Mechanics of single kinesin molecules measured by optical trapping nanometry

Abstract

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.