Distinct sites in tropomyosin specify shared and isoform-specific regulation of myosins II and V.

Muscle contraction, cytokinesis, cellular movement, and intracellular transport depend on regulated actin-myosin interaction. Most actin filaments bind one or more isoform of tropomyosin, a coiled-coil protein that stabilizes the filaments and regulates interactions with other actin-binding proteins, including myosin. Isoform-specific allosteric regulation of muscle myosin II by actin-tropomyosin is well-established while that of processive myosins, such as myosin V, which transport organelles and macromolecules in the cell periphery, is less certain. Is the regulation by tropomyosin a universal mechanism, the consequence of the conserved periodic structures of tropomyosin, or is it the result of specialized interactions between particular isoforms of myosin and tropomyosin? Here, we show that striated muscle tropomyosin, Tpm1.1, inhibits fast skeletal muscle myosin II but not myosin Va. The non-muscle tropomyosin, Tpm3.1, in contrast, activates both myosins. To decipher the molecular basis of these opposing regulatory effects, we introduced mutations at conserved surface residues within the six periodic repeats (periods) of Tpm3.1, in positions homologous or analogous to those important for regulation of skeletal muscle myosin by Tpm1.1. We identified conserved residues in the internal periods of both tropomyosin isoforms that are important for the function of myosin Va and striated myosin II. Conserved residues in the internal and C-terminal periods that correspond to Tpm3.1-specific exons inhibit myosin Va but not myosin II function. These results suggest that tropomyosins may directly impact myosin function through both general and isoform-specific mechanisms that identify actin tracks for the recruitment and function of particular myosins.

Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

Tropomyosin Structure, Function, and Interactions: A Dynamic Regulator.

Tropomyosin is the archetypal-coiled coil, yet studies of its structure and function have proven it to be a dynamic regulator of actin filament function in muscle and non-muscle cells. Here we review aspects of its structure that deviate from canonical leucine zipper coiled coils that allow tropomyosin to bind to actin, regulate myosin, and interact directly and indirectly with actin-binding proteins. Four genes encode tropomyosins in vertebrates, with additional diversity that results from alternate promoters and alternatively spliced exons. At the same time that periodic motifs for binding actin and regulating myosin are conserved, isoform-specific domains allow for specific interaction with myosins and actin filament regulatory proteins, including troponin. Tropomyosin can be viewed as a universal regulator of the actin cytoskeleton that specifies actin filaments for cellular and intracellular functions.

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation.

Tropomyosin (Tpm) is a two-chained α-helical coiled-coil protein that binds to filamentous actin (F-actin), and regulates its interactions with myosin by occupying three average positions on F-actin (blocked, closed, and open). Mutations in the Tpm are linked to heart diseases including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). To elucidate the molecular mechanisms of Tpm mutations (including DCM mutation E54K, HCM mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K, and a designed synthetic mutation D137L) in terms of their effects on Tpm flexibility and its interactions with F-actin, we conducted extensive molecular dynamics simulations for the wild-type and mutant Tpm in complex with F-actin (total simulation time 160 ns per mutant). The mutants exhibited distinct changes (i.e., increase or decrease) in the overall and local flexibility of the Tpm coiled-coil, with each mutation causing both local and long-range modifications of the Tpm flexibility. In addition, our binding calculations revealed weakened Tpm-F-actin interactions (except for L185R, D137L and A63V) involving five periods of Tpm, which correlate with elevated fluctuation of Tpm relative to the blocked position on F-actin that may lead to easier activation and increased Ca2+-sensitivity. We also simulated the αß/ßα-Tpm heterodimer in comparison with the αα-Tpm homodimer, which revealed greater flexibility and weaker actin binding in the heterodimer. Our findings are consistent with a complex mechanism underlying how different Tpm mutations perturb the Tpm function in distinct ways (e.g., by affecting specific sites of Tpm), which bear no simple links to the disease phenotypes (e.g., HCM vs. DCM).

A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics.

The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.

Evolutionarily conserved sites in yeast tropomyosin function in cell polarity, transport and contractile ring formation.

Tropomyosin is a coiled-coil protein that binds and regulates actin filaments. The tropomyosin gene in Schizosaccharomyces pombe, cdc8, is required for formation of actin cables, contractile rings, and polar localization of actin patches. The roles of conserved residues were investigated in gene replacement mutants. The work validates an evolution-based approach to identify tropomyosin functions in living cells and sites of potential interactions with other proteins. A cdc8 mutant with near-normal actin affinity affects patch polarization and vacuole fusion, possibly by affecting Myo52p, a class V myosin, function. The presence of labile residual cell attachments suggests a delay in completion of cell division and redistribution of cell patches following cytokinesis. Another mutant with a mild phenotype is synthetic negative with GFP-fimbrin, inferring involvement of the mutated tropomyosin sites in interaction between the two proteins. Proteins that assemble in the contractile ring region before actin do so in a mutant cdc8 strain that cannot assemble condensed actin rings, yet some cells can divide. Of general significance, LifeAct-GFP negatively affects the actin cytoskeleton, indicating caution in its use as a biomarker for actin filaments.

A systematic nomenclature for mammalian tropomyosin isoforms.

Tropomyosin, a ubiquitous protein in animals and fungi, is associated with the actin cytoskeleton and is involved with stabilising actin filaments and regulating the interaction of the filament with other actin binding proteins. The protein is best known for its role in regulating the interaction between actin and myosin in muscle contraction but in recent years its role as a major player in the organisation and dynamics of the cytoskeleton has been increasingly recognised. In mammals Tpm is expressed from four distinct genes and alternate splicing of each gene can produce a total of up to 40 different mRNA variants most of which are expressed as proteins. We are expecting a renaissance in the study of tropomyosins as the roles of these different isoforms are beginning to be deciphered. However, it is our belief that such a renaissance is being limited by confusion over the naming systems for the tropomyosin isoforms. These result in even experienced workers struggling to reconcile work done in different laboratories and at different times. We propose here a systematic nomenclature for tropomyosin based on the best current practice. We recommend the adoption of these names and a cross-reference to the table of alternate names and accession numbers for protein sequences is included here. The National Center for Biotechnology Information (NCBI) website has been amended to include the nomenclature for the human, mouse and rat genes.

Hugh E. Huxley: the compleat biophysicist.

The sliding filament model of muscle contraction, put forward by Hugh Huxley and Jean Hanson in 1954, is 60 years old in 2014. Formulation of the model and subsequent proof was driven by the pioneering work of Hugh Huxley (1924-2013). We celebrate Huxley's integrative approach to the study of muscle contraction; how he persevered throughout his career, to the end of his life at 89 years, to understand at the molecular level how muscle contracts and develops force. Here we show how his life and work, with its focus on a single scientific problem, had impact far beyond the field of muscle contraction to the benefit of multiple fields of cellular and structural biology. Huxley introduced the use of x-ray diffraction to study the contraction in living striated muscle, taking advantage of the paracrystalline lattice that would ultimately allow understanding contraction in terms of single molecules. Progress required design of instrumentation with ever-increasing spatial and temporal resolution, providing the impetus for the development of synchrotron facilities used for most protein crystallography and muscle studies today. From the time of his early work, Huxley combined electron microscopy and biochemistry to understand and interpret the changes in x-ray patterns. He developed improved electron-microscopy techniques, thin sections and negative staining, that enabled answering major questions relating to the structure and organization of thick and thin filaments in muscle and the interaction of myosin with actin and its regulation. Huxley established that the ATPase domain of myosin forms the crossbridges of thick filaments that bind actin, and introduced the idea that myosin makes discrete steps on actin. These concepts form the underpinning of cellular motility, in particular the study of how myosin, kinesin, and dynein motors move on their actin and tubulin tracks, making Huxley a founder of the field of cellular motility.

Regulation of nonmuscle myosin II by tropomyosin.

The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.

Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

A molecular evolution approach to study the roles of tropomyosin in fission yeast.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

A periodic pattern of evolutionarily conserved basic and acidic residues constitutes the binding interface of actin-tropomyosin.

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction.

To establish α-tropomyosin (Tm)'s structure-function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm-actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca(2+). Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm-actin interaction cooperatively affects actin to result in generation of greater force and velocity.

Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin.

Tropomyosin (Tm) is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3-4 sites mutated to Ala within the first half or the second half of periods 2-6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (> 4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤ 2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.

Systematic analysis of tropomodulin/tropomyosin interactions uncovers fine-tuned binding specificity of intrinsically disordered proteins.

An intriguing regulatory mechanism is the ability of some proteins to recognize their binding partners in an isoform-specific manner. In this study we undertook a systematic analysis of the specificity of the tropomodulin (Tmod) interaction with tropomyosin (TM) to show that affinities of different Tmod isoforms to TM are isoform-dependent. Intrinsic disorder predictions, alignment of sequences, and circular dichroism were utilized to establish a structural basis for these isoform-specific interactions. The affinity of model peptides derived from the N-terminus of different TM isoforms to protein fragments that correspond to the two TM-binding sites of different Tmod isoforms were analyzed. Several residues were determined to be responsible for the isoform-dependent differences in affinity. We suggest that changing a set of residues rather than a single residue is needed to alter the binding affinity of one isoform to mimic the affinity of another isoform. The general intrinsic disorder predictor, PONDR® VLXT, was shown to be a useful tool for analyzing regions involved in isoform-specific binding and for predicting the residues important for isoform differences in binding. Knowing the residues responsible for isoform-specific affinity creates a tool suitable for studying the influence of Tmod/TM interactions on sarcomere assembly in muscle cells or actin dynamics in non-muscle cells.

What makes tropomyosin an actin binding protein? A perspective.

Tropomyosin is a two-chained alpha-helical coiled coil that binds along the length of the actin filament and regulates its function. The paper addresses the question of how a "simple" coiled-coil sequence encodes the information for binding and regulating the actin filament, its universal target. Determination of the tropomyosin sequence confirmed Crick's predicted heptapeptide repeat of hydrophobic interface residues and revealed additional features that have been shown to be important for its function: a 7-fold periodicity predicted to correspond to actin binding sites and interruptions of the canonical interface with destabilizing residues, such as Ala. Evidence from published work is summarized, leading to the proposal of a paradigm that binding of tropomyosin to the actin filament requires local instability as well as regions of flexibility. The flexibility derives from bends and local unfolding at regions with a destabilized coiled-coil interface, as well as from the dynamic end-to-end complex. The features are required for tropomyosin to assume the form of the helical actin filament, and to bind to actin monomers along its length. The requirement of instability/flexibility for binding may be generalized to the binding of other coiled coils to their targets.

A peek into tropomyosin binding and unfolding on the actin filament.

BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

Structure of the N terminus of a nonmuscle alpha-tropomyosin in complex with the C terminus: implications for actin binding.

Tropomyosin is a coiled-coil actin binding protein that stabilizes the filament, protects it from severing, and cooperatively regulates actin's interaction with myosin. Depending on the first coding exon, tropomyosins are low molecular weight (LMW), found in the cytoskeleton and predominant in transformed cells, or high molecular weight (HMW), found in muscle and nonmuscle cells. The N- and C-terminal ends form a complex that allows tropomyosin to associate N terminus-to-C terminus along the actin filament. We determined the structure of a LMW tropomyosin N-terminal model peptide complexed with a smooth/nonmuscle tropomyosin C-terminal peptide. Using NMR and circular dichroism we showed that both ends become more helical upon complex formation but that the C-terminal peptide is partially unfolded at 20 degrees C. The first five residues of the N terminus that are disordered in the free peptide are more helical and are part of the overlap complex. NMR data indicate residues 2-17 bind to the C terminus in the complex. The data support a model for the LMW overlap complex that is homologous to the striated muscle tropomyosin complex in which the ends are oriented in parallel N terminus-to-C terminus with the plane of the N-terminal coiled coil perpendicular to the plane of the C terminus. The main difference is that the overlap spans 16 residues in the LMW tropomyosin complex compared to 11 residues in the HMW striated muscle overlap complex. We discuss the relevance of a stable but dynamic intermolecular junction for high-affinity binding to actin.

Tropomyosin period 3 is essential for enhancement of isometric tension in thin filament-reconstituted bovine myocardium.

Tropomyosin (Tm) consists of 7 quasiequivalent repeats known as "periods," and its specific function may be associated with these periods. To test the hypothesis that either period 2 or 3 promotes force generation by inducing a positive allosteric effect on actin, we reconstituted the thin filament with mutant Tm in which either period 2 (Delta2Tm) or period 3 (Delta3Tm) was deleted. We then studied: isometric tension, stiffness, 6 kinetic constants, and the pCa-tension relationship. N-terminal acetylation of Tm did not cause any differences. The isometric tension in Delta2Tm remained unchanged, and was reduced to approximately 60% in Delta3Tm. Although the kinetic constants underwent small changes, the occupancy of strongly attached cross-bridges was not much different. The Hill factor (cooperativity) did not differ significantly between Delta2Tm (1.79 +/- 0.19) and the control (1.73 +/- 0.21), or Delta3Tm (1.35 +/- 0.22) and the control. In contrast, pCa(50) decreased slightly in Delta2Tm (5.11 +/- 0.07), and increased significantly in Delta3Tm (5.57 +/- 0.09) compared to the control (5.28 +/- 0.04). These results demonstrate that, when ions are present at physiological concentrations in the muscle fiber system, period 3 (but not period 2) is essential for the positive allosteric effect that enhances the interaction between actin and myosin, and increases isometric force of each cross-bridge.