Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State
Fa-Qing Zhao 1, Raul Padron 2 and Roger Craig 1*
1 University of Massachusetts Medical School
2 IVIC
* To whom correspondence should be addressed. E-mail: roger.craig{at}umassmed.edu.
Submitted on May 9, 2008
Revised on June 2, 2008
Accepted on 13 June 2008
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Abstract |
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Blebbistatin is a small molecule, high affinity, non-competitive 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 µM blebbistatin, disordering is at least ten 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 induces 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.
Key Words:
electron microscopy, muscle, relaxation, structure, thick filament
