Molecular Motors

Molecular motors are special enzymes that catalyze a chemical reaction such as hydrolysis of ATP, capture the free energy released by the reaction, and use it to perform a mechanical work such as muscle contraction. An example of such a motor enzyme is kinesin, which binds to subcellular organelles such as chromosomes and transports them through the cytoplasm by pulling them along microtubules. Optical trapping has been used to study the process of movement of kinosin from site to site on the microtubule lattice (Visscher et al., 1999). In this work, they used a molecular force clamp method utilizing a feedback-driven optical trap, capable of maintaining a constant load (force) on a single kinosin molecule. The kinosin molecule is composed of two heavy chains, each consisting of a force generating a globular domain head (hence double-headed), a long a-helical coil, and a tail portion that is a small globular C-terminal domain. Microtubules are cylinders comprised of parallel protofilaments that are linear polymers of a- and b-tubulin dimers.

Visscher et al. (1999) used a kinosin-coated silica bead (diameter ~0.5 mm) that trapped in a focused 1064-nm beam from a Nd:YVO4 laser using the objective lens of an inverted microscope. The trap position within the speci-

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Figure 14.19. Schematic of the experimental geometry, which includes position measurement for kinosin-driven bead movement and the corresponding optical trap displacements at an ATP concentration of 2mM. (Reproduced with permission from Visscher et al., 1999.)

Figure 14.19. Schematic of the experimental geometry, which includes position measurement for kinosin-driven bead movement and the corresponding optical trap displacements at an ATP concentration of 2mM. (Reproduced with permission from Visscher et al., 1999.)

men plane was specified using two digitally computer-controlled acousto-optic deflectors. The bead positions were determined by focusing a low-power He-Ne laser beam onto the optically trapped kinosin-coated silica bead and measuring the deflected light in a plane conjugate to the back focal plane of the microscope condenser, using a quadrant photodiode arrangement (Visscher et al., 1996).

The schematics of the experimental geometry and the results are shown in Figure 14.19. The results indicate kinosin stepping tightly coupled to ATP hydrolysis over a wide range of forces. A single hydrolysis produces kinosin movement along a microtubule with an 8-nm step that coincides with the a, b-tubulin dimer repeat unit. The progressive movement of kinosin was explained by a hand-over-hand mechanism in which one head remains bound to the microtubule, while the other detaches and moves forward.

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