Two Rotary Motors of ATP Synthase - Single Molecule Dynamics in Life Science - page 240

three subunits exhibited different open and closed conformations and bound

different nucleotides - AMPPNP (a non-hydrolyzing ATP analog), ADP, or none.
Later, an intermediate half-closed conformation that binds ADP and sulfate
(mimicking Pi) was also reported [7]. The catalytic sites are located at the interfaces
of the

b

subunits; however, most of the residues responsible for catalysis are
associated with the

a and b

subunits
exhibited almost the same conformations. Furthermore, the amino- and carboxyl-
terminal residues of the

b subunit. In contrast to the b subunits, the three a

subunit formed an asymmetric coiled-coil structure and
extended into the central cavity of the

g

a b 3 ring. These structural features strongly 3

supported Boyers proposal.
Once rotation of F 1 was considered to be plausible, many researchers attempted to
prove it. Although the results of chemical cross-linking exchange experiments and
fluorescence polarization measurements proved the relative motion of the

g subunit

against the three subunits and were consistent with the rotation hypothesis, these

results were not the direct evidence of unidirectional rotation [8, 9]. Finally, in 1997,
single-molecule imaging by Yoshida and Kinositas group directly demonstrated
unidirectional successive rotation driven by ATP hydrolysis [10]. The direction of
rotation was counterclockwise when viewed from F o , which is consistent with the
expectation from the crystal structure.

b

10.2
Rotation of ATP Synthase

10.2.1
Single-molecule Imaging of Rotation of F 1 Driven by ATP Hydrolysis

10.2.1.1 Strategy for Visualization of Rotation
The

subcomplex of F 1 suf ces for ATP hydrolysis, and direct observation of
rotation was first carried out using this subcomplex [10] (hereafter, unless speci ed
otherwise, the

a 3 b 3 g

subcomplex is referred to as F 1 ). To visualize rotation, Yoshida
and Kinositas group executed the following strategies (Figure 10.4A) [11]. First, rapid
lateral and rotational Brownianmotion of F 1 (10 nm in diameter) in aqueous solution
was suppressed by fixing it on a Ni-nitrilotriacetic acid-modi ed glass surface via a
polyhistidine tag introduced into the amino terminus of the

a b g 3 3

b subunit. Next, the

small turning radius (approximately 1 nm) of the central subunit was magni ed by

attaching a fluorescent-labeled actin lament - a large probe having a length of
several microns. Using these strategies, the rotation of F 1 from thermophilic Bacillus
PS3 was first visualized under an optical microscope. Instead of the actin filament,
latex beads have often been used as a rotational probe in recent experiments.

g

10.2.1.2 Large Torque Generated by F 1
Due to the hydrodynamic friction against the large probe, the rotational speed
depends on the length of the actin filaments (Figure 9.2B) [12]. Long filaments rotate
slowly, while short ones rotate rapidly. However, the torque generated by F 1 calculated