Subfragment 1 Research Articles

HSP20 phosphorylation and airway smooth muscle relaxation

HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 μM isoproterenol or 10 μM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbachol-induced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.

A FERM domain autoregulates Drosophila myosin 7a activity

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V(max) approximately 1 s(-1) and K(ATPase) approximately 1 microM actin. We find that myosin 7a-FL has V(max) similar to S1 but K(ATPase) approximately 30 microM. Thus, at low actin concentrations (5 microM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K(ATPase) is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K(ATPase) to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K(d) approximately 30 microM regardless of ATP, similar to the K(ATPase) value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.

Suppressing effect of neocarrabiose 4-O-sulfate on thermal and freeze denaturation of myosin subfragment-1 and myofibrils

Suppressive effects of neocarrabiose 4-O-sulfate on the thermal and freeze denaturation of myosin subfragment-1 (S-1) and on myofibrils were investigated. The compound strongly suppressed the thermal denaturation of S-1. Its suppressive effect was greater than that of sorbitol and similar to that of maltose. However, it tended to accelerate the denaturation of myofibrils, suggesting a loss of protection by F-actin upon its addition. The compound suppressed the freeze denaturation of myofibrils and S-1. The effect was similar to that of sorbitol or maltose, and completely different from that of Na-sulfate. The compound solubilized myofibrils at concentrations similar to KCl. Therefore, it was concluded that neocarrabiose 4-O-sulfate behaved as an ionic salt in the thermal treatment process, whereas it behaved as a sugar in the frozen storage process.

Actin - Myosin Interaction

Muscle contraction is resulted from the interaction of myosin with actin and ATP. The study of kinetics of binding of myosin subfragment 1 (S1) to F-actin revealed the two step binding, which were modeled by initial binding of S1 to one actin monomer (state 1) and then to the second neighboring monomer (state 2). The results of time-resolved cross-linking of S1 and F-actin upon their rapid mixing in stopped flow apparatus directly demonstrated that myosin head initially binds through the loop 635–647 to the N-terminus of one actin and then through the loop 567–574 to the N-terminus of the second actin (Andreev & Reshetnyak, 2007, J. Mol. Biol. 365(3), 551–554). The computational docking of S1 with F-actin demonstrated that both actin monomers are located in the same strand of F-actin with the first and second actins being close to the pointed and barbed ends of F-actin, respectively. The closing of the main cleft in 50 kDa of S1 might prevent binding of S1 with two actins since the distance between loops 635–647 and 567–574 became too short to interact with N-termini of two actins simultaneously. Depending on degree of saturation of F-actin with S1s there are two structurally different complexes are formed: at complete saturation each S1 binds only one actin and its cleft is closed while at partial saturation S1 interacts with two actins and its cleft is opened. The transition between one- and two-actin binding states of myosin accompanying with opening the cleft in central domain of S1 might be associated with force generation. The formation of actin-myosin interface would be associated with the energy release that might be used in part for the generation of force in muscle.

Drug Effects and Mechanism Underlying the Force-velocity Relationship of Skeletal Muscle

Amrinone is a bipyridine drug with characteristic effects on the force-velocity relationship of fast skeletal muscle. Here we combined in vitro motility assays, transient biochemical kinetics and optical tweezers studies to elucidate the mechanisms underlying the drug effects. Amrinone (1-2 mM) reduced the sliding velocity of heavy meromyosin (HMM) propelled actin filaments by 31.0 ± 2.5% (n = 15) at different ionic strengths of the assay solution (20 - 160 mM). The drug also reduced (by 2 - 18%) the sliding velocity of actin filaments propelled by subfragment 1 (S1). Stopped-flow studies of myofibrils, acto-HMM and acto-S1 showed no amrinone-induced reduction in the rate of MgATP induced actomyosin dissociation and optical tweezers studies detected no changes in the working stroke length. In contrast, the ADP affinity of acto-HMM (but not acto-S1) was increased about two-fold by 1 mM amrinone. Our results are consistent with inhibition of a strain-dependent MgADP-release step as the basis for amrinone induced reduction in sliding velocity. Modeling suggests that such an effect may also account for most other amrinone-induced changes of the force-velocity relationship of muscle (e.g. in isometric force and in shape of the force-velocity curve). Moreover, the results point to the possible importance of cooperative interactions between the two myosin heads in muscle contraction.

Analysis of Conformation of Skeletal Muscle Myosin Cross-linked by pPDM Using FRET

Previously biochemical studies have demonstrated that the highly reactive cysteine residues SH1 and SH2 can be crosslinked by variety of bifunctional reagents with different spans (3-14 A) in the presence of nucleotides, suggesting that the region is highly flexible. The SH1-SH2 region is believed to play a key role in the conformational changes that occur in the myosin head during the force generation coupled to ATP hydrolysis. We have previously shown that the HMM, which SH1-SH2 was crosslinked by p-Phenylene-dimaleimide (pPDM) in the presence of ADP, have a novel conformation using quick freeze deep etch electron microscopy (QFDE-EM). We have also demonstrated that conformational change of the myosin motor domain during ATP hydrolysis can be monitored by measuring the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we analyzed the conformation of the myosin crosslinked by pPDM using the FRET between the ATP binding site and the A1 essential light chain (ELC) and compared with the 3D structure models of ATPase intermediates derived from electron microscopic analysis. We prepared the skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by 6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluorescent ADP analogue NBD-ADP was trapped in the ATPase site of S1 labeled by BD. The FRET efficiency was estimated by measuring the change of fluorescence intensity of BD comparing with control BD-S1. FRET efficiency of pPDM-S1-NBD-ADP was apparently different from other nucleotides and nucleotide analogues bound states.

Structural Basis for The Regulation of Drosophila Myosin 7a

Drosophila myosin-7a is a single-headed myosin with a tail containing two MyTH4-FERM domains separated by an SH3 domain. Myosin-7a is often found in regions of high actin density and mutations in human myosin-7a result in deafness and blindness. We have previously shown that a subfragment-1 (S1) from this myosin has a Vmax of 1s-1 and a KATPase of 1μM. Full length myosin-7a has a Vmax similar to that of S1, but its KATPase is about 30μM. Negatively-stained processed images of myosin-7a in the presence of ATP show that the tail is tightly folded back against the motor domain forming a compact structure which obscures the normally easily recognized features of the motor domain. Myosin-7a unfolds at either high ionic strength or in the absence of ATP, revealing a clearly recognizable motor domain, the lever arm and some features of the tail region. C-terminal truncations were made to determine which portions of the multi-domained tail are necessary for the regulation. Removal of the last 99 amino acids which are highly conserved in all myosin-7a molecules and form a subdomain (termed MyTH7) of the FERM domain, or mutation of two conserved amino acids in this region, is sufficient to prevent folding of the molecule in the presence of ATP and activates the enzymatic activity. A construct consisting of the second FERM domain binds actin in an ATP-insensitive manner with a Kd of 30μM which is similar to the KATPase value for the full length molecule. We propose that at low actin concentration myosin-7a is folded and inactive, but at high actin concentrations such exists in actin bundles, it binds first via its tail binding site which then frees the motor domain to functionally interact with actin.

Single Molecule Kinetic Measurements Of Non-muscle Myosin IIB Using Optical Tweezers

Non-muscle myosin IIB (NMIIB) is a cytoplasmic myosin which is ubiquitously expressed in eukaryotic cells, particularly in the central nervous system and cardiac tissues. Our previous solution kinetic study of the single headed, subfragment-1 (S1) construct of NMIIB showed that this myosin spends a significant amount of its actomyosin ATPase cycle in the strongly bound state. In the presence of actin, the rate of ADP release is slow (∼0.35 s−1), comparable to the steady-state ATPase rate (0.13 ± 0.01 s−1).

Myosin heavy chain genes expressed in juvenile and adult silver carp Hypopthalmichthys molitrix: Novel fast-type myosin heavy chain genes of silver carp

Silver carp Hypopthalmichthys molitrix is eurythermal temperate fish, whose muscle is considered to express several types of myosin heavy chain (MYH) genes at different stages of its growth and to adjust to the environmental temperature. In this study, MYH genes expressed in the muscles of juvenile and adult silver carp were investigated. Five types of MYH cDNA clone were isolated from silver carp ( H. molitrix) by RACE strategy using a set of fast-type MYH specific primers, and termed scMYH F1, scMYH F2, scMYH F3, scMYH F4 and scMYH F5 in the order of their abundance in cDNA libraries constructed from fast skeletal muscles of adult silver carp. scMYH F1, scMYH F3 and scMYH F5 showed high nucleotide sequence identities of 96, 98 and 96% to gcMYH F30, gcMYH F10 and gcMYH FI, respectively, that encode MYHs predominantly expressed in fast skeletal muscle of grass carp ( Ctenopharyngodon idella) acclimated to 30, 10 and 20 °C, respectively. scMYH F2 and scMYH F4 showed a high identity to A4-type MYH from rock cod ( Notothenia coriiceps) slow skeletal muscle. Phylogenetic analysis demonstrated that scMYH F1 and scMYH F5 were monophyletic with fish adult fast-type MYHs, whereas scMYH F2 and scMYH F4 formed a cluster with fish slow-like fast-type MYH, and scMYH F3 did with fish embryonic fast-type MYHs. Interestingly, juvenile silver carp predominantly expressed scMYH F3 irrespective of acclimation temperatures at 10, 18 or 26 °C. The comparison among scMYH F1, scMYH F2 and scMYH F3 in the deduced amino acid sequence revealed that the putative binding sites for ATP, actin, and essential and regulatory light chains in myosin subfragment-1 (S1) have high identities with each other (81–100%). However, their loop-1 and loop-2 regions in S1 were highly variable, suggesting their different functions. The deduced amino acid sequences of myosin subfragment-2 and L-meromyosin showed high identities of 90–91% and 86–90%, respectively, among the above three scMYHs.

Structural Dynamics of the Actomyosin Complex Probed by a Bifunctional Spin Label that Cross-Links SH1 and SH2

We have used a bifunctional spin label (BSL) to cross-link Cys 707 (SH1) and Cys 697 (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen–hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305–Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Cross-Linked Long-Pitch Actin Dimer Forms Stoichiometric Complexes with Gelsolin Segment 1 and/or Deoxyribonuclease I That Nonproductively Interact with Myosin Subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.

Characterization of Subfragment-2 Regions of Myosins from Invertebrate and Vertebrate Striated Muscles

Type II myosin, the major contractile component of muscle, is a hexamer composed of two heavy chains and four light chains (two essential and two regulatory light chains). The N terminal globular head portion of heavy chain subunit (called subfragment-1, S1) is connected to rod portion through subfragment-2 (S2) region, which causes the bends in the rod portion of myosin and might make S1 lift off the thick filament to facilitate its reaction with actin. In the present study, S2 regions from various striated muscle myosins were compared including recently sequenced ones from cephalopods to shed light on the uniqueness of these stretches. The sequences of myosins from eleven species were aligned, and the α-helical and coiled-coil formations of S2 regions were estimated. As a result, all the S2 regions were considered to have reduced propensity of coiled-coil structure, especially at around the hinge regions, suggesting these regions are structurally flexible. One skip residues was found for all the species, though glutamine was replaced by threonine for fly and vertebrate counterparts. Prediction of secondary structure revealed uniqueness of cephalopod sequence in the N terminal region of S2. This was also confirmed by coiled-coil propensity prediction. Phylogenetic tree drawn based on the amino acid sequence showed that both of scallop and cephalopod S2s formed clear clusters, and were clearly distant from the other invertebrate (fly, worm) and vertebrate counterparts. From the results obtained, it was suggested that S2 regions of muscle myosins are ingeniously tuned so as to adapt to the contraction speed of each myosin or contractile performance of each muscle.

Structure and Dynamics of the Force-Generating Domain of Myosin Probed by Multifrequency Electron Paramagnetic Resonance

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4GHz and 94GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94GHz reveals that the EPR spectrum of the S1.ADP.Vi-bound biochemical state of myosin, which presumably mimics the S1.ADP.Pi state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.Vi biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

Similarities and Differences between Frozen-Hydrated, Rigor Acto–S1 Complexes of Insect Flight and Chicken Skeletal Muscles

The structure and function of myosin crossbridges in asynchronous insect flight muscle (IFM) have been elucidated in situ using multiple approaches. These include generating “atomic” models of myosin in multiple contractile states by rebuilding the crystal structure of chicken subfragment 1 (S1) to fit IFM crossbridges in lower-resolution electron microscopy tomograms and by “mapping” the functional effects of genetically substituted, isoform-specific domains, including the converter domain, in chimeric IFM myosin to sequences in the crystal structure of chicken S1. We prepared helical reconstructions (∼ 25 Å resolution) to compare the structural characteristics of nucleotide-free myosin0 S1 bound to actin (acto–S1) isolated from chicken skeletal muscle ( CSk) and the flight muscles of Lethocerus ( Leth) wild-type Drosophila ( wt Dros) and a Drosophila chimera ( IFI-EC) wherein the converter domain of the indirect flight muscle myosin isoform has been replaced by the embryonic skeletal myosin converter domain. Superimposition of the maps of the frozen-hydrated acto–S1 complexes shows that differences between CSk and IFM S1 are limited to the azimuthal curvature of the lever arm: the regulatory light-chain (RLC) region of chicken skeletal S1 bends clockwise (as seen from the pointed end of actin) while those of IFM S1 project in a straight radial direction. All the IFM S1s are essentially identical other than some variation in the azimuthal spread of density in the RLC region. This spread is most pronounced in the IFI-EC S1, consistent with proposals that the embryonic converter domain increases the compliance of the IFM lever arm affecting the function of the myosin motor. These are the first unconstrained models of IFM S1 bound to actin and the first direct comparison of the vertebrate and invertebrate skeletal myosin II classes, the latter for which, data on the structure of discrete acto-S1 complexes, are not readily available.

Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers

In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed. Dissociation and equilibrium binding of apo-TnC (i.e., lacking divalent cations) under different conditions were monitored by a standard test for maximum tension (P (o)). After 10 min in low-Mg2+ relaxing solution, rTnC dissociation (i.e., tension loss) was 80% vs only 45% in rigor. In rigor, adding myosin subfragment 1 (S1) reduced dissociation approximately twofold, whereas stretching to reduce filament overlap increased dissociation to nearly the value for relaxed fibers. Dissociation of rTnC after addition of Pi or MgADP to form A.M.Pi or A.M.ADP cross-bridges was significantly greater than with rigor (A.M) bridges. The increase in P (o) during equilibration with different concentrations of rTnC showed that the affinity for rTnC binding to the thin filament increased progressively with stronger cross-bridges: rTnC concentrations for half-maximal reconstitution (K (0.5)) were 8.1, 3.7, 2.9, and 1.1 microM for A + M.ADP.Pi, A.M.Pi, A.M, and A.M + S1. Cross-bridges containing MgADP(-) (A.M.ADP) were also less effective than rigor bridges in promoting rTnC binding. We conclude that cross-bridge state and number both modulate TnC affinity for the thin filament and that the TnC C-domain is a central element in this pathway.

Small heat shock protein Hsp27 protects myosin S1 from heat-induced aggregation, but not from thermal denaturation and ATPase inactivation

We applied different methods, such as turbidity measurements, dynamic light scattering, differential scanning calorimetry and co-sedimentation assay, to analyze the interaction of small heat shock protein Hsp27 with isolated myosin head (myosin subfragment 1, S1) under heat-stress conditions. Upon heating at 43 °C, Hsp27 effectively suppresses S1 aggregation, and this effect is enhanced by mutations mimicking Hsp27 phosphorylation. However, Hsp27 was unable to prevent thermal unfolding of myosin heads and to maintain their ATPase activity under heat-shock conditions. Structured summary MINT- 6490863, MINT- 6490872: LC1 (S1) (uniprotkb: P02602), Myosin subfragment 1 (S1) (uniprotkb: P02562) and Hsp27 (uniprotkb: P04792) physically interact (MI: 0218) by dynamic light scattering (MI: 0038) MINT- 6490833: LC1 (S1) (uniprotkb: P02602), Myosin subfragment 1 (S1) (uniprotkb: P02562) and Hsp27 (uniprotkb: P04792) physically interact (MI: 0218) by cosedimentation (MI: 0027) MINT- 6490770, MINT- 6490782: LC1 (S1) (uniprotkb: P02602), Myosin subfragment 1 (S1) (uniprotkb: P02562) and Hsp27 (uniprotkb: P04792) physically interact (MI: 0218) by light scattering (MI: 0067)