Orientation and position of strongly and weakly bound heads in the kinesin-microtubule complex

Kinesin is a molecular motor that transports intracellular cargoes along microtubules. Kinesin moves in an asymmetric hand-over-hand mechanism as the two heads alternatively adopt the strongly and weakly bound states during turn-overs of one ATP per 8 nm step. While there is general agreement concerning the configuration (position and orientation) of the strongly bound head, the configuration of the weakly bound head is controversial. In this thesis the positional and orientational difference relative to the microtubule lattice between the strongly and weakly bound heads were investigated in detail using a single molecule fluorescence tracking experiment. A new kinesin construct was built with a highly photostable DNA-Cy3 on one head and a fluorescent bead on the neck. The positional difference between the strongly and weakly heads was measured by precisely tracking both the parallel and perpendicular displacement (relative to the microtubule long axis) of Cy3 associated with each 8 nm step the bead indicated. The orientational difference between the strongly and weakly bound heads was measured by putting Cy3 at different positions along the DNA 'lever arm'. The measurement results reveal both positional and orientational differences between the strongly and weakly bound heads. Based on these results a 3-dimensional structure model of the strongly and weakly bound heads in the kinesin-microtubule complex is proposed.