The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies

Figure 1. SDS-PAGE of muscle fibers (rabbit psoas) and tropomyosins used in the assays. Verification of myosin and Tpm–TN extraction from muscle fibers during the preparation of ghost fibers was carried out for each experiment (a,b). (4–11, 15–18) Analysis of the binding of wild-type (WT) and mutant Tpms to actin in ghost fibers shows that E173A, R90P, E150A, and A155T substitutions do not affect the binding of Tpm to actin. Seven to ten fibers each 15–20 mm in length were loaded per gel. Designations: Myosin HC—myosin heavy chains; LC1, LC3, and RLC—myosin light chains; MyBP-C—myosin-binding protein C; TnT, TnI, and TnC—troponin subunits.

Figure 2. The values for the angle ΦE (a,c) and for the bending stiffness ε (b,d) determined on the basis of analysis of the polarized fluorescence from FITC-phalloidin bound to F-actin (A*, FITC-actin) and from 5-IAF bound to Cys190 of wild-type tropomyosin (WTTpm*, AF-WTTpm) and the values of the parameters ΦE (a,c) and N (e,f) of the polarized fluorescence from 1,5-IAEDANS bound to Cys707 of myosin SH1 helix (S1*, AEDANS-S1) revealed in ghost fibers at simulating various steps of the ATPase cycle at high (a,b,e) and low Ca2+ (c,d,f). ΦE is the angle between the emission dipole of the probe and the filament axis; ε is the bending stiffness and N is a number of disorderly oriented fluorophores. Calculations of the ΦE, ε, and N values, the preparation of the fibers, their composition, and the conditions of the experiments are described in Section 4. The red and blue columns show data for the “pure” FITC-actin filaments and those decorated by Tpm-TN, respectively. Brown and grey columns show data for the filaments decorated by AF-Tpm-TN. Green columns show data for the thin filaments decorated by AEDANS-S1. The data represent means of 8–10 ghost fibers for each experimental condition. ΦE,…

Figure 3. The presumed relationship between changes in the polarized fluorescence parameter ΦE and spatial rearrangements of the proteins in the complex F-actin–Tpm–TN at high (A) and low (B) Ca2+, and in the complex F-actin–Tpm–TN–S1 at simulation of strong (E) and weak (F) binding of S1 to F-actin. Fluorescent probes are denoted as yellow stars. (C,D) Changes in the ΦE values (in degrees) for FITC-actin, AF-Tpm, and AEDANS-S1 and corresponding spatial rearrangements of actin monomers, Tpm, and the myosin heads induced by transition between ON and OFF states of thin filament and between weak- and strong-binding states of S1 with actin.

Figure 4. The effect of the E173A, R90P, E150A, and A155T mutations in γ-Tpm on the values of the corrected ΦE angle (a) and the bending stiffness ε (b) of the polarized fluorescence from 5-IAF bound to Cys190 of tropomyosin revealed in actin filaments. (c) Estimated relationship between the changes in the value of ΦE for AF-Tpm and FITC-Actin and spatial rearrangements of Tpm and actin monomer in the presence of the mutant Tpms with respect to the control. Red numbers indicate those changes that lead to the activation of thin filaments. The conditions of the measurements and abbreviations are as in Figure 2. The data represent means of 8–10 ghost fibers for each experimental condition. The corrected ΦE and ε values are significantly altered by point mutations in Tpm (p < 0.05). Error bars indicate ± SEM.

Figure 5. The effect of the Tpm mutations E173A (a), R90P (b), E150A (c) and A155T (d) on actin-S1 ATPase activity. Ca2+-dependence for the ATPase activity for fully regulated reconstituted thin filaments is shown. The error bars indicate the average SD data for each sample were fitted to the Hill equation using GraphPad Prism 5 software, and the average pCa50 values were obtained from the fitted curves. The experimental conditions are described in Section 4.

Figure 6. The effect of the E173A, R90P, E150A, and A155T mutations in γ-Tpm on the changes in the polarized fluorescence parameters observed at transition between the mimicked stages of the ATPase cycle at low Ca2+. The changes in the values for the angle ΦE (a,c,e) from AF-Tpm, FITC-Actin, and AEDANS-S1, respectively, the changes in the values for the bending stiffness ε (b,d) determined on the basis of analysis of the polarized fluorescence from 5-IAF and FITC-phalloidin, respectively, and the changes in the values for N (f) from 1,5-IAEDANS are shown. The data represent differences in the values of ΦE (ΔΦE), ε (Δε), and N (ΔN) between WTTpm and each of the mutant Tpms. Yellow, green, blue, and lilac columns show data for the filaments decorated with the E173A, R90P, E150A, and A155T mutant Tpms, respectively. They represent means of 8–10 ghost fibers for each experimental condition (see Section 4). The values of the ΔΦE, Δε and ΔN for the E173A, R90P, E150A, and A155T mutant Tpms are significantly different from the corresponding values for WTTpm both in the absence and in the presence of nucleotides (p < 0.05). Error bars indicate ± SEM. The asterisks were used to indicate…

Figure 7. Schematic representation of rearrangements of Tpm, actin monomers and S1 induced by the Tpm mutations E173A, R90P, E150A, and A155T in different structural–functional states of thin filament. All the changes are depicted in comparison with the WT Tpm (dotted lines). The composition of the fiber: actin–Tpm–TN at low Ca2+ in Blocked state; actin–Tpm–TN at high Ca2+ in Closed state; S1 bound strongly to actin-Tpm–TN at high Ca2+ in M–state; S1 bound weakly to actin–Tpm–TN at low Ca2+ in MATP-state. TN is not shown for simplicity. Tpm is shown in different colors depending on the mutation; strongly and weakly bound S1 is shown in various shades of green, depending on strength of its binding to F-actin. The direction of the presumed actin monomer rotation, axial shift of Tpm strands and translocation of S1 induced by the mutation are shown by arrows. The digits indicate changes in the ΦE values (in degrees) for the fluorescent probe attached to each protein (see Section 4).

Figure 8. The effect of the E173A, R90P, E150A, and A155T mutations in γ-Tpm on the changes in the polarized fluorescence parameters observed at transition between the mimicked stages of the ATPase cycle at high Ca2+. The changes in the values for the angle ΦE (a,c,e) from AF-Tpm, FITC-actin, and AEDANS-S1, respectively, the changes in the values for the bending stiffness ε (b,d) determined on the basis of analysis of the polarized fluorescence from 5-IAF and FITC-phalloidin, respectively, and the changes in the values for N (f) from 1,5-IAEDANS are shown. The data represent differences in the values of ΦE (ΔΦE), ε (Δε), and N (ΔN) between WTTpm and each of the mutant Tpms. Yellow, green, blue, and lilac columns show data for the filaments decorated with the E173A, R90P, E150A, and A155T mutant Tpms, respectively. They represent means of 8–10 ghost fibers for each experimental condition (see Section 4). The values of the ΔΦE, Δε and ΔN for the E173A, R90P, E150A, and A155T mutant Tpms are significantly different from the corresponding values for WTTpm both in the absence and in the presence of nucleotides (p < 0.05). Error bars indicate ± SEM. The asterisks were used to indicate…

Figure 9. The presumed relationship between changes in the polarized fluorescence parameter ΦE and spatial rearrangements of the proteins in the complex F-actin–Tpm–TN–S1 at high Ca2+ at simulation of strong and weak binding of S1 to F-actin. Upper panel: a schematic explanation of the proposed conformational changes of the myosin heads at different skeletal myopathies—CFTD, Cap, and NM. The tilt of the myosin head is based on the values of ΦE parameter for S1 modified by 1,5-IAEDANS. ΦE is the angle between the emission dipole of the probe and the thin filament axis. Strong binding of the myosin head to actin is characterized by a decrease in the ΦE parameter and the tilt of the myosin head towards the muscle fiber axis. The increase in the ΦE parameter indicates a decrease in the population of strongly bound myosin heads. Depending on the disease, an increase or a decrease in the population of the myosin heads strongly associated with actin can be observed. Lower panel: A scheme representing the rearrangements of tropomyosin (Tpm), actin monomers, and myosin subfragment-1 (S1) at weak-to-strong transition at different skeletal myopathies. TN is not shown for simplicity. Tpm is shown in…

Figure 10. Diagrams explaining the calculation of the polarized fluorescence parameters. (A): The laboratory frame is taken as OXZY, where OY is the direction of propagation of incident and emitted light. OZ is the axis of the muscle fiber. θ1/2 is the angle between the actin filament axis (OW) and the fiber axis (OZ) at parallel (‖) or perpendicular (⊥) direction of excitation light. (B): The molecular frame for the thin filament is taken as OUVW, where OW is the filament axis. The direction of the absorption dipole A→ and emission dipole E→ for a fluorescent probe are defined by the angles ΦA and ΦE, respectively. γ and δ are the angles between A→ and E→, and A′→, and E′→ dipoles, respectively.