A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin

The molecular motor myosin is proposed to bind to actin and swing its light -chain binding region through a large angle to produce an similar to 10 nm step in motion coupled to changes in the nucleotide state at the active sit e. To date, however, direct dynamic measurements have largely failed to sho w changes of that magnitude. Here, we use a cysteine engineering approach t o create a high resolution, FRET-based sensor that reports a large, similar to 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence reso nance energy transfer measurements unexpectedly reveals two distinct prestr oke states. The measurements also show that bound Mg.ADP.P-i, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.