroscopy was used to investigate the rotational motion of the head domains of native and desensitized scallop myosin and its proteolytic subfragments. Scallop myosin was spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, which reacted with a heavy chain residue in the subfragment 1 domain. As previously shown for rabbit skeletal muscle myosin (Thomas et al., 1975), the two head domains of native scallop myosin appear to have independent motion (rotational correlation time, pi, = 0.8 X 10(-7) s for subfragment 1; 1.4 X 10(-7) s for myosin). However, removal of a regulatory light chain, to effect desensitization of the actin-activated ATPase, was associated with an increase in pi for myosin to a value of 2.4 X 10(-6) s. The Ca2+ sensitivity and initial correlation time were restored on recombination of the regulatory light chain in the presence of Mg2+. Sedimentation velocity profiles in an analytical ultracentrifuge indicated that the desensitized myosin preparations were largely monomeric and therefore the change in pi appears to reflect an intramolecular event. Addition of EDTA to spin-labelled scallop heavy meromyosin caused an immediate 2.5 to 4-fold increase in pi and a partial desensitization of the ATPase activity. Comparable experiments with subfragment 1 yielded a barely detectable increase in pi (1.5-fold) in the first ten minutes. The restricted rotational motion observed in desensitized myosin and heavy meromyosin could arise by a conformational change in the subfragment 1-subfragment 2 hinge region or by an association of one head with its partner. The latter mechanism, involving the exposed light chain binding site, would also explain the preferential release of one regulatory light chain from scallop myosin, and might account for some other co-operative effects observed in this molecule (Bagshaw, 1980).     

Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.

Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid was similar to that of skeletal muscle myosin. The dephosphorylation of the smooth light chain in the hybrid did not induce a conformational change in the hybrid from the 6 S to the 10 S state, thereby indicating that the conformational transition is dependent also on the nature of the heavy chain subunit. Exchange of the smooth light chain premodified at its Cys-108 by photolabile 4-(N-maleimido)benzophenone and photolysis resulted in crosslinking to the heavy chain subunit. Immunopeptide mapping using a monoclonal antibody against residues 1-23 at the N-terminus of the skeletal muscle myosin heavy chain identified the location of the photocrosslinking site to be beyond 92 kDa away from the N-terminus.     

Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.

We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.     

Flexibility of myosin attachment to surfaces influences F-actin motion.

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.     

Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction.

By decreasing ionic strength slowly, thick filaments of several micrometers in length were obtained from purified rabbit skeletal muscle myosin. Dark-field observation showed these filaments with their center scattering light extensively. Active movement of actin filaments complexed with tetramethyl rhodamine-phalloidin along the reconstituted myosin filaments was observed. Actin filaments moved towards the center of myosin filaments at a speed of 3.9 +/- 1.6 microns s-1 (mean +/- SD, n = 40) and often continued to move beyond the center towards the tip of the opposite side at a lower speed. The speed of the movement away from the center was 1.0 +/- 0.6 microns s-1 (n = 59). Thus, the functional bipolarity in terms of the movement speed which was first found in native thick filaments of molluscan smooth muscle is also seen in reconstituted filaments from purified rabbit skeletal muscle myosin. The difference of the speed between the two directions is considered to be due to properties of myosin molecules themselves.     

Rotational dynamics of the regulatory light chain in scallop muscle detected by time-resolved phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy (TPA) to study the rotational dynamics of chicken gizzard regulatory light chain (RLC) bound to scallop adductor muscle myofibrils in key physiological states. Native RLC from scallop myofibrils was extracted and replaced completely with gizzard RLC labeled specifically at Cys 108 with erythrosin iodoacetamide (ErIA). The calcium sensitivity of the ATPase activity of the labeled myofibril preparation was quite similar to that of the native sample, indicating that the ErIA-labeled RLC is functionally bound to the myosin head. In rigor (in the absence of ATP, when all the myosin heads are rigidly bound to the thin filament), a slight decay was observed in the first few microseconds, followed by no change in the anisotropy. This indicates small-amplitude restricted motions of the RLC or the entire LC domain of myosin. Addition of calcium to rigor restricts these motions further. Relaxation with ATP (no Ca) causes a large decay in the anisotropy, indicating large-amplitude rotational motion with correlation times of 5-50 micros. Further addition of calcium, to induce contraction, resulted in a decrease in the rate and amplitude of anisotropy decay. In particular, there is clear evidence for a slow rotational motion with a correlation time of approximately 300 micros, which is not present either in rigor or relaxation. This indicates rotational motion that specifically correlates with force generation. The changes in the rotational dynamics of the light-chain domain in rigor, relaxation, and contraction support earlier work based on probes of the catalytic domain that muscle contraction is accompanied by a disorder-to-order transition of the myosin head. However, the motions of the LC domain are different from those of the catalytic domain, which indicates rotation of the two domains relative to each other.