Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin

Figures

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  Figure 1. Blebbistatin probing the mechanism for phosphate release and force generation. (A) The molecular structure of S-(−) blebbistatin is shown. (B) The motor domain of dictyostelium myosin II (48) shows blebbistatin at its binding site and ADP at the active site. The further enlarged image to the right illustrates the proposed molecular model for phosphate release (inorganic phosphate molecule indicated by star). The figure was produced by PyMOL 2.0 using the deposited structure with Protein Data Bank: 1YV3 showing the myosin II motor domain with blebbistatin and ADP. Vanadate, magnesium and 1,2-ethanediol present in the deposited structure are excluded. (C) Two models (model 1 (29) and model 2 (19, 24)) were selected for final testing based on well-founded arguments from previous experimental and theoretical studies, as considered in detail in the Discussion. The schematic drawing illustrates the myosin motor domain either bound or not bound to actin (double helical structure) and with different lever arm (straight lines) positions. Hypothetical blebbistatin-affected transitions tested below are indicated by “B?.” The dashed rectangle encloses the myosin motor domain illustrated in…

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  Figure 2. Effects of blebbistatin on sliding velocities in the in vitro motility assay. (A) Concentration-response curve for effect of blebbistatin on sliding velocity at 130 mM ionic strength (1 mM MgATP) is shown using HMM with dephosphorylated regulatory light chains (dP-HMM). Each data point represents two to five flow cells and more than 20 filaments unless indicated in parentheses. Data from three different blebbistatin batches with mixed enantiomers are illustrated by different symbols, and two different dP-HMM preparations are illustrated by filled and open symbols, respectively. Two experiments using pure S-(−) blebbistatin are illustrated by open triangles. The sliding velocity in the control solution (0 μM blebbistatin) varied between 9.8 and 11.8 μm/s in the different experiments. (B) Blebbistatin effects on sliding velocity at different ionic strengths (1 mM MgATP) are shown. Fractional velocity at either 2 μM (circles) or 30 μM (squares) blebbistatin is shown, where velocity data are normalized to the velocity at the respective ionic strength in the absence of blebbistatin (control). In the absence of blebbistatin, the mean velocity was constant with changes in ionic…

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  Figure 3. Effects of different concentrations of blebbistatin on contractile parameters in experiments and simulations. (A) Force records show the effects of blebbistatin on isometric force and the force response to stretch of fast skinned muscle fibers of rabbit psoas muscle. Stretch ramps at velocity of 2.5 fiber lengths/s (2500 nm/half-sarcomere/s) were imposed between times of 4.8 and 4.95 s during otherwise steady-state isometric contraction. The records were obtained in the presence of 0–10 μM blebbistatin. The critical force during stretch, Fc, was estimated as indicated by crossing dashed lines. (B) Concentration-effect curves for experiments (filled symbols, full black lines) were compared to the model (blue) assuming blebbistatin-induced reduction of rate constant (kP+') for the transition from the AM∗'DP to the AM∗DP state from 1000 to 1.5 s−1. Parameter values are either assumed to apply at 25–30°C (Tables S1 and S2, open symbols, dashed lines) or 5°C (Table S3, dotted lines). (C) Concentration-effect curves for experiments (filled symbols, full lines) were compared to the model assuming reduction of ΔGon (Eq. S2) from 0.7 to −4 kBT (red). Other parameter values for 25–30°C…

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  Figure 4. Modeling of blebbistatin effects. (A) Kinetic scheme of model states shows myosin (M) free or bound to actin (A) with ATP (T), ADP (D), and/or inorganic phosphate (P, Pi) at the active site. Several states exist also in blebbistatin-bound form (blue; subscript “bleb”). Rate constants or rate functions are indicated by the generic form ki(x) and equilibrium constant by Ki. Only forward rates are indicated. (B) Free-energy diagrams illustrate the dependence on the variable x of the free energy of cross-bridges in the states without blebbistatin. Here, x is the distance between the nearest actin site and a reference point on the myosin filament with x = 0 nm at the free-energy minimum of the AM state. (C) Simulated velocity length plots in the in vitro motility assay with standard conditions (black) and 1 μM blebbistatin (colored circles) are shown, assuming changes in parameter values corresponding to different hypotheses for blebbistatin effects. Simulations for 1 μM blebbistatin assumed either one (filled symbols and full lines) or three (open symbols and dashed lines) myosin-binding sites for actin per 36 nm half-repeat of the actin filament. Conditions tested included the…