Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.     

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

A new property of twitchin to restrict the “rolling” of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin-myosin interaction by “freezing” tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.     

Caldesmon inhibits the rotation of smooth actin subdomain-1 and alters its mobility during the ATP hydrolysis cycle

Smooth muscle thin filaments have been reconstituted in muscle ghost fibers by incorporation of smooth muscle actin, tropomyosin and caldesmon. For the first time, rotation of subdomain-1 and changes of its mobility in IAEDANS-labeled actin during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs have been demonstrated directly. Binding of caldesmon altered the mobility and inhibited the rotation of actin subdomain-1 during the transition from AM∗∗·ADP·Pi to AM state, resulting in inhibition of both strong and weak-binding intermediate states. These new results imply that regulation of actomyosin interaction by caldesmon during the ATPase cycle is fulfilled via the inhibition of actin subdomain-1 rotation toward the periphery of the thin filament, which decreases the area of the specific binding between actin and myosin molecules and is likely to underlie at least in part the mechanism of caldesmon-induced contractility suppression.     

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM^∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

Caldesmon inhibits the actin-myosin interaction by changing its spatial orientation and mobility during the ATPase activity cycle

Orientation and mobility of acrylodan fluorescent probe specifically bound to caldesmon Cys580 incorporated into muscle ghost fibers decorated with myosin S1 and containing tropomyosin was studied in the presence or absence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. Modeling of various intermediate states of actomyosin has shown discrete changes in orientation and mobility of the dye dipoles which is the evidence for multistep changes in the structural changes of caldesmon during the ATPase hydrolysis cycle. It is suggested that S1 interaction with actin results in nucleotide-dependent displacement of the C-terminal part of caldesmon molecule and changes in its mobility. Thus inhibition of the actomyosin ATPase activity may be due to changes in caldesmon position on the thin filament and its interaction with actin. Our new findings described in the present paper as well as those published recently elsewhere might conciliate the two existing models of molecular mechanism of inhibition of the actomyosin ATPase by caldesmon.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Actin and temperature effects on the cross-linking of the SH1-SH2 helix in myosin subfragment 1

Past biochemical work on myosin subfragment 1 (S1) has shown that the bent alpha-helix containing the reactive thiols SH1 (Cys(707)) and SH2 (Cys(697)) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5-15 A in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25 degrees C, the rate constants for cross-linking in S1 alone are low, with values of approximately 0.010 min(-1) for all of the reagents. At 4 degrees C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min(-1). The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4 degrees C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.     

Characterization of three regulatory states of the striated muscle thin filament

The troponin-tropomyosin-linked regulation of striated muscle contraction occurs through allosteric control by both Ca(2+) and myosin. The thin filament fluctuates between two extreme states: the inactive "off" state and the active "on" state. Intermediate states have been proposed from structural studies and transient kinetic measurements. However, in contrast to the well-characterised, on and off states, the mechanochemical properties of the intermediate states are much less well understood because of the instability of those states. In the present study, we have characterized a myosin-induced intermediate that is stabilized by cross-linking myosin motor domains (S1) to actin filaments (with a maximum of one S1 molecule for 50 actin monomers). A single S1 molecule is known to interact with two adjacent actin monomers. A detailed analysis revealed that thin filaments containing S1 molecules cross-linked to just one actin monomer (actin(1)-S1 complexes) are regulated with a 79% inhibition of the ATPase in the absence of Ca(2+). In contrast, filaments containing S1 molecules cross-linked at two positions, to two adjacent actin monomers (actin(2)-S1 complexes) totally lose their regulation in a highly cooperative manner. This loss of regulation was due both to an enhancement of the ATPase activity without calcium and an inhibition of the ATPase with calcium. Filaments containing actin(2)-S1 complexes, with significant ATPase activity in the absence of calcium (about 50%), did not move on a myosin-coated surface unless calcium was present. This partial uncoupling between the ATPase activity and in vitro motility in the absence of calcium demonstrates that the mechanical steps require actin-myosin contacts, which take place only in the on state and not in the off or intermediate states. These data provide new insights concerning the difference in cooperativity of Ca(2+) regulation that exists between the biochemical and mechanical cycles of the actin-myosin motor.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.     

Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: insight into the mode of inhibition.

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.     

Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon.

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60 degrees . This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motion of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.     

Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.     

Structural dynamics of the actin-myosin interface by site-directed spectroscopy

We have used site-directed spin and fluorescence labeling to test molecular models of the actin-myosin interface. Force is generated when the actin-myosin complex undergoes a transition from a disordered weak-binding state to an ordered strong-binding state. Actomyosin interface models, in which residues are classified as contributing to either weak or strong binding, have been derived by fitting individual crystallographic structures of actin and myosin into actomyosin cryo-EM maps. Our goal is to test these models using site-directed spectroscopic probes on actin and myosin. Starting with Cys-lite constructs of both yeast actin (ActC) and the Dictyostelium myosin II motor domain (S1dC), site-directed labeling (SDL) mutants were generated by mutating residues to Cys in the proposed weak and strong-binding interfaces. This report focuses on the effects of forming the strong-binding complex on four SDL mutants, two located in the proposed weak-binding interface (ActC5 and S1dC619) and two located in the proposed strong-binding interface (ActC345 and S1dC401). Neither the mutations nor labeling prevented strong actomyosin binding or actin-activation of myosin ATPase. Formation of the strong-binding complex resulted in decreased spin and fluorescence probe mobility at all sites, but both myosin-bound probes showed remarkably high mobility even after complex formation. Complex formation decreased solvent accessibility for both actin-bound probes, but increased it for the myosin-bound probes. These results are not consistent with a simple model in which there are discrete weak and strong interfaces, with only the strong interface forming under strong-binding conditions, nor are they consistent with a model in which surface residues become rigid and inaccessible upon complex formation. We conclude that all four of these residues are involved in the strong actin-myosin interface, but this interface is remarkably dynamic, especially on the surface of myosin.     

Hyperthyreosis inhibits the ability of actin to form strong-binding with myosin

The effect of hyperthyreosis development induced by the increase in thyroid hormones in rats (during 2-4 weeks) on the orientation and mobility of fluorescent probe N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) specifically bound to Cys 374 of actin in ghost muscle fibers isolated from fast (EDL) and slow (SOL) rat muscles was studied. It was found that the binding of myosin subfragment-1 (S1) to F-actin induced the typical for the formation of strong binding actomyosin decrease in mobility of actin subdomain 1 and its rotation towards thin filament periphery. Development of hyperthyreosis markedly inhibited these phenomena. The maximal effect was observed after 21 days of disease development. It is suggested that one of the reasons of the contractile deficit of muscle in hyperthyreosis is inhibition of the strong binding between actin and myosin during ATPase cycle.