Kinesin-1 is a force-generating enzyme, or motor protein, which converts the free energy of the gamma phosphate bond of ATP into mechanical work. This work is used to power the transport of intracellular organelles along microtubules. The development of in vitro motility assays for studying motors, combined with very sensitive displacement and force-measuring apparatus, has yielded new insights into the mechanical workings of motor proteins.
Kinesin-1 driven motility can be directly monitored in cell-free assays by observing, under the light microscope, the gliding of individual microtubules across glass surfaces coated with purified Kinesin-1. The polar microtubules always glide such that one end leads: this, together with assays using microtubules fluorescently marked at the minus ends, indicates that Kinesin-1 always moves toward the plus, or fast-growing, end of the microtubule. This polarity and the orientation of microtubules within cells show that Kinesin-1 is an anterograde motor. Interestingly, other kinesin proteins, whose motor domains have high sequence homology and nearly identical structure to Kinesin-1, move in the opposite direction.
By decreasing the density of Kinesin-1 on the surface, it has been shown that a single Kinesin-1 molecule suffices to move a microtubule through distances of several microns. Interestingly, a single motor can move a microtubule as quickly – at a rate of about 1 micron per second – as ten or one hundred motors. This processive movement and the high speed of even a single motor likely reflect the fact that in cells only a limited number of motors can bind to the surface of a small organelle, such as a synaptic vesicle, in order to move it along a microtubule.
A large body of structural, biochemical and biophysical evidence shows that Kinesin-1 has just one binding site per tubulin dimer, and that the motor takes 8-nm steps from one tubulin dimer to the adjacent one in a direction parallel to the protofilaments. Since the isolated motor domain of Kinesin-1 can hydrolyze up to 100 ATP molecules per second, it is likely that each step corresponds to one cycle of the ATPase reaction.
By tethering single molecules of Kinesin-1 to tiny tensiometers – a flexible glass rod or an optical trap – it is possible to measure the force exerted by a single Kinesin-1 molecule.
The trace in the adjacent figure shows a single Kinesin-1 molecule pulling on a microtubule attached to a glass rod. The elastic force acting on the motor via the glass fiber and microtubule can be calculated from the stiffness of the fiber, the displacement of the tip of the fiber to which the microtubule is attached, and the displacement of the tip of the fiber when the fiber is free. The average maximum displacement for the motor whose trace is shown corresponds to a maximum force of 5 pN (piconewtons).
As the opposing force is increased, the forward speed of Kinesin-1 decreases until, at a force of ~5 pN, the motor stalls. Given that mechanical work equals force times distance, these micromechanical experiments indicate that Kinesin-1 can do ~40 x 10-21 J of work per step, and if there is just one ATP consumed per step, then the efficiency is ~40%.
The current model for how motor proteins generate force is that the motor contains an elastic element, a spring, that becomes strained as a result of one of the transitions between chemical states: this strain is the force that the motor puts out, and the relief of this internal strain is the driving force for the forward movement.
Contributed by Joe Howard
- Howard, J. (2001) Mechanics of motor proteins and the cytoskeleton. Sinauer, Sunderland, MA.
- Howard, J. (1996) The movement of kinesin along microtubules. Ann. Rev. Physiol. 58: 703-729.
View links to recent articles on biophysics and biochemistry of the kinesin motor proteins from the MEDLINE database.
Learn how to build a moveable beam optical trap by Steve Block.
Learn more about optical tweezers by Alex Knight and Justin Molloy.
Learn about Total Internal Reflection Fluorescence Microscopy (TIRFM) by Alex Knight and Justin Molloy.
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Created 7 July 1996 20:00 GMT
Modified 29 August 2006 12:40 GMT