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Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 μm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15–0.275 nm−1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.Accepted versio

Pseudo-phosphorylation of essential light chains affects the functioning of skeletal muscle myosin

The work aimed to investigate how the phosphorylation of the myosin essential light chain of fast skeletal myosin (LC1) affects the functional properties of the myosin molecule. Using mass-spectrometry, we revealed phosphorylated peptides of LC1 in myosin from different fast skeletal muscles. Mutations S193D and T65D that mimic natural phosphorylation of LC1 were produced, and their effects on functional properties of the entire myosin molecule and isolated myosin head (S1) were studied. We have shown that T65D mutation drastically decreased the sliding velocity of thin filaments in an in vitro motility assay and strongly increased the duration of actin-myosin interaction in optical trap experiments. These effects of T65D mutation in LC1 observed only with the whole myosin but not with S1 were prevented by double T65D/S193D mutation. The T65D and T65D/S193D mutations increased actin-activated ATPase activity of S1 and decreased ADP affinity for the actin-S1 complex. The results indicate that pseudo-phosphorylation of LC1 differently affects the properties of the whole myosin molecule and its isolated head. Also, the results show that phosphorylation of LC1 of skeletal myosin could be one more mechanism of regulation of actin-myosin interaction that needs further investigation

Functional and Structural Properties of Cytoplasmic Tropomyosin Isoforms Tpm1.8 and Tpm1.9

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex

Calculated intensity of the A1 actin layer line.

<p>The blue line corresponds to 40% of the total number of myosin heads bound to actin stereo-specifically by only one of the two heads of a myosin molecule. The black line corresponds to 60% of myosin heads stereo-specifically bound to actin: 20% of myosin molecules with one head only and the other 20% with both their heads. The purple and red lines correspond to non-stereo-specific attachment of the same 60% of heads with random uniform distribution of the azimuthal orientation angles within ranges of 60° or 80°, respectively. The vertical lines show the integration ranges for meridian (Mer), and the 10 and 11 row lines used for the experimental data shown in Fig. 3. Inset shows an actin filament (cyan, viewed along the filament axis) with a pair of stereo-specifically bound myosin heads (red heavy chains, magenta light chains). The same pair rotated by ±60° is shown in gray.</p

Meridional profiles of the meridional (top) and off-meridional intensities during isometric contraction (blue lines) and ramp stretch (red lines) in the 1^st series of experiments.

<p>The intensities were integrated in the reciprocal radii regions of ±0.018 nm⁻¹ (meridian), 0.018–0.035 nm⁻¹ (10 row line), and 0.035–0.06 nm⁻¹ (11 row line) after correction for the change in specimen volume in the x-ray beam and mirroring four quadrants of the x-ray diffraction pattern. Background was subtracted as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085739#pone.0085739-Bershitsky2" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085739#pone.0085739-Tsaturyan1" target="_blank">[14]</a>. The positions of some X-ray reflections of interest are marked.</p

Experimental protocol.

<p>Averaged records (from top to bottom): calculated temperature, motor position (in % of bundle length), tension and an example of the x-ray exposure framing (signal from a pin diode in a run of the experimental protocol). Noise on the pin diode signal, 30 ms from the beginning of the recording is caused by the high voltage heating pulse of the T-jump apparatus.</p

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin&ndash;myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A&gt;T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the &ldquo;open&rdquo; state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic