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Crystal structure of dimeric kinesin (Adapted from Kozielski et al., Cell 1997)
Kinesin (seen above in a structure adapted from Kozielski et al., Cell 1997) is one of the world's tiniest motors. A single (dimeric) protein molecule -- consisting of only ~800 amino acids, or roughly ten thousand atoms -- is sufficient to move long distances along a microtubule, even against considerable load. By studying the motion of single molecules of kinesin, our aim is to understand how this tiny machine works. Kinesin proteins are essential for vesicle transport in neurons, and chromosome movement in dividing cells. By learning how kinesin works, we take an important step toward comprehending the dynamics of living cells.
Cartoon of a kinesin experiment. The kinesin walks along the microtubule towards its plus-end, while being subjected to a retarding force by the optical trap.
In order to track kinesin motion, we attach the molecules to microscopic beads. Kinesin itself is much too small to see in the optical microscope, so the beads serve as markers that can be tracked with very high precision (to 1 nm or better). The beads also act as "handles", through which we can apply force using an optical trap. Applying tension reduces Brownian motion of the bead, making it clear that kinesin moves in a stepwise fashion, in 8-nm increments (see below, and Svoboda et al., Nature 1993). For each 8-nm step, kinesin uses a single fuel molecule, hydrolyzing one ATP molecule into ADP and inorganic phosphate (Schnitzer and Block, Nature 1997).
Kinesin steps under constant forward load. The position of a kinesin motor in nanometers versus time at low ATP concentration. In this graph, the 8-nm steps that kinesin takes along the microtubule can readily be seen. This step size corresponds to the spacing of the tubulin dimers that make up the microtubule (Figure from Lang et al., Biophys. J. 2002).
Our optical traps have become very sophisticated, with automatic control over both the specimen stage and the optical trap (Lang et al, Biophys. J. 2002). Feedback control allows us to apply forces of constant magnitude and direction (any direction in the plane of the coverslip) to the kinesin motors while they move. Studying the effect of load on the speed and regularity of kinesin's stepping motion provides clues about how it is coupled to the biochemical events of ATP hydrolysis. For example, our most recent data suggests that at least 3 nm of each 8-nm step occurs all at once, during a single biochemical transition (Block et al., Proc. Natl. Acad. Sci. 2003). This "working stroke" must be well-aligned with the microtubule. The remaining 5 nm may occur simultaneously, or may occur after a slight (< 1 ms) delay. If there is a delay, it might be possible to resolve substeps in the motion by improving the time-resolution of our instrument. We also find evidence for smaller (< 1 nm) side-to-side motions occurring elsewhere in the biochemical cycle.
Read about this work in: Optical trap provides new insights into motor molecules -- nature's ultimate nanomachines in the Stanford Report.
3D cartoon of the effect of sideways loads. By using a 2D optical force clamp, we have been able to probe the multi-dimensional potential energy landscape that governs the kinetics of kinesin motion.
New tools will be required to determine exactly which biochemical steps correspond to physical motions. Toward this end, we are developing a combined instrument for optical trapping and single molecule fluorescence. By attaching fluorescent reporter molecules to kinesin or ATP, we hope to determine the timing of biochemical events relative to the mechanical steps.