Myosin's powerstroke transitions define atomic scale movement of cardiac thin filament tropomyosin.

Dynamic interactions between the myosin motor head on thick filaments and the actin molecular track on thin filaments drive the myosin-crossbridge cycle that powers muscle contraction. The process is initiated by Ca2+ and the opening of troponin-tropomyosin-blocked myosin-binding sites on actin. The ensuing recruitment of myosin heads and their transformation from pre-powerstroke to post-powerstroke conformation on actin produce the force required for contraction. Cryo-EM-based atomic models confirm that during this process, tropomyosin occupies three different average positions on actin. Tropomyosin pivoting on actin away from a TnI-imposed myosin-blocking position accounts for part of the Ca2+ activation observed. However, the structure of tropomyosin on thin filaments that follows pre-powerstroke myosin binding and its translocation during myosin's pre-powerstroke to post-powerstroke transition remains unresolved. Here, we approach this transition computationally in silico. We used the myosin helix-loop-helix motif as an anchor to dock models of pre-powerstroke cardiac myosin to the cleft between neighboring actin subunits along cardiac thin filaments. We then performed targeted molecular dynamics simulations of the transition between pre- and post-powerstroke conformations on actin in the presence of cardiac troponin-tropomyosin. These simulations show Arg 369 and Glu 370 on the tip of myosin Loop-4 encountering identically charged residues on tropomyosin. The charge repulsion between residues causes tropomyosin translocation across actin, thus accounting for the final regulatory step in the activation of the thin filament, and, in turn, facilitating myosin movement along the filament. We suggest that during muscle activity, myosin-induced tropomyosin movement is likely to result in unencumbered myosin head interactions on actin at low-energy cost.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents.

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7.

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin.

We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in ß-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.

Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation.

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

The molecular mechanism of muscle dysfunction associated with the R133W mutation in Tpm2.2.

Ghost muscle fibres reconstituted with myosin heads labeled with the fluorescent probe 1,5-IAEDANS were used for analysis of muscle fibre dysfunction associated with the R133W mutation in ß-tropomyosin (Tpm2.2). By using polarized microscopy, we showed that at high Ca2+ the R133W mutation in both αß-Tpm heterodimers and ßß-Tpm homodimers decreases the amount of the myosin heads strongly bound to F-actin and the number of switched-on actin monomers, with this effect being stronger for ßß-Tpm. This mutation also inhibits the shifting of the R133W-Tpm strands towards the open position and the efficiency of the cross-bridge work. At low Ca2+, the amount of the strongly bound myosin heads is lower for R133W-Tpms than for WT-Tpms which may contribute to a low myofilament Ca2+-sensitivity of the R133W-Tpms. It is concluded that freezing of the mutant αß- or ßß-Tpm close to the blocked position inhibits the strong binding of the cross-bridges and the switching on of actin monomers which may be the reason for muscle weakness associated with the R133W mutation in ß-tropomyosin. The use of reagents that activate myosin may be appropriate to restore muscle function in patients with the R133W mutation.

The molecular mechanisms of a high Ca2+-sensitivity and muscle weakness associated with the Ala155Thr substitution in Tpm3.12.

Substitution of Ala for Thr residue in 155th position in γ-tropomyosin (Tpm3.12) is associated with muscle weakness. To understand the mechanisms of this defect, we studied the Ca2+-sensitivity of thin filaments in solution and multistep changes in mobility and spatial arrangement of actin, Tpm, and myosin heads during the ATPase cycle in reconstituted muscle fibres, using the polarized fluorescence microscopy. It was shown that the Ala155Thr (A155T) mutation increased the Ca2+-sensitivity of the thin filaments in solution. In the absence of the myosin heads in the muscle fibres, the mutation did not alter the ability of troponin to switch the thin filaments on and off at high and low Ca2+, respectively. However, upon the binding of myosin heads to the thin filaments at low Ca2+, the mutant Tpm was found to be markedly closer to the open position, than the wild-type Tpm. In the presence of the mutant Tpm, switching on of actin monomers and formation of the strong-binding state of the myosin heads were observed at low Ca2+, which indicated a higher myofilament Ca2+-sensitivity. The mutation decreased the amount of myosin heads bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation. It is suggested that direct binding of myosin to Tpm may be one оf the reasons for muscle weakness associated with the A155T mutation. The use of reagents that decrease the Ca2+-sensitivity of the troponin complex may not be adequate to restore muscle function in patients with the A155T mutation.

The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies.

Point mutations in genes encoding isoforms of skeletal muscle tropomyosin may cause nemaline myopathy, cap myopathy (Cap), congenital fiber-type disproportion (CFTD), and distal arthrogryposis. The molecular mechanisms of muscle dysfunction in these diseases remain unclear. We studied the effect of the E173A, R90P, E150A, and A155T myopathy-causing substitutions in γ-tropomyosin (Tpm3.12) on the position of tropomyosin in thin filaments, and the conformational state of actin monomers and myosin heads at different stages of the ATPase cycle using polarized fluorescence microscopy. The E173A, R90P, and E150A mutations produced abnormally large displacement of tropomyosin to the inner domains of actin and an increase in the number of myosin heads in strong-binding state at low and high Ca2+, which is characteristic of CFTD. On the contrary, the A155T mutation caused a decrease in the amount of such heads at high Ca2+ which is typical for mutations associated with Cap. An increase in the number of the myosin heads in strong-binding state at low Ca2+ was observed for all mutations associated with high Ca2+-sensitivity. Comparison between the typical conformational changes in mutant proteins associated with different myopathies observed with α-, ß-, and γ-tropomyosins demonstrated the possibility of using such changes as tests for identifying the diseases.

The reason for the low Ca2+-sensitivity of thin filaments associated with the Glu41Lys mutation in the TPM2 gene is "freezing" of tropomyosin near the outer domain of actin and inhibition of actin monomer switching off during the ATPase cycle.

The E41K mutation in TPM2 gene encoding muscle regulatory protein beta-tropomyosin is associated with nemaline myopathy and cap disease. The mutation results in a reduced Ca2+-sensitivity of the thin filaments and in muscle weakness. To elucidate the structural basis of the reduced Ca2+-sensitivity of the thin filaments, we studied multistep changes in spatial arrangement of tropomyosin (Tpm), actin and myosin heads during the ATPase cycle in reconstituted fibers, using the polarized fluorescence microscopy. The E41K mutation inhibits troponin's ability to shift Tpm to the closed position at high Ca2+, thus restraining the transition of the thin filaments from the "off" to the "on" state. The mutation also inhibits the ability of S1 to shift Tpm to the open position, decreases the amount of the myosin heads bound strongly to actin at high Ca2+, but increases the number of such heads at low Ca2+. These changes may contribute to the low Ca2+-sensitivity and muscle weakness. As the mutation has no effect on troponin's ability to switch actin monomers on at high Ca2+ and inhibits their switching off at low Ca2+, the use of reagents that increase the Ca2+-sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

The primary cause of muscle disfunction associated with substitutions E240K and R244G in tropomyosin is aberrant behavior of tropomyosin and response of actin and myosin during ATPase cycle.

Using the polarized photometry technique we have studied the effects of two amino acid replacements, E240K and R244G, in tropomyosin (Tpm1.1) on the position of Tpm1.1 on troponin-free actin filaments and the spatial arrangement of actin monomers and myosin heads at various mimicked stages of the ATPase cycle in the ghost muscle fibres. E240 and R244 are located in the C-terminal, seventh actin-binding period, in f and b positions of the coiled-coil heptapeptide repeat. Actin, Tpm1.1, and myosin subfragment-1 (S1) were fluorescently labeled: 1.5-IAEDANS was attached to actin and S1, 5-IAF was bound to Tpm1.1. The labeled proteins were incorporated in the ghost muscle fibres and changes in polarized fluorescence during the ATPase cycle have been measured. It was found that during the ATPase cycle both mutant tropomyosins occupied a position close to the inner domain of actin. The relative amount of the myosin heads in the strongly-bound conformations and of the switched on actin monomers increased at mimicking different stages of the ATPase cycle. This might be one of the reasons for muscle dysfunction in congenital fibre type disproportion caused by the substitutions E240K and R244G in tropomyosin.

Molecular mechanisms of dysfunction of muscle fibres associated with Glu139 deletion in TPM2 gene.

Deletion of Glu139 in ß-tropomyosin caused by a point mutation in TPM2 gene is associated with cap myopathy characterized by high myofilament Ca2+-sensitivity and muscle weakness. To reveal the mechanism of these disorders at molecular level, mobility and spatial rearrangements of actin, tropomyosin and the myosin heads at different stages of actomyosin cycle in reconstituted single ghost fibres were investigated by polarized fluorescence microscopy. The mutation did not alter tropomyosin's affinity for actin but increased strongly the flexibility of tropomyosin and kept its strands near the inner domain of actin. The ability of troponin to switch actin monomers "on" and "off" at high and low Ca2+, respectively, was increased, and the movement of tropomyosin towards the blocked position at low Ca2+ was inhibited, presumably causing higher Ca2+-sensitivity. The mutation decreased also the amount of the myosin heads which bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation; this may contribute to contractures and muscle weakness.

The reason for a high Ca2+-sensitivity associated with Arg91Gly substitution in TPM2 gene is the abnormal behavior and high flexibility of tropomyosin during the ATPase cycle.

Substitution of Arg for Gly residue in 91th position in ß-tropomyosin caused by a point mutation in TPM2 gene is associated with distal arthrogryposis, characterized by a high Ca2+-sensitivity of myofilament and contracture syndrome. To understand the mechanisms of this defect, we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres, using the polarized fluorescence microscopy. The mutation was shown to markedly decrease the bending stiffness of ß-tropomyosin in the thin filaments. In the absence of the myosin heads the mutation did not alter the ability of troponin to shift tropomyosin to the blocked position and to switch actin monomers off at low Ca2+. During the ATPase cycle the movement of the mutant tropomyosin is restrained, it is located near the open position, which allows strong binding of the myosin heads to actin even at low Ca2+. This may be the reason for both high Ca2+-sensitivity and contractures associated with the Arg91Gly mutation. The use of reagents that decrease the Ca2+sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

Deviations in conformational rearrangements of thin filaments and myosin caused by the Ala155Thr substitution in hydrophobic core of tropomyosin.

Effects of the Ala155Thr substitution in hydrophobic core of tropomyosin Tpm1.1 on conformational rearrangements of the components of the contractile system (Tpm1.1, actin and myosin heads) were studied by polarized fluorimetry technique at different stages of the actomyosin ATPase cycle. The proteins were labelled by fluorescent probes and incorporated into ghost muscle fibres. The substitution violated the blocked and closed states of thin filaments stimulating abnormal displacement of tropomyosin to the inner domains of actin, switching actin on and increasing the relative number of the myosin heads in strong-binding state. Furthermore, the mutant tropomyosin disrupted the major function of troponin to alter the distribution of the different functional states of thin filaments. At low Ca2+ troponin did not effectively switch thin filament off and the myosin head lost the ability to drive the spatial arrangement of the mutant tropomyosin. The information about tropomyosin flexibility obtained from the fluorescent probes at Cys190 indicates that this tropomyosin is generally more rigid, that obviously prevents tropomyosin to bend and adopt the appropriate conformation required for proper regulation.

Molecular mechanisms of deregulation of the thin filament associated with the R167H and K168E substitutions in tropomyosin Tpm1.1.

Point mutations R167H and K168E in tropomyosin Tpm1.1 (TM) disturb Ca2+-dependent regulation of the actomyosin ATPase. To understand mechanisms of this defect we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres using the polarized fluorescence microscopy. It was found that both mutations disturbed the mode of troponin operation in the fibres. At high Ca2+, troponin increased the fraction of actin monomers that were in the "switched on" state, but both mutant tropomyosins were shifted toward the outer actin domains, which decreased the fraction of strongly bound myosin heads throughout the ATPase cycle. At low Ca2+, the R167H-TM was located close to the outer actin domains, which reduced the number of strongly-bound myosin heads. However, under these conditions troponin increased the number of actin monomers that were switched on. The K168E-TM was displaced far to the outer actin domains and troponin binding decreased the fraction of switched on actin monomers, but the proportion of the strongly bound myosin heads was abnormally high. Thus, the mutations differently disturbed transmission of conformational changes between troponin, tropomyosin and actin, which is essential for the Са2+-dependent regulation of the thin filament.

Abnormal movement of tropomyosin and response of myosin heads and actin during the ATPase cycle caused by the Arg167His, Arg167Gly and Lys168Glu mutations in TPM1 gene.

Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions.

Myopathy-causing Q147P TPM2 mutation shifts tropomyosin strands further towards the open position and increases the proportion of strong-binding cross-bridges during the ATPase cycle.

The molecular mechanisms of skeletal muscle dysfunction in congenital myopathies remain unclear. The present study examines the effect of a myopathy-causing mutation Q147P in ß-tropomyosin on the position of tropomyosin on troponin-free filaments and on the actin­myosin interaction at different stages of the ATP hydrolysis cycle using the technique of polarized fluorimetry. Wild-type and Q147P recombinant tropomyosins, actin, and myosin subfragment-1 were modified by 5-IAF, 1,5-IAEDANS or FITC-phalloidin, and 1,5-IAEDANS, respectively, and incorporated into single ghost muscle fibers, containing predominantly actin filaments which were free of troponin and tropomyosin. Despite its reduced affinity for actin in co-sedimentation assay, the Q147P mutant incorporates into the muscle fiber. However, compared to wild-type tropomyosin, it locates closer to the center of the actin filament. The mutant tropomyosin increases the proportion of the strong-binding myosin heads and disrupts the co-operation of actin and myosin heads during the ATPase cycle. These changes are likely to underlie the contractile abnormalities caused by this mutation.

Aberrant movement of ß-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle.

We have investigated the effect of the E41K, R91G, and E139del ß-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluorimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle.

The E117K mutation in ß-tropomyosin disturbs concerted conformational changes of actomyosin in muscle fibers.

The effect of the skeletal myopathy-causing E117K mutation in human ß-tropomyosin on actomyosin structure during the ATPase cycle was studied using fluorescent probes bound to actin subdomain 1 and the myosin head. Multistep changes in flexural rigidity of actin filament and in spatial arrangement of actin subdomain 1 and myosin SH1 helix in troponin-free ghost muscle fibers were revealed. During the ATPase cycle E117K tropomyosin inhibited the rotation of subdomain 1 by 46% and the tilt of the SH1 helix by 49% compared with wild-type. At strong-binding stages the proportion of strong binding sub-states in the actomyosin population is decreased by the mutation. At weak-binding stages abnormally high numbers of switched-on actin monomers were observed, thus indicating a disturbance in concerted conformational changes of actomyosin. These structural alterations are likely to underlie the contractile deficit observed with this mutation.

Gly126Arg substitution causes anomalous behaviour of α-skeletal and ß-smooth tropomyosins during the ATPase cycle.

To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle ß-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of ß-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and ß-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.

The nemaline myopathy-causing E117K mutation in ß-tropomyosin reduces thin filament activation.

The effect of the nemaline myopathy-causing E117K mutation in ß-tropomyosin (TM) on the structure and function of this regulatory protein was studied. The E117K mutant was found to have indistinguishable actin affinity compared with wild-type (WT) and similar secondary structure as measured by circular dichroism. However the E117K mutation significantly lowered maximum activation of actomyosin ATPase. To explain the molecular mechanism of impaired ATPase activation, WT and E117K TMs were covalently labeled at Cys-36 with 5-iodoacetimido-fluorescein and incorporated into ghost muscle fibers. The changes in the position and flexibility of tropomyosin strands on the thin filaments were observed at simulation of weak and strong binding states of actomyosin at high or low Ca(2+) by polarized fluorescence techniques. The E117K mutation was found to shift the tropomyosin strands towards the closed position and restrict the tropomyosin displacement during the transformation of actomyosin from weak to strong binding state thus leading to a reduction in thin filament activation.