Engineering of a novel Ca²⁺-regulated kinesin molecular motor using a calmodulin dimer linker.
The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have "on-off" control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca(2+)-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca(2+)-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.
Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
Hideki
H
Shishido
Shinsaku
S
Maruta
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D009038:Motion; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization
D002147:Calmodulin; D016547:Kinesins; D002118:Calcium