Photo-Control Of Kinesin Dimerization And Motor Activity Using Photochromic Molecules

Kinesin is an ATP driven dimeric motor protein carrying cellular cargoes along microtubules. The stalk region of kinesin is necessary for dimerization with coiled-coil interaction. The formation of dimer is essential for kinesin to perform processive movement along microtubules. The aim of this study is to control kinesin dimerization and its motor activity utilizing photochromic molecules. We synthesized three bifunctional azobenzene derivatives, azobenzene-dimaleimide (ABDM), water soluble azobenzene (WSA), and azobenzene-dinitoriro tri acetic acid (AB-NTA). All of the crosslinking spans are altered significantly by cis-trans photo-isomerization of azobenzene moiety upon ultraviolet and visible light irradiations reversibly. We have previously demonstrated that the two reactive cysteine residues SH1(707) and SH2(697) in α-helix of myosin, which region is believed to have a energy transducing role, were crosslinked by ABDM. And the cis-trans isomerization of the crosslinked ABDM induced disordered α-helix in the SH1-SH2 region resulting in global conformational change of myosin head. Therefore, it is expected that the two cysteine residues located in the coiled-coil stalk region of kinesin are also crosslinked. In this study, we prepared two kinesin mutants. One of them is the dimer kinesin having two reactive cysteine residues at its coiled-coil stalk region and the other is the monomer kinesin having a His-tag at its stalk region. ABDM and WSA crosslink two cysteine residues located on the surface of kinesin inter- or intra-molecularly. On the other hand, AB-NTA crosslinks inter-molecularly His-tagged kinesin. Photo-reversible alteration in the microtubule dependent ATPase activities and motor activity of cross-linked kinesins were examined.