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. 2008 Oct;95(7):3322-9.
doi: 10.1529/biophysj.108.137067. Epub 2008 Jul 3.

Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State

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Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State

Fa-Qing Zhao et al. Biophys J. .
Free PMC article

Abstract

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 microM blebbistatin, disordering was at least 10 times slower. In the M.ADP state, myosin heads are also disordered. When blebbistatin was added to M.ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.

Figures

FIGURE 1
FIGURE 1
Effects of different nucleotides on helical ordering of myosin heads. (a, Top) tarantula thick filament negatively stained in relaxing conditions (M·ADP·Pi state), showing helical ordering of myosin heads. Ordering along helical tracks is most apparent when viewing along the filament axis at a glancing angle, and is confirmed by the appearance of layer lines at orders of 43.5 nm in averaged Fourier transform (bottom). (b) Filament negatively stained after removal of solution ATP and allowing time for ADP and Pi to dissociate (apo state). Heads are disordered and project away from the filament backbone (compare with the compact structure of a); the averaged Fourier transform lacks significant layer lines, confirming disorder. (c) Filament negatively stained in the presence of MgADP (M·ADP state). The filament image and averaged Fourier transform show disorder. Weak meridional reflections at the 6th order (7.2 nm) of the 43.5 nm repeat in (b) and (c) probably originate from the filament backbone, which is more exposed in the disordered filaments. Averaged transforms in ac are from 40 filament segments, all of the same length.
FIGURE 2
FIGURE 2
Blebbistatin maintains head ordering in the absence of free nucleotide. (ah) Time points at 0, 5, 60, and 90 min after treatment of thick filament suspension with apyrase (to remove ATP and ADP from solution) in the absence (a–d) and presence (e–h) of 100 μM blebbistatin. Average Fourier transforms of filaments confirm visual impression of order or disorder. Helical order is retained for at least 1 h in the presence of blebbistatin, but is lost in its absence. At 90 min (d and h) and 120 min (not shown), only the 43.5 and 14.5 nm reflections are prominent, showing that over long time periods helical order weakens even in the presence of blebbistatin.
FIGURE 3
FIGURE 3
Blebbistatin restores helical order to disordered M·ADP thick filaments. (a) In rigor solution containing 1 mM MgADP, heads are disordered and project away from the filament backbone. (b) After treatment with blebbistatin, helical ordering is restored.
FIGURE 4
FIGURE 4
Blebbistatin does not restore order to apo thick filaments. (a) Under rigor conditions, in which all ATP and ADP have been removed from solution, and all bound nucleotide has had time to dissociate, myosin heads are disordered and project away from the filament backbone. (b) Treatment with blebbistatin fails to restore helical ordering to such rigor filaments.
FIGURE 5
FIGURE 5
Inhibition of thick-thin filament interaction by blebbistatin. (a) Relaxed muscle homogenate containing thick and thin filaments showing very few interactions. Thick filaments reveal helical order by eye and in the averaged Fourier transform. (b) When a relaxed muscle homogenate containing thick and thin filaments is activated by Ca2+, numerous densely stained filament aggregates are formed, suggesting thick-thin filament interaction. These aggregates are difficult to image due to the density of staining. Smaller aggregates, like the one shown, reveal many filament interactions and a generally disordered array of heads. (c) When treated with Ca2+ in the presence of blebbistatin, thick and thin filaments show minimal interaction with each other, aggregates are absent, and the heads have a degree of helical ordering similar to that of relaxed filaments (compare the transform with that in a).

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