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

Abstract

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.

Directed attachment of antibodies to kinesin‐powered molecular shuttles

“…2) compared with the untreated control, which suggests that SHTH-functionalization of the MT surface did not disrupt the motility characteristics of the MTs over the 6-day period. While the velocity of certain treatments were significantly higher than the control, the observed velocities across all treatments and days were within the normal variance for kinesin motors (Coy et al, 1999;Greene et al, 2008;Hess et al, 2004), and likely explained by normal variation within assays (e.g., temperature) and kinesin preparations. Overall, these data are also consistent with negligible effects on velocity based on modification of MTs with small molecules such as biotin (Korten and Diez, 2008).…”

Soft, Biological and Composite Nanomaterials

“…CINT has developed the capability for engineering kinesin (and other biomolecular motors) to address issues of both control (e.g., on/off switch) and stability, as well as to incorporate "chemical handles" on motors to enable integration. [44][45][46][47][48] In addition, a number of functionalization schemes to enable the attachment of cargo (e.g., NPs to microtubules) have been achieved and have been used to establish a means of actively assembling nanocomposite structures [49][50][51][52] . [53][54][55][56] For example, Bachand and Paxton used this transport system to actively assemble networks of block copolymer nanotubes.…”

Engineering tubulin: microtubule functionalization approaches for nanoscale device applications

“…One approach to date involves sequestering and releasing ATP to achieve an “on/off” switch (Hess et al 2001; Yokokawa et al 2003; Tucker et al 2008). Greene and coworkers engineered a zinc binding site into conventional kinesin and showed that increasing the zinc concentration inhibited motility (Greene et al 2008). Tomishige and Vale engineered cysteines into the neck linker domain and showed that the motor can be regulated by reversible disulfide linking (Tomishige and Vale 2000).…”