Development of Molecular Shuttle Regulated by External Stimulation Utilizing Kinesin ATP Driven Motor

Abstract

Kinesin is known as a dimeric motor protein, which carries cellular cargoes along microtubules by hydrolyzing ATP. Its structure and the molecular mechanism of energy transduction to move along microtubule are well studied. The kinesin has many possibility of application to the molecular machines. Previously, we introduced photochromic molecules into the functional key region of the kinesin as a photoswitch and tried to control the function of kinesin by ultraviolet (UV) and visible (VIS) light irradiations. The kinesin mutant S275C modified with thiol reactive azobenzene derivative exhibited photocontrolled ATPase activity correlating to photo isomerization. We have also demonstrated that kinesin fused with CaM at the C-terminal binds reversibly to M13-Qdots in a calcium dependent manner. As mentioned above, kinesin works as a nanodevice to be a component of bionanomachines. In this study, we tried to prepare the molecular shuttles regulated by external stimulation utilizing kinesin and other functional proteins. We designed the two chimeric kinesins. One is the kinesin motor domain (K355) fused with calmodulin (CaM) and the other is K355 fused with calmodulin target peptide M13. The chimeras were expressed by E. coli. and purified by Co-Chelate column. These kinesin chimeras showed normal ATPase activities. K355-CaM bound to K355-M13 in a calcium dependent manner and formed dimer. The dimer composed of the two chimeras induced exhibited motor activity to induce microtubule gliding. Moreover, we also tried to prepare the chimeric kinesins, K355-CaM-split GFP C-terminal domain and K355-split GFP N-terminal domain to be dimerized and fluorescently labelled spontaneously.