Coupling between phosphate release and force generation in muscle actomyosin.

Area of Science:

• Muscle physiology and biophysics
• Molecular mechanisms of muscle contraction
• Biochemistry of force generation

Background:

• Previous studies suggested phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibers is linked to force generation and reversible.
• The transition to the force-generating state and Pi release were hypothesized as separate but closely linked steps.
• Early myosin crystal structures suggested rigid coupling, but didn't explain myosin's affinity for actin and nucleotides.

Purpose of the Study:

• To investigate the role of phosphate release in muscle force generation.
• To reconcile structural data with kinetic experiments on actomyosin interactions.
• To elucidate the sequence of events in the myosin motor cycle.

Main Methods:

• Energetic, kinetic, and oxygen exchange experiments.
• Isometric optical clamp measurements of single actomyosin interactions.
• Analysis of newer myosin crystal forms and structural data.

Main Results:

• Phosphate (Pi) release from AM.ADP.Pi is reversible and linked to force generation in muscle fibers.
• Pi shortens actomyosin interactions only if they last long enough (20-40 ms) for Pi to dissociate.
• Newer structural data suggest actin-binding cleft closure opens switch I, decreasing nucleotide affinity.

Conclusions:

• The findings support a stepwise model of muscle contraction: myosin.ADP.Pi binds weakly, then strongly to actin, generating force.
• Phosphate dissociation may further increase force or sliding.
• Structural and kinetic data are consistent with a myosin motor cycle involving sequential binding, force generation, and Pi release.