Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

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. laments to lose their helical ordering. In the presence of 100 mM blebbistatin, disordering was at least 10 times slower. In the M center dot ADP state, myosin heads are also disordered. When blebbistatin was added to M center dot ADP thick. laments, helical ordering was restored. However, blebbistatin did not improve the order of thick. laments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and. lament 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.