Tropomyosin (Tm) is an a-helical coiled-coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled-coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca 2+ concentrations but also increases Ca 2+ sensitivity of the actin-myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca 2+ -regulated actin-myosin interaction can be accounted for not only by decreased flexibility of actin-bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.

Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T). The results of our structural studies show that all these mutations affect, although differently, the thermal stability of the C-terminal part of the Tpm molecule: mutations E180G and I172T destabilize this part of the molecule, whereas mutation E180V strongly stabilizes it. Moreover, various HCM-causing mutations have different and even opposite effects on the stability of the Tpm-actin complexes. Studies of reconstituted thin filaments in the in vitro motility assay have shown that those HCM-associated mutations that lie within the troponin T-binding region of Tpm similarly increase the Ca sensitivity of the sliding velocity of the filaments and impair their relaxation properties, causing a marked increase in the sliding velocity in the absence of Ca, while mutation I172T decreases the Ca sensitivity and has no influence on the sliding velocity under relaxing conditions. Finally, our data demonstrate that various HCM mutations can differently affect the structural and functional properties of Tpm and cause HCM by different molecular mechanisms.

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We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са2+ concentrations and the Са2+-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin–Tm–myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.