Ca2+ Dependent Dimerization of Kinesin-CaM and Kinesin-M13 Fusion Proteins

Kinesin is known as a dimeric motor protein, which carries cellular cargoes along microtubules by hydrolyzing ATP. Calmodulin (CaM) is a calcium binding protein that participates in cellular regulatory processes. CaM undergoes a conformational change upon binding to calcium, which enables it to bind to specific proteins for a specific response. We have previously demonstrated that kinesin fused with CaM at the C-terminal binds reversibly to M13-Qdots in a calcium dependent manner. In this study, we tried to make the calcium dependent reversible dimerization of kinesin utilizing CaM- target peptide M13 binding system in order to control motility of kinesin. First we designed and prepared the cDNA of the truncated kinesin (355 amino acids) that does not form dimer. We prepared the cDNA encoding two kinesin chimeric proteins in which C-terminal of kinesin355 was fused with calmodulin (K355-CaM) and fused with M13-Cys (K355-M13-cys). The cDNAs of the kinesin chimeras were cloned into expression vector pET21a and transformed into E.coli BL21(DE3). The kinesin chimeras were successfully expressed and purified by Co-Chelate column. These kinesin chimeras showed normal ATPase activities. Furthermore, K355-CaM bound to M13-YFP in a calcium dependent manner. The calcium dependent interaction between K355-CaM and K355-M13-Cys was examined using Size-exclusion chromatography (SEC)-HPLC, whereas two chimeras did not bind in the absence of Ca2+. In vitro motility assay demonstrated that the dimerized chimera induced microtubule gliding in the presence of Ca2+.