Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Figure 1. SDS-PAGE gels (right) and the time course of tryptic digestion (left) of A134L and E218L tropomyosin (Tpm) in comparison with WT Tpm. Trypsin:Tpm weight ratio was 1:150. Tpm concentration was 0.5 mg/mL; the reaction was carried out at 30 °C in 30 mM Hepes-Na buffer, 100 mM NaCl, 1mM MgCl2, pH 7.3.

Figure 2. Heat sorption curves and deconvolution analysis of DSC profiles for A134L/C190A (B) and E218L/C190A (C) mutants in comparison with control C190A Tpm (A). Solid lines represent the experimental curves after subtraction of chemical and instrumental baselines, dotted curves represent the calorimetric domains obtained from experimental data by analysis with the non-two-state model. The experiments were done at a constant heating rate of 1 K/min and protein concentration 2 mg/mL.

Figure 3. The affinity of Tpm species A134L/C190A, E218L/C190A, and control C190A Tpm for F-actin determined by co-sedimentation assay. The Tpm concentration of F-actin half-saturation (K50%) was 1.42 ± 0.18 μM for A134L/C190A, 1.96 ± 0.11 μM for E218L/C190A, and 1.68 ± 0.32 μM for C190A Tpm species. The K50% values are the average ± SD for three experiments. All experimental points were plotted.

Figure 4. Normalized temperature dependencies of light scattering obtained for F-actin complexes with A134L/C190A, E218L/C190A, and C190A Tpm species. The standard experiment was done using 20 μM F-actin stabilized by an equal molar amount of phalloidin and 10 μM of Tpm in 30 mM Hepes-Na buffer containing 100 mM NaCl, pH 7.3. The decrease in light scattering reflects the dissociation of Tpm from F-actin.

Figure 5. Ca2+-dependent sliding velocities of thin filaments containing E218L/C190A (A) and A134L/C190A (B) Tpm mutants in comparison with C190A Tpm obtained in the in vitro motility assay. Each data point represents the mean ± SD from three independent experiments. Data were fitted with the Hill equation as described in Material and Methods. The fitting parameters are shown in Table 2.

Figure 6. The dependence of the sliding velocity of thin filaments containing A134L/C190A, E218L/C190A, and C190A Tpm on the NEM-modified myosin concentration in the in vitro motility assay at pCa 4. The fraction of NEM-myosin required to stop the filament movement is a measure of relative force, which myosin can develop. Each data point represents the mean ± SD of three experiments. To stop the movement of thin filaments with C190A Tpm, A134L/C190A Tpm, and E218L/C190A Tpm, 32 ± 4%, 15 ± 2%, and 31 ± 3% of NEM-myosin were needed, respectively.

Figure 7. The result of the molecular dynamics (MD) simulation of the WT Tpm and the Ala134Leu and Glu218Leu Tpm mutants. Left: characteristic of the bending stiffness (see Materials and Methods section, less steep dependency corresponds to higher stiffness) (A); right: the occupancies of the backbone h-bonds (B).

Figure 8. Structural models illustrating possible involvement of the Glu218 Tpm residue in the interaction with myosin (A) and actin (B). The models were obtained by minor side-chain modifications using a rotamer library with UCSF Chimera [37] from the structure of the F-actin–Tp–myosin complex in the open state (A, [35], PDB entry 4A7H) and the F-actin-Tpm complex in the closed state (B, [36], 6KN8).