In muscle, the
myosin head ('crossbridge') performs the 'working
stroke', in which
ATP is hydrolysed to generate the sliding of actin and
myosin filaments. The
myosin head consists of a globular motor domain and a long lever-arm domain. The 'lever-arm hypothesis' predicts that during the working
stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working
stroke in operation, we constructed fusion
proteins by connecting
Aequorea victoria green fluorescent protein and blue fluorescent
protein to the amino and carboxyl termini of the motor domain of
myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent
proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the
ATP hydrolysis cycle, and then swings back at the subsequent step in which
inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the
phosphate-release step may correspond to the working
stroke, and the swing at the isomerization step to the recovery
stroke.