Chemistry Reference
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biochemical cycle of ATP hydrolysis. Myosin has two heads, each of which has ATP
and actin binding sites. Therefore, for simplicity, one-headed sub-fragments of
myosin were used. It was shown that one-headed sub-fragments of myosin can
move actin
filaments at the same velocity as two-headed myosin in a myosin-coated
surface assay [22].
2.2.2
Mechanical Events
The mechanical events occurring in a myosin motor were measured by optical
trapping nanometry. Two ends of an actin
filament, which had been visualized by
labeling with
fluorescent phalloidin [23], were attached to optically-trapped beads.
The suspended
filament was brought into contact with a single myosin head
xed on
the surface of a pedestal made on a glass surface. Individual mechanical events such
as displacement and force due to interactions between actin and the myosin head
were determined by measuring the displacement of a bead. The bead displacements
were determined with nanometer accuracy by projecting the bead onto a quadrant
photodiode and measuring its differential outputs [15, 17].
2.2.3
Simultaneous Measurements
Individual ATP hydrolysis cycles and mechanical events associated with a myosin
motor were simultaneously measured by combining the single molecule imaging
technique with optical trapping nanometry [24] (Figure 2.3a). Figure 2.3b shows time
traces of displacements (upper trace) and changes in the
fluorescence intensity from
Cy3
-
nucleotides (ATP or ADP) bound to the myosin, which have beenmeasured by a
photon counter (lower trace). When an ATP molecule binds to the myosin head, the
myosin head dissociates from the actin and the displacement decreases to zero. The
myosin head, dissociated from the actin by ATP, rebinds to the actin and generates
displacement and force. Thus, each displacement corresponds to a single ATPase
turnover.
2.3
Resolving the Process of a Displacement by Scanning Probe Nanometry
Next we attempted to resolve the process of a displacement occurring during a single
ATP hydrolysis cycle, in order to make clear how the myosin motor works using the
chemical energy provided by ATP [17, 25]. Since the displacements take place rapidly,
within 1ms, it was impossible to resolve the rising phase of a displacement by optical
trapping nanometry due to a poor signal to noise ratio. This is because the
displacements were measured by observing the movement of actin, not myosin,
and thus the compliance (1/stiffness) of the linkage between the optically trapped
beads and the actin
filament damped the signal to noise ratio. To overcome this
