The myosin inhibitor blebbistatin stabilizes the super-relaxed state in skeletal muscle

Biophys J. 2014 Oct 7;107(7):1637-46. doi: 10.1016/j.bpj.2014.07.075.


The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Binding Sites
  • Heterocyclic Compounds, 4 or More Rings / pharmacology*
  • Kinetics
  • Muscle Relaxation / drug effects
  • Muscle, Skeletal / drug effects*
  • Muscle, Skeletal / metabolism*
  • Muscle, Skeletal / physiology
  • Myosins / antagonists & inhibitors*
  • Myosins / chemistry*
  • Myosins / metabolism
  • Protein Conformation / drug effects
  • Protein Stability / drug effects
  • Rabbits
  • Spin Labels


  • Heterocyclic Compounds, 4 or More Rings
  • Spin Labels
  • blebbistatin
  • Adenosine Triphosphate
  • Myosins