Single-molecule studies of kinesin biophysics using DNA-kinesin chimeras

Kinesins are a family of motor proteins that have a wide range of functions including transport of molecular cargoes within cells, assembly and disassembly of networks of rigid microtubules that are used to organize cell contents, and the separation of chromosomes when cells divide. Conventional kinesin has two identical heads that can each bind to a microtubule. It 'walks' hand-over-hand, which requires coordination - it is important that at least one head remains bound at all times, otherwise the motor is likely to lose contact with its track. The mechanisms by which the motor coordinates its heads and generates force are still not clear. It is important to understand them, because defective kinesins can cause disease and because kinesin and microtubules are drug targets in some cancer therapies. We are studying the mechanism of kinesin by creating artificial motors incorporating elements of kinesin attached to frameworks assembled from short strands of DNA. This allows us to alter the number of kinesin heads that are working together, or the length or the elasticity of the link between them, in ways that are not possible when working with the natural protein alone. Our aims are to improve the precision of measurements of single kinesin molecules walking, to study how kinesin's heads are coordinated, and to investigate how many kinesins work together to create larger forces.

We will address key problems in the kinesin mechanism by using DNA self-assembly to produce assemblies of motors containing a defined number of kinesin units (single heads or dimers) arranged with a defined attitude, spacing and mechanical coupling. No other system that provides this degree of architectural control currently exists. We have developed a system for attaching kinesin molecular motors to double stranded DNA using Zn-finger DNA recognition domains fused to the C-terminus of kinesin. We can programme the number, spacing and relative orientation of binding sites into the DNA template. We have assembled teams of kinesin dimers and single kinesin heads and have used gliding assays, in which motor teams are anchored to a cover slip and fluorescently labelled microtubules move over them, to measure gliding velocity for teams of different compositions over a wide range of temperatures. We have also demonstrated the inverted assay in which motor teams labelled with single fluorescent quantum dots are observed to move along immobilized microtubules. We will use an optical trap to measure force-velocity and force-displacement curves for single motors and motor teams. We will use self-assembled DNA templates to achieve precise control of the number of motors attached to the trapped bead and the geometry with which they interact with a microtubule. These structures will be designed to improve the mechanical properties of the linkage between the trapped bead and motor, increasing the temporal and spatial resolution of the trap. Velocity and run length of fluorescently labelled motor teams on immobilized microtubules, and microtubule gliding velocities on surface-bound teams, will also be measured. We will thus be able to investigate the basic mechanisms by which kinesin motors generate force, how individual kinesin heads are coordinated and how motors cooperate - including the structural requirements and limits for effective function within multimotor teams.