Photo-Regulation of Kinesin ATPase Using Microtubules Modified with Photochromic Molecule

Abstract

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously we have introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin and succeeded to regulate the ATPase activity of kinesin reversibly by light irradiation. However, the regulation efficiency of the ATPase activity was not significant. It is well known that the contraction of skeletal muscle myosin is regulated by calcium binding to the rail protein actin. The regulating system achieves high efficient switching. Therefore, it is expected that introducing artificial switching system into the microtubules enables high efficient regulation of kinesin motor activity. In this study, we have done the experiments on incorporation of photochromic molecule into wild type microtubules and gliding assay of them under UV and VIS light irradiation. The gliding speed of the wild type microtubules modified with PAM or 3,3-dimethyl-6’-nitro −1-[2- [3-(3- pyrroline −2,5-dion) −1-yl] propanoyloxy]ethylindoline −2-spiro-2’(2H)- chromene (MASP) did not change. The results suggested that modification of the intrinsic reactive cysteine residues on the surface of microtubules does not affect motor activity of kinesin. Therefore, currently we are trying to prepare mutant microtubules that have an additional single reactive cysteine residue at the region near the kinesin binding site.