Effects of Cardiomyopathic Mutations on the Cytoplasmic Tropomyosin Isoform Tpm1.7

Figure 1. The exon composition of Tpm1.1 and Tpm1.7 isoforms of the human TPM1 gene. The positions of E40K and E54K mutations in exon 2b are marked by arrows.

Figure 2. Thermal stability of Tpm1.7 WT, Tpm1.7 E40K, and Tpm1.7 E54K measured by circular dichroism (CD). (a) Representative CD melting curves of Tpms recorded at 222 nm. The SDS-PAGE represents the purity of protein preparations. (b) Content of α-helical structures in Tpms. (c) The first derivative analysis of the CD melting curves presented in panel (a).

Figure 3. Temperature dependences of the excess heat capacity (ΔCp) of Tpm1.7 WT (a), Tpm1.7 E40K (b), and Tpm1.7 E54K (c) obtained by differential scanning calorimenty (DSC). Representative experimental DSC curves after subtracting instrumental and chemical baselines are shown by solid black lines, solid red lines are obtained by fitting the data to the non-two-state model, and grey lines represent the calorimetric domains (individual thermal transitions) obtained by the deconvolution analysis. The calculated values of Tm and ΔHcal are presented in Table 1.

Figure 4. The affinity of Tpm1.7 WT, Tpm1.7 E40K, and Tpm1.7 E54K for F-actin estimated by co-sedimentation at 100,000× g. The experiment was performed in a 30 mM HEPES-NaOH buffer (pH 7.3), with 200 mM NaCl. (a) The fractions of Tpm associated with F-actin were plotted against the concentration of unbound Tpm in the supernatant. (b) The representative SDS-PAGEs of cosedimentation of F-actin with Tpm1.7 WT, Tpm1.7 E40K, and Tpm1.7 E54K. The total concentrations of Tpm in samples are presented under the gels. The k50% (presented as mean ± SD) were 1.3 ± 0.3 µM for Tpm1.7 WT, 0.8 ± 0.1 µM for Tpm1.7 E40K, and 1.3 ± 0.4 µM for Tpm1.7 E54K. There is no statistically significant difference between the k50% for Tpm1.7 E40K or Tpm1.7 E54K, and Tpm1.7 WT. Statistical differences were assessed using the nonparametric Mann–Whitney U test at a significance level of p < 0.05.

Figure 5. F-actin severing in the presence of cofilin-1 (cof-1). Filament length distributions of (a) bare F-actin; (b) F-actin with cof-1; (c) F-actin with Tpm1.7; (d) F-actin with Tpm1.7 and cof-1; (e) F-actin with Tpm1.7 E40K; (f) F-actin with Tpm1.7 E40K and cof-1; (g) F-actin with Tpm1.7 E54K; (h) F-actin with Tpm1.7 E54K and cof-1. Fluorescence micrographs of TRITC-rhodamine phalloidin stained filaments used for quantification. The histograms for distribution of length of filaments were obtained from 1000 filaments for each sample. Bar is 10 µm. To determine the significance of the differences, Kolmogorov–Smirnov test was applied. All samples, except F-actin/Tpm1.7 E54K and F-actin/Tpm1.7 E54K/cof-1 pair, significantly differed from each other, with p ≤ 0.01.

Figure 6. The effect of cof-1 on the viscosity of the solutions of actin filaments decorated with Tpm1.7 WT, Tpm1.7 E40K, and Tpm1.7 E54K. All values are presented as the mean ± 95% confidence interval. (a) The ratio of the excess viscosity (ΔηX) of F-actin solutions with different Tpm1.7 variants (orange) and/or Tpm1.7 variants with cof-1 (green) to the excess viscosity of bare F-actin (ΔηF-actin). (b) The ratio of the excess viscosity of F-actin solutions with or without Tpm1.7 variants in the presence of cof-1 (ΔηX+cof-1) to the viscosity of the corresponding solutions in the absence of cof-1 (ΔηX). To determine the significance of the differences in the viscosities, Student’s t-test was used (n = 24). The viscosity of the samples F-actin ± cofilin, F-actin ± Tpm1.7, F-actin + cofilin ± Tpm, differed significantly (p < 0.05).