Chemistry Reference
In-Depth Information
tweezers exert forces in the piconewton range on the particles. Biomolecules are too
small to be directly trapped by optical tweezers, so they are generally attached to an
optically-trapped bead. Microneedles or a bead trapped by a laser act as a spring that
expands in proportion to the applied force. Thus, the force and the displacement
caused by the biomolecules can bemeasured. The displacement of amicroneedle and
a bead has been determined with sub-nanometer accuracy, which is considerably
more sensitive than the diffraction limit of an optical measurement [15
-
19]. This
accuracy of displacement corresponds to the sub-piconewton accuracy in the force
measurements. Thus, the mechanical property of biomolecules can be determined
directly at the single-molecule level. Furthermore, combined with the single-mole-
cule imaging technique, simultaneous measurements of mechanical and chemical
reactions of single biomolecules are possible.
We have used SMD techniques to uncover the unique operation of a typical
molecular machine, a molecular motor, and studied how the
flexible and adaptive
nature speci
c to biological systems is generated by molecular machines operating
under the in
uence of strong thermal agitation.
2.2
Simultaneous Measurements of Individual ATP Hydrolysis Cycles and Mechanical
Events by a Myosin Motor
We have chosen the myosin motor as our model of a molecular machine. There are
several types of myosinmotors, all of whichmove along actin
filaments by converting
the chemical energy produced from ATP hydrolysis into mechanical energy to
generate cellular motility such as muscle contraction. Important functions of
proteins such as enzymatic action, energy transduction, molecular recognition and
self-assembly are integrated into the myosin motor [21]. Therefore, elucidating the
molecular mechanism of the myosin motor should provide the general principles
used by molecular machines. The most fundamental problem regarding the mecha-
nism of the myosin motor is its conversion of chemical energy into mechanical
energy. In order to solve this problem, we simultaneously measured individual ATP
hydrolysis cycles and single myosin motor mechanical events.
2.2.1
ATP Hydrolysis Cycles
Individual ATP hydrolysis cycles by a myosin motor were measured by the single-
molecule imaging technique TIRFM in combinationwith the
uorescent ATP analog
Cy3-ATP (Figure 2.2) [2, 4, 5]. It was con
rmed that Cy3-ATP was hydrolyzed by
myosin in the same way as ATP. The biochemical cycle rate of ATP hydrolysis
averaged over many events for individual myosin molecules was consistent with
that obtained by a conventional biochemical method using a suspension of myosin.
Cy3-ATP (or -ADP) free in solution does not produce clear
uorescent spots on a
detector, because of its rapid Brownianmotion. However, when Cy3-ATP (or -ADP) is
