Actomyosin energy transduction and ortophosphate release from myosin

Eukaryotic cells use actin and myosin II to develop force and motion powered by ATP hydrolysis.

Release of the formed inorganic phosphate (Pi) from the myosin active site is central in energy transduction, being coupled to the force-generating structural change, the power-stroke. However, the temporal relationship between Pi-release and the power-stroke is unclear.

To reconcile conflicting findings in this regard we recently proposed a “three-step model” where Pi is assumed to pause at external sites on myosin, on its way from the active site to bulk solution. Whereas we found convincing evidence for the external sites we could not verify slow Pi-release required by the model.

Our purpose is to address this critical issue, using a unique single molecule transient biochemical kinetics approach.

The method will be applied to human cardiac myosin produced by a recently developed non-viral expression system with point mutations modifying Pi-binding at the external sites. We also propose to compare the three-step model to another model that has gained recent attention.

The work will elucidate long-term critical issues in actin-myosin energy transduction as well as enzymatic function beyond motor enzymes.

The results are of key importance in drug discovery with myosin as emerging drug target and the new single molecule transient kinetics method will be of great value by appreciably reduced requirement of precious proteins in fundamental studies and drug discovery.