Controlling kinesin motor proteins in nanoengineered systems through a metal-binding on/off switch.

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.

Physical factors affecting kinesin-based transport of synthetic nanoparticle cargo.

Recently, kinesin biomolecular motors and microtubules filaments (MTs) were used to transport metal and semiconductor nanoparticles with the long-term goal of exploiting this active transport system to dynamically assemble nanostructured materials. In some cases, however, the presence of nanoparticle cargo on MTs was shown to inhibit transport by interfering with kinesin-MT interactions. The primary objectives of this work were (1) to determine what factors affect the ability of kinesin and MTs to transport nanoparticle cargo, and (2) to establish a functional parameter space in which kinesin and MTs can support unimpeded transport of nanoparticles and materials. Of the factors evaluated, nanoparticle density on a given MT was the most significant factor affecting kinesin-based transport of nanoparticles. The density of particles was controlled by limiting the number of available linkage sites (i.e., biotinylated tubulin), and/or the relative concentration of nanoparticles in solution. Nanoparticle size was also a significant factor affecting transport, and attributed to the ability of particles < 40 nm in diameter to bind to the "underside" of the MT, and block kinesin transport. Overall, a generalized method of assembling and transporting a range of nanoparticle cargo using kinesin and MTs was established.