Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α‐tropomyosin molecule

Tropomyosin (Tm) is an α‐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 Ca2+ concentrations but also increases Ca2+ sensitivity of the actin–myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca2+‐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. We showed that substitution of non‐canonical residues G126 and D137 in the middle of tropomyosin (Tm) with canonical ones, Arg and Leu, and especially double mutation G126R/D137L, stabilized Tm and increased myosin ATPase and sliding velocity of regulated thin filaments in vitro. A model explaining the involvement of these Tm residues in the interaction with myosin on F‐actin is proposed.