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

ABSTRACT Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electronmicroscopy tostudy theeffectsof blebbistatinon theorganizationof themyosinheadsonmuscle thick filaments. LossofADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100mMblebbistatin, disordering was at least 10 times slower. In theMADPstate,myosin heads are also disordered.Whenblebbistatinwas added toMADP thick filaments, helical orderingwas 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