Genetic engineering of a Ca²⁺ dependent chemical switch into the linear biomotor kinesin

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca²⁺-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca²⁺-dependency in ATPase activity at Ca²⁺ concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca²⁺-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca²⁺-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.