Rotation of a motor protein, F(1)-ATPase, was demonstrated using a unique single-molecule observation system. This paper reviews what has been clarified by this system and then focuses on the role of residues at the hinge region of the beta subunit. We have visualised rotation of a single molecule of F(1)-ATPase by attaching a fluorescent actin filament to the top of the beta subunit in the immobilised F(1)-ATPase, thus settling a major controversy regarding the rotary catalysis. The rotation of the beta subunit was exclusively in one direction, as could be predicted by the crystal structure of bovine heart F(1)-ATPase. Rotation at low ATP concentrations revealed that one revolution consists of three 120 degrees steps, each fuelled by the binding of an ATP to the beta subunit. The mean work done by a 120 degrees step was approximately 80 pN nm, a value close to the free energy liberated by hydrolysis of one ATP molecule, implying nearly 100% efficiency of energy conversion. The torque is probably generated by the beta subunit, which undergoes large opening-closing domain motion upon binding of AT(D)P. We identified three hinge residues, betaHis179, betaGly180 and betaGly181, whose peptide bond dihedral angles are drastically changed during domain motion. Simultaneous substitution of these residues with alanine resulted in nearly complete loss (99%) of ATPase activity. Single or double substitution of the two Gly residues did not abolish the ATPase activity. However, reflecting the shift of the equilibrium between the open and closed forms of the beta subunit, single substitution caused changes in the propensity to generate the kinetically trapped Mg-ADP inhibited form: Gly180Ala enhanced the propensity and Gly181Ala abolished the propensity. In spite of these changes, the mean rotational torque was not changed significantly for any of the mutants.

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