The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm(-1) for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s(-1) at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.

During the last 25 years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is presented that uses this information to simulate the motion of a bead carried by a kinesin molecule along a microtubule, with and without a weak optical trap present. A video sequence made from individual steps of the simulation gives a three-dimensional visual insight into these types of experiment at the molecular level.

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