Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps.
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.
Suzuki
Y
Y
Department of Life Sciences, School of Arts and Sciences, University of Tokyo, Japan.
Yasunaga
T
T
Ohkura
R
R
Wakabayashi
T
T
Sutoh
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D004023:Dictyostelium; D004735:Energy Transfer; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008958:Models, Molecular; D020409:Molecular Motor Proteins; D009218:Myosins / Q000378:metabolism / Q000502:physiology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins
D008164:Luminescent Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D009218:Myosins