Subfragment-2 Region Research Articles

Abstract 915: Dysregulation of the Myosin and Myosin Binding Protein-C interaction in Hypertrophic Cardiomyopathy

Rationale: Affecting 1 in 300 individuals, hypertrophic cardiomyopathy (HCM) is a genetic heart disease characterized by left ventricular hypertrophy, myocardial disarray, and sudden cardiac death. Often, HCM is associated with myocardial hypercontractility. The subfragment-2 (S2) of beta-myosin heavy chain contains a cluster of missense and deletion mutations associated with severe HCM. Interestingly, myosin S2 interacts with the C0-C2 region of cardiac myosin binding protein C (cMyBP-C) in a phosphorylation-dependent manner to regulate sarcomere contractility. However, the nature of myosin S2 and cMyBP-C interactions and the mechanism(s) by which mutations in myosin S2 cause HCM remain to be elucidated. Objective: To determine whether mutations in myosin S2 weaken its interaction with cMyBP-C, resulting in enhanced myofilament contractility. Methods and Results: Myosin S2 proteins (126 amino acids) containing three clinically relevant mutations (R870H, E924K, E930del, or wild type), and recombinant C0-C2 region of cMyBP-C were produced and purified by metal affinity chromatography. Solid-phase binding assays and isothermal calorimetry experiments revealed a significantly dampened binding to C0-C2 for these three mutants in myosin S2 (30% lower than wild type, p<0.002), suggesting that mutations in S2 regions reduce their bindings to cMyBP-C. Conversely, upon protein kinase A phosphorylation of C0C2, these S2 mutants displayed an increased affinity to cMyBP-C, an effect opposite that of wild-type S2. Structural analyses of these mutations within myosin S2 are predicted to reduce the alpha-helical content, thereby reducing stability of the critical coiled-coil structure. Conclusions: Mutations in myosin S2 result in reduced binding to cMyBP-C. Functionally, this would result in a greater attachment of cross-bridges, and thus enhance myofilament contractility. Strikingly, these mutations increase their affinity to cMyBP-C upon phosphorylation, demonstrating fundamental changes to the regulation of contractile function.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Evidence for the Regulation of Actin-myosin Binding Strength by Lever Arm and Subfragment-2 Regions of Myosin Molecule in Contracting Skinned Muscle Fibers as Revealed by the Effect of Antibodies

It is generally believed that myosin heads (M) extending from myosin filaments form weak binding with actin filament (A) in the form of A-M-ADP-Pi, and strong binding with A in the form of A-M-ADP and A-M. During muscle contraction, M first attaches weakly to A in the form of M-ADP-Pi, and then release Pi to attach strongly with A in the form of A-MADP to exert power stroke producing myofilament sliding. The weak to strong binding transition between M and A is therefore essential for producing muscle contraction. After power stroke, associated with release of ADP, M detaches from A on binding with next ATP to form M-ADP-Pi. Thus, M repeats attachment-detachment cycle with A, coupled with ATP hydrolysis. Using antibodies to myosin lever arm domain (anti-LD antibody) and to myosin subfragment-2 region (anti-S-2 antibody), we have found that, if these antibodies attach to LD or S-2 regions, myosin heads no longer generate force, but still hydrolyse ATP at a normal rate without contributing to muscle fiber stiffness and internal resistance against fiber shortening. The puzzling effect of antibodies can be accounted for by assuming that in myosin molecules, with antibodies attached to their LD or S-2 region, (1) the strength of A-M binding is markedly reduced to be readily broken by small length perturbations or muscle fiber shortening, and (2) such myosin heads still repeat attachmentdetachment cycle coupled with ATP hydrolysis without passing through weak to strong A-M binding transition. Thus, the present work indicates functional influence of LD and S-2 regions on the A-M binding of myosin molecule, suggesting essential role of these regions in muscle contraction.

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.

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

'Artificial' myosin isozymes: preparation and characteristics.

Using precisely monitored proteolytic digestion conditions rabbit fast skeletal muscle myosin could be selectively modified in different ways. A myosin isozyme with a 20-kDa alkali light chain 1 (A1) could be obtained by digesting with papain in the presence of Ca2+. Under these conditions alkali light chain 2 (A2) was cleaved at Lys-17 and lost a 2.3-kDa N-terminal fragment including the strongly basic N terminus and about half of the characteristic (Ala-Pro) sequence. The Nbs2-[5,5' dithiobis(2-nitrobenzoic acid)-]light chain and A2 were left unmodified and less than 5% of the myosin heavy chain presented a break in the subfragment-2 region. EDTA and Ca2+ ATPase activities were unchanged. A myosin isozyme with an 18-kDa Nbs2-light chain was obtained by limited digestion with trypsin in the presence of Ca2+. The 18.9 leads to 18-kDa conversion was nearly 100% whereas less than 10% of the heavy chain was fragmented and only about 5% of A1 was converted to A1. The Nbs2-light chain was cleaved at Arg-7 preserving Ser-15 and consequently a phosphorylated modified myosin could be obtained. A quasi-elastic light-scattering study showed that this modified myosin in high-ionic-strength solutions exhibited physicochemical characteristics quite similar to those of unmodified myosin.

Phosphorylation-dependent regulation is absent in a nonmuscle heavy meromyosin construct with one complete head and one head lacking the motor domain.

To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V(max) by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot be down-regulated by a regulatory domain (without the motor domain) on the partner head. Two motor domains are required for regulation. This result is predicted by a structural model (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390) showing interaction between the motor domains for unphosphorylated smooth muscle myosin, if motor-motor interaction is the basis for down-regulation.

Innocuous Labeling of the Subfragment-2 Region of Skeletal Muscle Heavy Meromyosin with a Fluorescent Polyacrylamide Nanobead and Visualization of Individual Heavy Meromyosin Molecules

We have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.

Unfolding Domains in Smooth Muscle Myosin Rod

Gizzard smooth muscle myosin rod, an alpha-helical coiled coil, exhibits two cooperative thermal or denaturant-induced helix unfolding transitions in solutions containing 0.6 M NaCl at neutral pH, when monitored by circular dichroism at 222 nm. The first smaller transition unfolds part of the subfragment 2 (S2) domain, and the main transition unfolds the remaining helix including the light meromyosin (LMM) domain. These unfolding domains were identified by monitoring the fluorescence of acrylodan, an environmentally sensitive fluorescence probe, and the ESR signal of a maleimide spin-label, sensitive to motion, both specifically attached to Cys 43 in the S2 region of the rod sequence. The identities of the domains were verified by studying the unfolding of the S2 and LMM coiled-coil peptides obtained by proteolytic cleavage of spin-labeled and unlabeled rod. The fluorescence of acrylodan-labeled rod indicated that although the S2 intermediate is unfolded, it is not in a random-coil conformation. The unfolded S2 region stabilized the LMM domain against unfolding, possibly by a direct interaction with the LMM region. Such an interaction may be involved in the salt- and phosphorylation-dependent 6S to 10S shift in configuration of the myosin molecule.

Pressure effects and thermal stability of myosin rods and rod minifilaments: fluorescence and circular dichroism studies.

In the present study hydrostatic pressure was applied upon both skeletal myosin rod molecules and rod minifilaments to learn more of the intra- and intermolecular interaction behavior of myosin. Applied pressure disassembled the rod minifilaments into individual rod molecules and dissociated each myosin rod molecule into two chains of alpha-helix. The dissociation and disassembly profiles of these systems were obtained by measuring their fluorescent anisotropy under pressure. The mid-disassembly pressure of rod minifilaments at 0.4 mg/mL concentration was 430-490 bar. However, dissociation of two helical strands of rod molecules occurred at a much higher pressure, with a mid-disassembly pressure of 1300 bar at this concentration. These results indicate that the intramolecular interactions occurring between two alpha-helical chains of a rod molecule are much more stable under pressure than the intermolecular interactions that occur among rod molecules in a minifilament. The regions in the rod molecules involved in filament assembly were investigated through usage of both the intrinsic fluorescence of tryptophan residues and the extrinsic fluorescence of 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) labeled cysteine residues. The blue spectral shifts upon minifilament formation suggest the participation of both light meromyosin (LMM) and subfragment-2 (S-2) regions of myosin rods in the filament formation. Profiles of thermal unfolding of myosin rod molecules and rod minifilaments were obtained by circular dichroism measurement. The multiple transitions exhibited upon unfolding profiles indicated the presence of more than one structural domain, each correlating with a cooperative transition. The domain transitional temperatures were found to be 1-4 degrees C higher for rods in minifilaments than those for rod molecules in a solution of similar ionic composition, indicating that all structural domains are involved in filament assembly. Furthermore, the domain transitional temperatures for rod molecules in a buffer containing 0.6 M NaCl were 6-8 degrees C higher than those for rod molecules in 5 mM sodium pyrophosphate buffer, suggesting that each structural domain of a rod molecule becomes stabilized at 0.6 M NaCl solution.

Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards.

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.

Contraction of myofibrils in the presence of antibodies to myosin subfragment 2.

In a muscle-based version of in vitro motility assays, the unloaded shortening velocity of rabbit skeletal myofibrils has been determined in the presence and absence of affinity-column-purified polyclonal antibodies directed against the subfragment-2 region of myosin. Contraction was initiated by photohydrolysis of caged ATP and the time dependence of shortening was monitored by an inverted microscope equipped with a video camera. Antibody-treated myofibrils undergo unloaded shortening in a fast phase with initial rates and half-times comparable to control (untreated) myofibrils, despite a marked reduction in the isometric force of skinned muscle fibers in the presence of the antibodies. In antibody-treated myofibrils, this process is followed by a much slower phase of contraction, terminating in elongated structures with well-defined sarcomere spacings (approximately 1 micron) in contrast to the supercontracted globular state of control myofibrils. These results suggest that although the unloaded shortening of myofibrils (and in vitro motility of actin filaments over immobilized myosin heads) can be powered by myosin heads, the subfragment-2 region as well as the myosin head contributes to force production in actively contracting muscle.

Suppression of contractile force in muscle fibers by antibody to myosin subfragment 2.

Polyclonal antibody directed against the subfragment-2 region of myosin was purified by affinity chromatography. Skinned muscle fibers that had been preincubated with antibody were able to sustain only 7% of the active isometric force generated by control fibers. The effect of antibody on force production could not be accounted for by inhibition of ATP turnover.

Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge

We have used α-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 °C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 M r and 64,000 M r (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 m m), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant ( k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 m m) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 °C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.

Local melting in the subfragment-2 region of myosin in activated muscle and its correlation with contractile force

Local melting within the subfragment-2 region of activated rabbit skeletal glycerinated muscle fibers has been investigated over the temperature range 5 to 37 °C, using an enzyme (chymotrypsin)-probe method. The cleavage rates were determined from the time-course of formation of digestion products by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. We found the cleavage sites to be localized in a restricted region M r = 64,000 to 90,000/polypeptide chain, measured from the C terminus of the myosin rod (the subfragment-2 hinge domain). The cleavage rate constant for activated muscle fibers in the presence of an ATP-regenerating system was about 100 times larger at each temperature than that for rigor or for relaxed muscle fibers and showed a marked increase in magnitude with increasing temperature. Comparative plots of the apparent rate-constant for cleavage within the subfragment-2 hinge domain and the isometric force generated by active fibers versus MgATP concentration gave closely similar profiles suggesting a strong positive correlation. Thus, there appears to be a close coupling between the conformational transition within the subfragment-2 hinge domain and contractile force when the cross-bridges undergo cycling.

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 M r and 53,000 M r per polypeptide chain) and a short subfragment-2 particle (34,000 M r per polypeptide chain) by three different enzymes (α-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 °C. We followed the time-course of digestion at specific sites at high ( I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 M r) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 °C a restricted region at M r = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 °C local melting was detected by all three enzymes at several specific sites within the hinge domain ( M r = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.

Stability and melting kinetics of structural domains in the myosin rod

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 ( M r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 ( M r = 16,000 to 17,000) and 24,000 to 26,000 ( M r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range ( τ f) and the other in the millisecond range ( τ s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast ( τ f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.

Structural studies of isolated native thick filaments from rabbit psoas muscle with AMP deaminase decoration.

AMP deaminase (adenylate deaminase; AMP aminohydrolase, EC 3.5.4.6), a large flat tetrameric enzyme found in skeletal muscle, binds strongly and specifically to the subfragment-2 region of rabbit skeletal muscle myosin. This allows its use as a structural probe in myosin and myosin rod aggregation studies. When mixed with a preparation of isolated native thick filaments, AMP deaminase decorates the entire filament backbone except for the central bare zone. Binding is particularly ordered in the banded region, where 11 stripes of about 43-nm spacing on either side of the bare zone have been observed in studies of isolated A-bands. No systematic absence of deaminase is seen here, indicating that the presence of the C-protein and H-protein bands does not make the binding site inaccessible to the tetramer. Optical diffraction patterns of the decorated filaments show the expected 42.9-nm periodicities and a reflection indexing at 28.6 nm. Because of the bulkiness of the tetramer relative to the number of available binding sites at each 14.3-nm interval along the filament shaft, the helix net is being sampled at a lower frequency than is the underlying myosin organization. As a result, reflections on layer lines other than orders of 42.9 nm are also observed (e.g., 57.2); these reflections strongly indicate a structure based on a 12/1 primitive helix. The results appear to eliminate the symmetric double two-fold and three-fold models of thick filament structure but are consistent with an asymmetric four-fold structure.

7] Preparation of myosin and its subfragments from rabbit skeletal muscle

This chapter presents a procedure for the preparation of myosin and its sub-fragments from rabbit skeletal muscle. The discussion is limited to rabbit skeletal muscle myosin because this particular myosin is the most widely used protein in studies of muscle contraction. At a salt concentration approximating physiological conditions, myosin aggregates to form bipolar thick filaments with a central bare zone; S1 heads protrude from the filament on either side of the bare zone. The insolubility of myosin at μ < 0.3 derives from the rod region, which is insoluble in low salt. The catalytic and actin-binding properties of myosin reside in the water-soluble S1 heads. Both the rod and S1 can be obtained in relatively homogeneous form by proteolytic digestion of myosin. In addition to the above, a number of other subfragments of myosin can be obtained: digestion with trypsin results in light meromyosin, which like the rod retains the solubility properties of myosin, and in heavy meromyosin (HMM), which consists of the two S1 heads attached to the flexible subfragment-2 region. Single-headed HMM and single-headed myosin have also been prepared by limited digestion of myosin with papain.

Cross-bridge movement and the conformational state of the myosin hinge in skeletal muscle

We have modified and extended our earlier procedure for cross-linking myosin segments in the thick filaments of muscle so as to: (1) follow the time-course of cross-linking the subfragment-2 and light meromyosin regions as a function of pH; and (2) eliminate the contribution of intramolecular cross-linking in the analysis of the sodium dodecyl sulfate/polyacrylamide gels. From the kinetics of cross-linking the S-2 † † Abbrviations used: S-2, subfragment-2; LMM, light meromyosin; HMM, heavy meromyosin; S−1, subfragment-1; SDS, sodium dodecyl sulfate; DMS, dimethyl suberimidate; k 0 S − 1, the ratio of the 2 crosslinking rate constants ( k S−1 k rod ); k 0 S − 2, the ratio of the 2 cross-linking rate constants ( k S − 2 k LMM ). and LMM regions of myosin in glycerinated rigor myofibrils of rabbit we find that the normalized rate ( k S − 2 k LMM ) of cross-linking falls over a narrow range of pH (7.4 to 8.4) with a sigmoidal profile closely similar to that observed earlier for the pH dependence of cross-linking of the subfragment-1 subunits. This result is contrary to our earlier conclusion (Sutoh et al., 1978 a; Chiao & Harrington, 1979) that a major fraction of the S-2 link is immobilized on the surface under these conditions, and suggests that this segment, like the S-1 subunit, is released and swings away from the thick filament surface when the pH is raised. This release of S-2 is highly co-operative and appears to be accompanied by a conformational transition of the polypeptide chains within the light meromyosin-heavy meromyosin hinge to a more open structure, as shown by an increased susceptibility to chymotryptic proteolysis. Treatment of the co-operative release process as a two-state equilibrium reveals that a change in charge equivalent to release of only about two protons occurs when the cross-bridge is detached from the surface of the thick filament.