Abstract
It is believed that the loop L5 of kinesin is important region for motor function. Interestingly mitotic kinesin Eg5 has a several times longer L5 in comparison with other kinesins. It has been demonstrated that the L5 of Eg5 performed as a stabilizer for the Eg5-specific inhibitors (STLC, monastrol) complexes. Aim of our study is to control the function of Eg5 photo-reversibly using photochromic molecules incorporated into L5. Previously, we have prepared Eg5 mutants (E116C,E118C,T125C,W127C,D130C) which have a single cysteine residue in L5 in order to incorporate photochromic molecules. We also synthesized thiol reactive photochromic molecules 4-phenylazomaleinanil(PAM) and Iodoacetyl-spiropyran (IASP). PAM and IASP were incorporated into the mutants stoichiometrically. Some of the Eg5 mutants modified with PAM and IASP showed reversible alteration of ATPase activity upon ultraviolet (UV) and visible (VIS) light irradiations. In this study, we synthesized a novel thiol reactive photochromic molecules monoiodoacetyl-flugide(IAFG). Fulgimide performs photoreversible isomerzation between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Althought the modified Eg 5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photoreversible alteration upon UV and visible light irradiations. Alteration in the ATPase activity of W127C-IAFG in the presence of STLC upon UV and VIS light irradiations was also examined.
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