Incorporation of Photochromic Molecule into the Functional Loop L5 of Kinesin for the Purpose of Photo-Regulation

Abstract

ATP driven molecular motor kinesin has several unique loops, which may determine the characteristic properties of kinesin. L5 is one of the unique loops locates in the vicinity of ATP binding site. We have previously demonstrated that the point mutations in the loop drastically affect ATPase activity. Moreover, it seems that the plus-end directed kinesins have longer L5 loop than that of minus-end directed kinesins. Therefore, the loop may determine the directionality of kinesin. Rice plant kinesin K16 has much shorter L5 than that of conventional kinesin. We have prepared the K16 mutant Q101C and H102C that have a single reactive cysteine reside in the L5. SH group reactive photochromic molecule composed of azobenzene derivative, N- (4-phenylazophenyl) maleimide (PAM) was incorporated into the cysteine residue in L5 to induce conformational change of L5 by ultraviolet (UV) and visible (VIS) light irradiation. The kinesin modified by PAM slightly altered the ATPase activity by UV and VIS light irradiation reversibly. We have also focused on the mitotic kinesin Eg5 that has an unusually elongated L5. It is known that the L5 of Eg5 has an important role to stabilize sterically its specific inhibitors (monastrol, STLC) that bind to the pocket near the ATPase site. We designed Eg5 mutants, E116C, E118C, D130C, A133C and Y125C to introduce photochromic molecule into the L5 of Eg5. The Effect of photoisomerization of azobenzene derivatives incorporated into L5 upon UV-VIS light irradiation for the inhibition of ATPase activity by monastrol or STLC was studied.