Effects of tropomyosin, troponin and Ca on the interaction between F-actin and heavy meromyosin

1. In the absence of ATP, H-meromyosin (heavy meromyosin) bound with the F-actin-tropomyosin-troponin complex up to the molar ratio of H-meromyosin to actin of 1:1, independently of the concentration of Ca²⁺.

2. In the presence of free Ca²⁺ above about 1 μM, with an increasing amount of H-meromyosin bound to a fixed amount of the F-actin-tropomyosin-troponin complex, the degree of flow birefringence decreased and the extinction angle increased. The minimum value of the birefringence and the maximum value of the extinction angle were found at the molar ratio of H-meromyosin to actin of 1:2. A further increase of bound H-meromyosin to actin restored both the degree of birefringence and the extinction angle to nearly the same level as the F-actin-tropomyosin-troponin complex only. In the absence of free Ca²⁺, the birefringence did not change with the binding of H-meromyosin.

3. This sensitivity of birefringence to the concentration of Ca²⁺ appeared only in the presence of tropomyosin and troponin. At a fixed ratio of H-meromyosin, actin and tropomyosin, the birefringence in the absence of Ca²⁺ increased with increasing amount of added troponin up to the weight ratio of troponin to actin of 1:6; whereas the birefringence in the presence of Ca²⁺ did not change.

4. At a fixed ratio of H-meromyosin to actin, the birefringence changed with increasing amount of tropomyosin added up to the weight ratio of tropomyosin to actin of 1:6; above this ratio, the birefringence was constant.

5. Subfragment S-1, prepared by the chymotryptic digestion of myosin, bound to F-actin, but the birefringence did not change even in the presence of tropomyosin and troponin.     

Electric birefringence studies of rabbit skeletal tropomyosin

The electric birefringence of rabbit skeletal tropomyosin and its nonpolymerizable derivative was studied as a function of protein concentration, pulse length, and electric field strength. Analyses of the zero field birefringence decay curves show that nonpolymerizable tropomyosin, which has had on average six C-terminal residues removed, and tropomyosin are both well approximated by rigid cylinders in solution at low salt concentrations at 20°C. The measured relaxation times for the monomers of polymerizable and nonpolymerizable tropomyosin are 1.5 ± 0.4 μs and 1.30 ± 0.2 μs, respectively, in good agreement with the values calculated from the known dimensions. For tropomyosin the electrical pulse induces the formation of linear dimers. Orientation occurs primarily by a permanent dipole mechanism. Permanent and induced dipole moments were calculated from reversing field experiments and from the saturation of the birefringence. Removal of the six C-terminal residues decreases the measured permanent dipole moment by 9.5%, from 6300 to 5700 Debyes, which is in good agreement with the 7% decrease calculated for permanent dipole contributions arising from the peptide dipoles and from the asymmetric distribution of the formal charges. This change is due primarily to the removal of Asp 280.     

Rate and mechanism of the assembly of tropomyosin with actin filaments

The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM MgCl2, 100 mM KCl, pH 7.5, 25 degrees C) and at the protein concentrations used (2.5-24 microM actin, 0.2-3.4 microM tropomyosin) the half-life time of assembly of tropomyosin with actin filaments was found to be less than 1 s. The results were analyzed quantitatively by a model in which tropomyosin initially binds to isolated sites. Further tropomyosin molecules bind contiguously to bound tropomyosin along the actin filaments. Good agreement between the experimental and theoretical time course of assembly was obtained by assuming a fast preequilibrium between free and isolatedly bound tropomyosin.     

Flexibility of smooth and skeletal tropomyosins

The rotational relaxation times of nonpolymerizable skeletal and smooth muscle tropomyosin were measured by analysis of the decay of the zero-field birefringence at different temperatures and salt concentrations. Skeletal tropomyosin in solution is equally well modeled as a rigid rod or as a semiflexible rod with a persistence length of 150 nm. Smooth muscle tropomyosin does not fit the rigid rod model but is well approximated by a semiflexible rod model with a persistence length of 55 nm. The results indicate that smooth muscle tropomyosin is either a more flexible molecule than skeletal muscle tropomyosin or is a curved structure with an end-to-end length shorter than the coiled-coil contour length. Smooth muscle tropomyosin controls the actomyosin ATPase differently from skeletal muscle tropomyosin and it had been suggested that the reason is because it is more rigid; clearly, another explanation must be sought.     

Equilibrium of the actin-tropomyosin interaction

The actin-tropornyosin interaction was studied by means of light-scattering. The experimental data were analysed on the basis of the model of co-operative binding of large ligands to a one-dimensional lattice with overlapping binding sites. The affinity of tropomyosin for actin filaments was dependent on the magnesium concentration. A fivefold increase of the magnesium concentration (from 0·5 mm to 2·5 mm) enhanced the equilibrium constant twofold (from 700 to 1600 m^?1) for the isolated binding of tropomyosin molecules to actin filaments. At low magnesium concentrations (0·5 mm), tropomyosin molecules were bound to isolated binding sites on an actin filament about 600 times more weakly than to contiguous binding sites. At increased magnesium concentrations (2·5 mm), the tendency of tropomyosin to bind contiguously increased twofold. Due to the co-operative nature of the actin-tropomyosin interaction, a small change in the magnesium concentration may cause a great change of the structural organisation of the complex. A small enhancement of the magnesium concentration (from 1 mm to 1·5 mm) caused bare filaments to be covered almost completely with tropomyosin. The length of tropomyosin clusters and the number of gaps on actin filaments depended strongly on the magnesium concentration. From the values of the experimentally determined equilibrium constants, it was concluded that the end-to-end interaction of tropomyosin was not strong enough to bring about all-or-none behaviour, where actin filaments of physiological length (~1000 nm) are either completely covered with or completely free of tropomyosin.     

Interactions of the inhibitory component of troponin, F-actin, and tropomyosin.

The interaction of the inhibitory component (TN I) of troponin and F-actin in the presence and absence of tropomyosin was studied by a number of physico-chemical techniques: i.e., gel filtration, ultracentrifugation, flow birefringence, viscosity and dynamic viscoelasticity measurements, and electron microscopy. The results indicated that TN I and F-actin interact with each other more strongly in the presence of tropomyosin than in its absence. The physiological implication of this finding is discussed.     

A new model of cooperative myosin-thin filament binding

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.     

Effect of ethanol on tropomyosin in solution and in reconstituted thin filaments

The effects of ethanol at concentrations below 10% on the conformation of tropomyosin, its end-to-end polymerization, its binding to F-actin, and its effects on actomyosin ATPase activity were studied. Ethanol stabilized the tropomyosin conformation by shifting the helix thermal unfolding profile to higher temperatures, and increased the end-to-end polymerization of tropomyosin. Ethanol-induced changes in the excimer fluorescence of pyrene-tropomyosin indicated that its conformation was stabilized by ethanol both free and bound to F-actin. Effects of tropomyosin and tropomyosin-troponin on actomyosin ATPase activity were measured under conditions for which tropomyosin binding to F-actin increases the activity. Under conditions for which the binding of tropomyosin to F-actin is optimum, in the presence of tropomyosin, the actomyosin ATPase activity decreased as the ethanol concentration increased, further indicating that ethanol induces a structural change in the tropomyosin-F-actin complex. Under conditions for which the binding of tropomyosin to F-actin is weak (low salt or high temperature), addition of ethanol increased the ATPase activity due to increased binding of tropomyosin to F-actin. Thus, ethanol appears to modify actomyosin ATPase activity by increasing the binding of tropomyosin to F-actin and affecting the structure of tropomyosin in the tropomyosin-F-actin filament.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Analysis of edge birefringence.

We present an experimental and theoretical study of the phenomenon of edge birefringence that appears near boundaries of transparent objects which are observed with high extinction and high resolution polarized light microscopy. As test objects, thin flakes of isotropic KCl crystals were immersed in media of various refractive indices. The measured retardation near crystal edges increased linearly with both the crystal thickness (tested between 0.3 and 1 micron), and the difference in refractive indices n between crystal (n = 1.49) and immersion liquids (n between 1.36 and 1.62). The specific edge birefringence, i.e., the retardation per thickness and per refractive index difference, is 0.029 on the high refractive index side of the boundary and -0.015 on the low refractive index side. The transition through zero birefringence specifies the position of a boundary at a much higher precision than predicted by the diffraction limit of the optical setup. The theoretical study employs a ray tracing procedure modeling the change in phase and polarization of rays passing through the specimen. We find good agreement between the model calculations and the experimental results indicating that edge birefringence can be attributed to the change in polarization of light that is refracted and reflected by dielectric interfaces.     

Similarities and differences of the alpha and beta components of tropomyosin.

Rabbit skeletal tropomyosin was separated into two components, alpha and beta, by CM cellulose column chromatography in the presence of urea. The two components are apparently different from TN-T, since, 1) upon addition of the components to F-actin solutions, they increase the degree of flow birefringence delta n, while TN-T does not, 2) the reduced mean residue elipticities [theta] at 220 nm are about 2.5-fold higher than for TN-T, and they contain no proline. These features are similar to those of intact tropomyosin, but the two components are not identical for the following reasons; 1) leucine is the C-terminus of the beta component and isoleucine is the C-terminus of the alpha component, 2) the beta component has a lower helicity and a somewhate lower capacity to increase delta n of F-actin solutions than the alpha component, and 3) the beta component has a higher content of glutamic acid and methionine than the alpha component. The two components can be crystallized into paracrystals in the presence of magnesium. Electron micrographs of the paracrystals of both components show a band pattern with 400 A periodicity. Bovine cardiac tropomyosin migrates on SDS gels as two poorly resolved bands, which could be separated by CM cellulose column chromatography. The C-terminus of the slower moving component was leucine, and that of the faster moving component was isoleucine, corresponding to the beta and alpha components of skeletal tropomyosin.     

A long-lasting birefringence change recorded from a tetanically stimulated squid giant axon.

A long-lasting birefringence change (the delayed response) was found to be produced in a tetanically stimulated squid giant axon. The change was independent of the concurrent membrane potential change, summated on repetitive stimulation, and always had a sign representing a decrease in resting birefringence. The axons was placed between a polarizer and an analyzer with their polarizing axes crossed, making an angle of 45 degrees with the longitudinal direction of the axon. The light beam that passed through the axon and the other optical elements was received by a photodiode. The change in light intensity evoked by repetitive stimulation was composed of brief initial responses, which took place in response to individual stimuli, and a delayed response, which developed gradually and lasted for several hundred msec. It was necessary to differentiate the effect of birefringence change from that of turbidity change. Formulas were derived on the assumption that the optical properties of the axon could be represented by a model of a uniaxial crystal that was not only birefringent but also dichroic, its extinction coefficients and the angle of retardation being changed independently on excitation. Calculations with them yielded the resting retardation, which agreed well with those obtained by the Senarmont's method, and the change in birefringence, which agreed well with the other calculated value derived from experiments using a quarter-wave plate. The results of the calculation confirmed the existence of the long-lasting birefringence change in the tetanically stimulated axon.     

A long-lasting birefringence change recorded from a tetanically stimulated squid giant axon

A long-lasting birefringence change (the delayed response) was found to be produced in a tetanically stimulated squid giant axon. The change was independent of the concurrent membrane potential change, summated on repetitive stimulation, and always had a sign representing a decrease in resting birefringence. The axon was placed between a polarizer and an analyzer with their polarizing axes crossed, making an angle of 45° with the longitudinal direction of the axon. The light beam that passed through the axon and the other optical elements was received by a photodiode. The change in light intensity evoked by repetitive stimulation was composed of brief initial responses, which took place in response to individual stimuli, and a delayed response, which developed gradually and lasted for several hundred msec. It was necessary to differentiate the effect of birefringence change from that of turbidity change. Formulas were derived on the assumption that the optical properties of the axon could be represented by a model of a uniaxial crystal that was not only birefringent but also dichroic, its extinction coefficients and the angle of retardation being changed independently on excitation. Calculations with them yielded the resting retardation, which agreed well with those obtained by the Sénarmont's method, and the change in birefringence, which agreed well with the other calculated value derived from experiments using a quarter-wave plate. The results of the calculation confirmed the existence of the long-lasting birefringence change in the tetanically stimulated axon.     

Co-operative regulation mechanism of muscle contraction: Inter-tropomyosin co-operation model

A new model is presented on the basis of our experimental data and the “tropomyosin-blocking theory” of muscle relaxation to explain the regulation of certain characteristics of muscle contraction, namely that the relation of contraction to pCa is co-operative while calcium-binding is essentially non-cooperative. Our experiments show that end-to-end interactions between adjacent tropomyosin molecules in the groove of the actin helix are essential for the co-operative regulation. The blocking theory says that the tropomyosin molecule in relaxed muscle sterically blocks the myosin attachment site on actin, whereas in contracting muscle it moves to a position away from the attachment site. In this model a concerted movement of tropomyosin molecules, brought about by their end-to-end interactions, is considered to be the essential mechanism of co-operative regulation, and it is assumed that the positional changes of tropomyosin occur primarily when the four calcium binding sites of troponin on the tropomyosin are saturated with calcium. Theoretical analysis of the model, based upon the two-state allosteric model, leads to a Michaelis-Menten equation for the Ca-binding function together with a co-operative equation for the state function, proportional to the contraction or ATPase activity. These two functions fit well the experimental data. With cardiac muscle the slope of the contraction versus pCa curve is slightly less steep than that obtained with skeletal muscle. This difference can be explained by the difference in the number of Ca-binding sites of troponins.     

Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units

Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding. In contrast, under high pressure, unlabeled and labeled tropomyosin molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.     

Intrinsic Birefringence of Poly-γ-Benzyl-l-Glutamate, A Helical Polypeptide, and the Theory of Birefringence

The intrinsic birefringence of macromolecules can be obtained directly from flow birefringence measurements in a solvent whose refractive index matches that of the solute. A small and positive value (approximately 0.01) was found for the helical polypeptide, poly-γ-benzyl-L-glutamate. The birefringence in solvents of varying index calculated from the Peterlin-Stuart theory using this value of the intrinsic birefringence did not agree with experimental values. Considerations of polydispersity and shear deformation indicated that the discrepancy could not be attributed to these effects. Also it could not be explained in terms of specific solvent effects. It is concluded that optical properties cannot be derived from the continuum model employed by Peterlin and Stuart. Much better agreement was obtained with a helical dipole necklace model.     

Modeling the rod outer segment birefringence change correlated with metarhodopsin II formation.

A rapid birefringence loss associated with metarhodopsin II formation, delta (delta n) MII, is produced when frog rod outer segments are exposed to a bleaching light flash. To analyze the nature of the underlying structure change, measurements of delta (delta n) MII were made in rod outer segments perfused with glycerol solutions to increase the refractive index of the cytoplasmic and intradisk spaces. Comparisons of experimental results with computed changes in the form birefringence component using two- and three-dielectric outer segment models for several putative structure changes were made. It is concluded that delta (delta n) MII can be due to either a change in the intrinsic birefringence component caused by the reorientation of anisotropic molecules, or to a change in the form birefringence component caused by small changes in the cytoplasmic and/or intradisk volumes.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

Three-dimensional image reconstruction of actin-tropomyosin complex and actin-tropomyosin-troponin T-troponin I complex.

The structure of the actin-tropomyosin complex, which represents on active form of the thin filaments of skeletal muscle and the actin-tropomyosin-troponin T-troponin I complex, which represents an inhibited form, have been studied by three-dimensional reconstruction from electron micrographs.A model of the three-dimensional structure of the actin-tropomyosin complex obtained by averaging the twelve “best” sets of data showed that the structure of the helix was polar and that the actin-tropomyosin contact was relatively loose. The detailed shape of the actin monomer and tropomyosin strands could be observed.A model of the three-dimensional structure of the actin-tropomyosin-troponin T-troponin I complex obtained by averaging the nine “best” sets of data showed that the contact between the actin and tropomyosin was very close in the inhibited filament, where the position of tropomyosin differed by approximately 10 Å from that in the active filament.The biological significance of the change in the extent of the actin-tropomyosin contact and of the movement of tropomyosin is discussed with reference to the mechanism of the regulation of muscle contraction by the tropomyosin-troponin-calcium system.