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.     

Modification of interface between regulatory and essential light chains hampers phosphorylation-dependent activation of smooth muscle myosin

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.     

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head–head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head–head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837–854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.     

Essential light chain modulates phosphorylation-dependent regulation of smooth muscle myosin

To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.     

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle

The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeF_x complex (stable analog of S1¹-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeF_x complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.     

Molecular modeling of the myosin-S1(A1) isoform

Type II myosin is the molecular motor which drives contraction upon cyclic interaction with filamentous actin while consuming ATP. The contemporary crystallographic structure of the myosin subfragment-1 (S1) of myosin covers both the motor domain of the heavy chain (MHC) as well as the essential (ELC) and regulatory light chains (RLC). A part of the N-terminus of the ELC is, however, missing in the 3D-models of Type II myosin. The N-terminal domain of the ELC comprises interesting functional features since it binds to actin thus controlling myosin motor activity. For the first time, we modeled the missing 46 N-terminal amino acid of the ELC to the contemporary actin-myosin-S1 complex. We show a rod-like 91 A structure being long enough to bridge the gap between the ELC core of myosin-S1 and the appropriate binding site of the ELC on the actin filament.     

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.     

On the relationship between distance information derived from cross-linking and from resonance energy transfer, with specific reference to sites located on myosin heads.

The techniques of fluorescence resonance energy transfer (FRET) and cross-linking can provide complementary information concerning the relative separation of a pair of sites. Cross-linking experiments provide an assessment of the distance of closest approach between a pair of sites. FRET measurements, by contrast, yield information about the average distance between the pair of sites. We have taken advantage of hybrid myosins to understand the relationship between distances obtained for a pair of equivalent sites, one on each myosin head, using both FRET (steady-state and time-decay) and cross-linking techniques. The rigid cross-linker, 4-4'-dimaleimidyl-stilbene-2-2'-disulfonic acid (DMSDS), can efficiently cross-link the two myosin regulatory light-chains, each at residue Cys50 of the Mercenaria regulatory light chain (Chantler, P.D., and S. M. Bower. 1988. J. Biol. Chem. 263:938-944), indicating that these sites can come within 18 +/- 2 A of each other. In a complementary set of experiments, steady-state and time-decay measurements using fluorescence donor/acceptor pairs located at these same sites indicate transfer efficiencies of somewhat less than 20%, suggesting an average separation of greater than 50 A between sites (Chantler, P. D., and T. Tao. 1986. J. Mol. Biol. 192:87-99). Here, we present theoretical calculations which show that efficient cross-linking can be achieved readily in dynamic systems such as the heads of myosin, even though the necessary subpopulation of proximate molecules at any instant may be below the detection limits of time-decay-FRET.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.     

Dynamic modulation of the regulatory domain of myosin heads by pH, ionic strength, and RLC phosphorylation in synthetic myosin filaments

The position of the myosin head with respect to the filament backbone is thought to be a function of pH, ionic strength (micro) and the extent of regulatory light chain (RLC) phosphorylation [Harrington (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5066-5070]. The object of this study is to examine the dynamics of the proximal part of the myosin head (regulatory domain) which accompany the changes in head disposition. The essential light chain was labeled at Cys177 with the indanedione spin-label followed by the exchange of the labeled proteins into myosin. The mobility of the labeled domain was investigated with saturation transfer electron paramagnetic resonance in reconstituted, synthetic myosin filaments. We have found that the release of the heads from the myosin filament surface by reduction of electrostatic charge is accompanied by a 2-fold increase in the mobility of the regulatory domain. Phosphorylation of the RLC by myosin light chain kinase resulted in a smaller 1. 5-fold increase of motion, establishing that the head disordering observed by electron microscopy [Levine et al. (1996) Biophys. J. 71, 898-907] is due to increased mobility of the heads. This result indirectly supports the hypothesis that the RLC phosphorylation effect on potentiation of force arises from a release of heads from the filament surface and a shift of the heads toward actin.     

Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.     

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K–associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.     

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (β) of 120-125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with β ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at β ∼105°, similar to that determined previously for the RLC region.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

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.     

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.     

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x that mimic S1 ATPase intermediate states S1**-ADP-P_i and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.