Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation

Figure 1. (A). Pedigree of the family T. The affected proband is shown with the closed symbol and is marked by the black arrow. The age of family members is shown at the last appointment (early 2023). CMP—cardiomyopathy, NMD—neuromuscular disease, PM—pacemaker. (B). Fragment of the Sanger electropherogram of the TPM1 gene. Visualization is given by the Chromas2.5 software (Technelysium Pty. Ltd., South Brisbane, Australia). (A—adenine, green peaks; C—cytosine, blue peaks; G—guanine, black peaks; T—thymine, red peaks). Heterozygous variant c.292G > A (p.E98K) is marked by the red arrow.

Figure 2. CD measurements of the thermal unfolding of E98K Tpm compared with that of WT Tpm. (A) The temperature dependencies of α-helix content were measured as the ellipticity at 222 nm at a constant heating rate of 1 °C/min. (B) First-derivative profiles for the data shown in panel (A).

Figure 3. Temperature dependences of the excess heat capacity (Cp) monitored by DSC and deconvolution analysis of the heat sorption curves for WT Tpm in the fully reduced state (A) and Tpm with mutation E98K (B,C) both in reduced (B) and in cross-linked (C) states. Solid lines represent the experimental curves after the subtraction of instrumental and chemical baselines, and dotted red lines represent the individual thermal transitions (calorimetric domains 1–4) obtained from fitting the non-two-state model [19] to the data.

Figure 4. The time-average occupancies of the backbone h-bonds for WT Tpm (black) and E98K Tpm (red). The average occupancy values in the two chains of the Tpm coiled coil are shown against the residue number. The vertical dashed line shows the position of the 98th residue.

Figure 5. The effect of the E98K substitution in the Tpm molecule on the Tpm affinity for F-actin estimated by the co-sedimentation assay (A) and on the thermal stability of the Tpm-actin complex determined by light scattering measurements (B).

Figure 6. Dependence of the sliding velocity of reconstructed thin filaments containing WT Tpm or E98K Tpm over cardiac myosin in the in vitro motility assay on pCa at myosin concentrations of 300 µg/mL (A) on myosin concentration at saturating calcium concentration (pCa 4) (B). Each data point represents the mean ± SD from three experiments. The data were fitted with the Hill equation. Characteristics of the pCa–velocity relationship are presented in Table 2.

Figure 7. A snapshot of the MD simulation trajectory. Actin monomers (sea green), WT Tpm (cornflower blue and purple), and Tn (Tn-I C-terminal fragment, green) are shown. During the simulation, Tpm Glu98 formed the h-bonds with Ser199 and Leu198 of TnI, while C-terminal residues 208–210 of TnI were anchored in the actin monomer. H-bonds are shown in red.