Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7

Figure 1. Composition of the muscle fibers (lane 1), ghost fibers (lane 2), and ghost fibers reconstituted with the wild-type (WT, lane 3) or R90P-mutant (lane 4) tropomyosins (Tpm). The unbound Tpm was removed by exposing the fibers to the washing solution for 15 min. Here, 8–10 fibers were used to prepare the probe loaded per gel. The preparations of F-actin, as well as the mutant and WT Tpms, were loaded on lanes 5, 6, and 7, respectively. According to the calculation using Image Lab 6.0, ghost fibers are composed of F-actin by 86.0 ± 0.4%. The ratio of the R90P-mutant Tpm bound to actin is 19 ± 2% lower than that of the WT-Tpm. Designations: Tpm1.1, Tpm2.2, and Tpm3.12—tropomyosin isoforms; MyBP-C—myosin-binding protein C; LC1—myosin light chain 1; TnT and TnI—troponin subunits.

Figure 2. The effects of the increasing Ca2+ concentration on the actin-activated ATPase activity of S1. Ca2+ dependence was determined for fully regulated reconstituted thin filaments in the presence of WT-Tpm (blue circles) or R90P-Tpm (red triangles). The figure shows one of the typical graphs. The average values for pCa50 from 3–4 independent experiments are presented. Errors indicate ± SEM. Other conditions are as described in Materials and Methods.

Figure 3. A schematic view of the localization of 5-IAF, FITC-phalloidin, and 1,5-IAEDANS in Tpm, F-actin (Actin), and myosin subfragment-1 (S1), respectively, in the ghost muscle fibers.

Figure 4. The effect of troponin (TN) and Ca2+ on the values of ΦE (a) and ε (b) of the polarized fluorescence for 5-IAF linked with the WT-Tpm and R90P-Tpm, revealed in ghost fibers under conditions simulating the sequential steps of the actomyosin ATPase cycle. The values of ΦE were corrected in order to take into account the changes in the conformation of Tpm strands. The first and second entries from the left in each panel present the data obtained in the absence of S1. The data represent the mean values for 5–7 fibers for each experiment (see Materials and Methods). The ΦE and ε values in the absence and in the presence of nucleotides are significantly altered by TN and Ca2+ for both WT-Tpm and R90P-Tpm (p < 0.05). Mark “ns” indicates unreliable differences in the values of ε between the WT and mutant Tpms. Error bars indicate ± SEM. The values of N are close to zero.

Figure 5. Schematic explanation of the changes in the tropomyosin (Tpm) position, angular orientation of the actin monomer, and persistence lengths of the Tpm strands and actin filaments due to the R90P substitution in Tpm. The information was obtained from the calculation of the value for ΦE (the angle of orientation of the emission dipoles of the fluorescent probes bound to Tpm or actin) and of the value for ε (the bending stiffness; shown here as the alterations in the persistence length of the Tpm strands and actin filament drawn to scale). Ghost fibers were composed of actin and Tpm (a) or of actin, Tpm, and TN (b). The Tpm position and actin monomers orientation are affected by the R90P mutation and are typical for the shift of Tpm to the inner domains of actin, and for the switching of actin monomers on (clockwise rotation). The mutation decreases the persistence lengths of Tpm and actin. The R90P replacement reverses the changes in the persistence lengths of Tpm and actin that occur in response to the rising Ca2+ concentration (b). Designations: the changes in ΦE between the states at a high and a low Ca2+ are shown by numbers, and the direction of the rearrangements is depicted by…

Figure 6. Schematic view of the alterations in the Tpm position, conformational rearrangements in actin and S1, and the changes in the persistence lengths of the Tpm strands and actin filaments due to the R90P substitution in Tpm, as follows from the polarized fluorimetry data. The changes in the value for ΦE are interpreted as the shift of Tpm relative to the inner and outer domains of actin, the rotation of actin monomers in the actin filament and the azimuthal tilt of the myosin motor domain. The left and right panels show the transition of the Actin-Tpm-TN-S1 complex from the state in the presence of ADP at a high Ca2+ to the state in the presence of ATP at a high Ca2+ (a), and further to the state at a low Ca2+ concentration (b). The mutant Tpm strands have a lower persistence length and are shifted towards the inner domains of actin as compared with WT-Tpm. The persistence length changes are drawn to scale. The mutation increases the persistence length of actin, inhibits the actin monomer rotation, and causes the switching of actin monomers off at the transition from the state in the presence of ADP to that in the presence of ATP and the switching of actin monomers on at a low Ca2+.…

Figure 7. The effect of troponin and Ca2+ on the values of ΦE (a) and ε (b) of the polarized fluorescence for FITC-phalloidin-Actin, in the presence of the WT-Tpm or R90P-Tpm under conditions simulating the sequential steps of the myosin ATPase cycle. The first and second entries from the left in each panel present the data obtained in the absence of S1. Calculations of ΦE and ε values, preparation of the fibers, their composition, and the conditions of the experiments are described in Materials and Methods. The data represent the mean values for 8–10 fibers for each experimental condition. Both in the absence and in the presence of nucleotides, the ΦE and ε values for the WT-Tpm and R90P-Tpm are significantly altered by troponin and Ca2+ (p < 0.05). Index “ns” marks unreliable difference in the value for ε between the WT and mutant tropomyosins. Error bars indicate ± SEM. The values of N are close to 0.1.

Figure 8. The effect of troponin and Ca2+ on the values of ΦE (a), ε (b), and N (c) of the polarized fluorescence of N-(iodoacetaminoethyl)-1-naphthyl-amine-5-sulfonic acid (1,5-IAEDANS) bound to S1 (AEDANS-S1), in the presence of WT-Tpm or R90P-Tpm, revealed in ghost fibers under conditions simulating the sequential steps of the actomyosin ATPase cycle. The data represent the mean values for 5–7 fibers for each experimental condition. The ΦE, ε, and N values in the absence and in the presence of TN, Ca2+, and nucleotides are significantly altered by the R90P substitution in Tpm (p < 0.05). Statistically insignificant differences in the values of ΦE, ε, and N between the WT and mutant Tpms are marked by the symbol “ns”. Error bars indicate ± SEM.

Figure 9. The effect of 20 mM BDM (a) and 100 µM W7 (b) on the sensitivity of the thin filaments containing the R90P mutation in γ-Tpm to activating Ca2+ concentrations. Ca2+-dependence is determined for fully regulated reconstituted thin filaments. The actin-S1 ATPase is measured in the presence of R90P-Tpm and 20 mM BDM or 0.1 mM W7 at 25 °C. Each panel shows one of the typical graphs. pCa values are calculated from the data averaged from three experiments. Errors indicate ± SEM.

Figure 10. The effect of troponin and Ca2+ on the values of ΦE (a), ε (b), and N (c) of the polarized fluorescence of 1,5-IAEDANS bound to S1 (AEDANS-S1), in the absence and in the presence of 50 mM BDM, revealed in ghost fibers containing R90P-Tpm under conditions simulating the sequential steps of the actomyosin ATPase cycle. The experimental conditions and designations are described in Materials and Methods. The data represent the mean values for 5–7 fibers for each experimental condition. The ΦE, ε, and N values in the absence of TN and in the presence of Ca2+ and nucleotides are significantly altered by BDM (p < 0.05). The index “ns” indicates statistically insignificant differences in the values of ΦE, ε, and N between the mutant tropomyosin in the absence or presence of BDM. Error bars indicate ± SEM.

Figure 11. The effect of troponin and Ca2+ on the values of ΦE (a), ε (b), and N (c) of the polarized fluorescence of 1,5-IAEDANS bound to S1 (AEDANS-S1), in the absence and in the presence of 100 µM W7, revealed in ghost fibers, containing R90P-Tpm under conditions simulating the sequential steps of the actomyosin ATPase cycle. The experimental conditions and designations are described in Materials and Methods. The data represent the mean values for 5–7 fibers for each experimental condition. The ΦE, ε, and N values in the absence of troponin and in the presence of Ca2+ and nucleotides are significantly altered by W7 (p < 0.05). Index “ns” indicates statistically insignificant differences in the values for ΦE and N between the WT and mutant tropomyosin. Error bars indicate ± SEM.

Figure 12. The effect of BDM and W7 on the changes in the R90P-Tpm position, in actin–myosin conformational rearrangements, and in the persistence lengths of the Tpm strands and actin filaments. The value for ε is the bending stiffness. The upper and bottom panels show the transition of the actin-Tpm-TN-S1 complex from the state in the presence of ADP at a high Ca2+ to the state in the presence of ATP at a high Ca2+ (a) and further to the state at a low Ca2+ concentration (b). BDM stimulates the formation of strong myosin binding with actin in the presence of ADP and the formation of weak myosin binding with actin under the relaxing conditions. W7 does not affect the conformation of the myosin head at a high Ca2+, however, it allows for the weak binding of the myosin heads to actin in the presence of ATP at a low Ca2+. The mobility of the myosin heads is increased significantly in the presence of BDM and W7, as follows, from the changes in the number of disordered probes (parameter N). The changes in the actin rotation and in the Tpm position are derived from the data on the myosin conformational changes. BDM and W7 increases the persistence length of actin (drawn to scale), and decreases…