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.     

Isolation and some structural and functional properties of macrophage tropomyosin

Tropomyosin purified from rabbit lung macrophages is very similar in structure to other nonmuscle cell tropomyosins. Reduced and denatured, the protein has two polypeptides which migrate during electrophoresis in sodium dodecyl sulfate on polyacrylamide gels with slightly different mobilities corresponding to apparent Mr's of about 30 000. Following cross-linking by air oxidation in the presence of CuCl2, electrophoresis under nonreducing conditions reveals a single polypeptide of Mr 60 000. Macrophage tropomyosin has an isoelectric point of 4.6 and an amino acid composition similar to other tropomyosins. It contains one cysteine residue per chain. In the electron microscope, macrophage tropomyosin molecules rotary shadowed with platinum and carbon are slender, straight rods, 33 nm in length. Macrophage tropomyosin paracrystals grown in high magnesium concentrations have an axial periodicity of 34 nm. On the basis of yields from purification and from two-dimensional electrophoretic analyses of macrophage extracts, tropomyosin comprises less than 0.2% of the total macrophage protein, a molar ratio of approximately 1 tropomyosin molecule to 75 actin monomers in the cell. Macrophage tropomyosin binds to actin filaments. Macrophage, skeletal muscle, and other nonmuscle cell tropomyosins inhibit the fragmentation of actin filaments by the Ca2+-gelsolin complex. The finding implies that tropomyosin may have a role in stabilizing actin filaments in vivo.     

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.     

Effect of tropomyosin on formin-bound actin filaments

Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly.     

Effect of the state of oxidation of cysteine 190 of tropomyosin on the assembly of the actin-tropomyosin complex

Tropomyosin, cross-linked at cysteine 190, was found to bind more weakly to actin filaments than uncross-linked tropomyosin. Cross-linking of tropomyosin can cause actin filaments nearly completely covered with tropomyosin to be uncovered almost completely. The critical monomer concentration of actin is not significantly changed by binding of cross-linked or uncross-linked tropomyosin to actin filaments. The binding curves were analyzed quantitatively, thereby taking into account the polar end-to-end contact of tropomyosin molecules bound by actin and the overlap of the seven subunit binding sites along the actin filament. Under the conditions of the experiment (80 mM KCl, 1 mM MgCl2, pH 7.5, 38-42 degrees C), the equilibrium constant for isolated binding of tropomyosin to actin filaments is in the range 1 x 10(3)-3 x 10(3) M-1. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for cross-linked and uncross-linked tropomyosin differ by a factor of only about two. Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyosin at cysteine 190.     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.     

Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.     

The effect of caldesmon on dynamic properties of F-actin alone and bound to heavy meromyosin and/or tropomyosin

The effect of caldesmon on the rotational dynamics of actin filaments alone or conjugated with heavy meromyosin and/or tropomyosin has been measured by the electron paramagnetic resonance (EPR) technique using a maleimide spin label rigidly bound to Cys374 of actin. The rotation of actin protomers in filaments and the angular distribution of spin probes on actin were determined by conventional EPR spectroscopy, while torsional motions within actin filaments were detected by saturation transfer EPR measurements. Binding of caldesmon to F-actin resulted in the reduction of torsional mobility of actin filaments. The maximum effect was produced at a ratio of about one molecule of caldesmon/seven actin protomers. Smooth muscle tropomyosin enhanced the effect of caldesmon, i.e. caused further slowing down of internal motions within actin filaments. Caldesmon increased the degree of order of spin labels on F-actin in macroscopically oriented pellets in the presence of tropomyosin but not in its absence. Computer analysis of the spectra revealed that caldesmon alone slightly changed the orientation of spin probes relative to the long axis of the filament. In the presence of tropomyosin this effect of caldesmon was potentiated and then approximately every twentieth protomer along the actin filament was affected. Caldesmon weakened the effect of heavy meromyosin both on the polarity of environment of the spin label attached to F-actin and on the degree of order of labels on actin in macroscopically oriented pellets. Whereas the former effect of caldesmon was independent of tropomyosin, the latter one was observed only in the absence of tropomyosin.     

Two general classes of cytoplasmic actin filaments in tissue culture cells: The role of tropomyosin

In the assembly of actin filaments that takes place during the spreading of a polulation of human lung cells, after trypsin detachment off the substratum and replating, tropomyosin exhibits a considrable lag in its association with the newly forming filament bundles; it begins to associate with them during the later stages of cell spreading as the actin filament bundles normally seen in interphase cells begin to organize. This lag is evident in a number of cell types that are spreading onto a substratum; it does not appear to be due to a selective degradation of this molecule during rounding up of the cells, since tropmyosin associates with the actin filament bundles after this lag even under conditions where the protein synthetic activity of the cell is inhibited to more than 95% by cycloheximide. The preferential binding of tropomyosin to fully assembled filament bundles but not to newly formed bundles of actin filaments suggests therefore the existence of two classes of action filaments: those that bind tropomyosin and those that do not. This selective localization of tropomyosin and those that do not. This selective localization of tropomyosin on actin filaments was further pursued by examining the localization of this molecule in membrane ruffles. The immunofluorescent results indicate that ruffling is an actin-filament-dependent, microtubule-independent phenomenon. Tropomyosin is absent from membrane ruffles under a variety of circumstances where ruffling is expressed and, more generally, from any other cellular activity where actin filaments are expected to be in a dynamic state of reorganization or are required to be in a flexible configuraion. It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype. The absence of tropomyosin from certain motile phenomena where actin filaments are involved indicates that these classes of actin filaments are regulated by cytoplasmic mechanisms distinct from that by which tropomyosin (and troponin) mediates contractility in skeletal mulscle; it opens the possibility that different types of actin filaments enagaged in different cellular motile phenomenon in tissue culture cells may be regulated by a host of coexisting regulatory mechanisms, some as yet undetermined.     

Purification of an 80 kDa Ca2+-dependent actin-modulating protein, which severs actin filaments, from bovine adrenal medulla

A protein with a molecular weight of 80 kDa, which binds Ca2+-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca2+, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca2+-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4 X 10(-7) M free Ca2+ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca2+. This result also indicates that the 80 kDa protein itself is a Ca2+-binding protein.     

Tropomyosin and gelsolin cooperate in controlling the microfilament system

Tropomyosin has been shown to cause annealing of gelsolin-capped actin filaments. Here we show that tropomyosin is highly efficient in transforming even the smallest gelsolin-actin complexes into long actin filaments. At low concentrations of tropomyosin, the effect of tropomyosin depends on the length of the actin oligomer, and the cooperative nature of the process is a direct indication that tropomyosin induces a conformational change in the gelsolin-actin complexes, altering the structure at the actin (+) end such that capping by gelsolin is abolished. At increased concentrations of tropomyosin, heterodimers, trimers, and tetramers are converted to actin filaments. In addition, evidence is presented demonstrating that gelsolin, once removed from the (+) end of the actin, can reassociate with the newly formed tropomyosin-decorated actin filaments. Interestingly, the binding of gelsolin to the tropomyosin-actin filament complexes saturates at 2 gelsolin molecules per 14 actin and 2 tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along the filament. These observations support the view that both tropomyosin and gelsolin are likely to have important functions in addition to those proposed earlier.     

Tropomyosin as a regulator of the sliding movement of actin filaments

We examined the capacity of tropomyosin molecules regulating the sliding movement of actin filaments on myosin molecules in the presence of ATP molecules to be hydrolyzed. For this objective, we prepared tropomyosin molecules modified to be a little bit stiffer compared to the intact ones by applying a fixed cross-linker between a pair of twisted tropomyosin monomers. The cross-linked tropomyosin molecules, when complexed with actin filaments, were found to inhibit the sliding movement of the filaments on myosin molecules even in the absence of calcium-regulated troponin molecules. It is then suggested that the mechanical flexibility of tropomyosin molecules may be instrumental to actualizing the proper functional regulation of the sliding movement of actin filaments.     

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.     

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin

Application of the myosin competition test (Lehman, W., and Szent-Gy?rgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

Do thin filaments of smooth muscle contain calponin? A new method for the preparation

A new method for the preparation of smooth muscle thin filaments which include calponin was established. We found that calponin readily separated from thin filaments in the presence of 10 mM ATP. By preventing thin filament extract from exposing to ATP, we obtained thin filaments which contained actin, tropomyosin, caldesmon and calponin in molar ratios of 7:0.9:0.6:0.7. We studied myosin Mg-ATPase activity by using the thin filaments in comparison with classical thin filaments prepared by the method of Marston and Smith, which contained the same amounts of caldesmon and tropomyosin as our thin filaments but lost almost all calponin. The presence of calponin reduced the Vmax value for thin filament-activated myosin Mg-ATPase activity by 33% without a significant change in Km value. These findings suggest that calponin inhibits myosin Mg-ATPase activity by modulation of a kinetic step as an integral component of smooth muscle thin filaments.     

Mutual dependence between tropomodulin and tropomyosin in the regulation of sarcomeric actin assembly in Caenorhabditis elegans striated muscle

Tropomodulin and tropomyosin are important components of sarcomeric thin filaments in striated muscles. Tropomyosin decorates the side of actin filaments and enhances tropomodulin capping at the pointed ends of the filaments. Their functional relationship has been extensively characterized in vitro, but in vivo and cellular studies in mammals are often complicated by the presence of functionally redundant isoforms. Here, we used the nematode Caenorhabditis elegans, which has a relatively simple composition of tropomodulin and tropomyosin genes, and demonstrated that tropomodulin (unc-94) and tropomyosin (lev-11) are mutually dependent on each other in their sarcomere localization and regulation of sarcomeric actin assembly. Mutation of tropomodulin caused sarcomere disorganization with formation of actin aggregates. However, the actin aggregation was suppressed when tropomyosin was depleted in the tropomodulin mutant. Tropomyosin was mislocalized to the actin aggregates in the tropomodulin mutants, while sarcomere localization of tropomodulin was lost when tropomyosin was depleted. These results indicate that tropomodulin and tropomyosin are interdependent in the regulation of organized sarcomeric assembly of actin filaments in vivo.     

Modulation of actin mechanics by caldesmon and tropomyosin

The ability of cells to sense and respond to physiological forces relies on the actin cytoskeleton, a dynamic structure that can directly convert forces into biochemical signals. Because of the association of muscle actin-binding proteins (ABPs) may affect F-actin and hence cytoskeleton mechanics, we investigated the effects of several ABPs on the mechanical properties of the actin filaments. The structural interactions between ABPs and helical actin filaments can vary between interstrand interactions that bridge azimuthally adjacent actin monomers between filament strands (i.e. by molecular stapling as proposed for caldesmon) or, intrastrand interactions that reinforce axially adjacent actin monomers along strands (i.e. as in the interaction of tropomyosin with actin). Here, we analyzed thermally driven fluctuations in actin's shape to measure the flexural rigidity of actin filaments with different ABPs bound. We show that the binding of phalloidin increases the persistence length of actin by 1.9-fold. Similarly, the intrastrand reinforcement by smooth and skeletal muscle tropomyosins increases the persistence length 1.5- and 2- fold respectively. We also show that the interstrand crosslinking by the C-terminal actin-binding fragment of caldesmon, H32K, increases persistence length by 1.6-fold. While still remaining bound to actin, phosphorylation of H32K by ERK abolishes the molecular staple (Foster et al. 2004. J Biol Chem 279;53387-53394) and reduces filament rigidity to that of actin with no ABPs bound. Lastly, we show that the effect of binding both smooth muscle tropomyosin and H32K is not additive. The combination of structural and mechanical studies on ABP-actin interactions will help provide information about the biophysical mechanism of force transduction in cells.     

Evidence for a direct but sequential binding of titin to tropomyosin and actin filaments

Titin is a giant molecule that spans half a sarcomere, establishing several specific bindings with both structural and contractile myofibrillar elements. It has been demonstrated that this giant protein plays a major role in striated muscle cell passive tension and contractile filament alignment. The in vitro interaction of titin with a new partner (tropomyosin) reported here is reinforced by our recent in vitro motility study using reconstituted Ca-regulated thin filaments, myosin and a native 800-kDa titin fragment. In the presence of the tropomyosin-troponin complex, the actin filament movement onto coated S1 is improved by the titin fragment. Here, we found that two purified native titin fragments of 150 and 800 kDa, covering respectively the N1-line and the N2-line/PEVK region in the I-band and known to contain actin-binding sites, directly bind tropomyosin in the absence of actin. We have also shown that binding of the 800-kDa fragment with filamentous actin inhibited the subsequent interaction of tropomyosin with actin, as judged by cosedimentation. However, this was not the case if the complex of actin and tropomyosin was formed before the addition of the 800-kDa fragment. We thus conclude that a sequential arrangement of contacts exists between parts of the titin I-band region, tropomyosin and actin in the thin filament.     

E93K charge reversal on actin perturbs steric regulation of thin filaments

Contraction in striated muscles is regulated by Ca2+-dependent movement of tropomyosin-troponin on thin filaments. Interactions of charged amino acid residues between the surfaces of tropomyosin and actin are believed to play an integral role in this steric mechanism by influencing the position of tropomyosin on the filaments. To investigate this possibility further, thin filaments were isolated from troponin-regulated, indirect flight muscles of Drosophila mutants that express actin with an amino acid charge reversal at residue 93 located at the interface between actin subdomains 1 and 2, in which a lysine residue is substituted for a glutamic acid. Electron microscopy and 3D helical reconstruction were employed to evaluate the structural effects of the mutation. In the absence of Ca2+, tropomyosin was in a position that blocked the myosin-binding sites on actin, as previously found with wild-type filaments. However, in the presence of Ca2+, tropomyosin position in the mutant filaments was much more variable than in the wild-type ones. In most cases (approximately 60%), tropomyosin remained in the blocking position despite the presence of Ca2+, failing to undergo a normal Ca2+-induced change in position. Thus, switching of a negative to a positive charge at position 93 on actin may stabilize negatively charged tropomyosin in the Ca2+-free state regardless of Ca2+ levels, an alteration that, in turn, is likely to interfere with steric regulation and consequently muscle activation. These results highlight the importance of actin's surface charges in determining the distribution of tropomyosin positions on thin filaments derived from troponin-regulated striated muscles.