Rotation of a motor protein, F-1-ATPase, was demonstrated using a unique si
ngle-molecule observation system. This paper reviews what has been clarifie
d by this system and then focuses on the role of residues at the hinge regi
on 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 gam
ma subunit in the immobilised F-1-ATPase, thus settling a major controversy
regarding the rotary catalysis, The rotation of the gamma subunit was excl
usively 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 bin
ding of an ATP to the beta subunit, The mean work done by a 120 degrees ste
p 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 und
ergoes large opening-closing domain motion upon binding of AT(D)P, We ident
ified three hinge residues, beta His179, beta Gly180 and beta Gly181, whose
peptide bond dihedral angles are drastically changed during domain motion.
Simultaneous substitution of these residues with alanine resulted in nearl
y complete loss (99%) of ATPase activity. Single or double substitution of
the two Gly residues did not abolish the ATPase activity. However, reflecti
ng the shift of the equilibrium between the open and closed forms of the be
ta subunit, single substitution caused changes in the propensity to generat
e the kinetically trapped Mg-ADP inhibited form: Gly180Ala enhanced the pro
pensity and Gly181Ala abolished the propensity. In spite of these changes,
the mean rotational torque was not changed significantly for any of the mut
ants.