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...