Function Control of Kinesin using Functionality Loop L5 of Kinesin Midified Photochromic Molecule

Abstract

L5 is one of the unique loops locates in the vicinity of ATP binding site of kinesin. We have previously demonstrated that the point mutation at the L5 dramatically alters ATPase activity and interaction with microtubules. Therefore, the loop may be functional key region. The novel rice plant specific kinesin K16 has several unique enzymatic characteristics comparing with conventional kinesin. The most interesting property is that the ADP-free K16 motor domain is very stable, contrast to conventional kinesin that is very labile in ADP-free state. Recently, we have determined crystal structure of the novel rice kinesin K16 motor domain. The crystal structure revealed that the length of the loop L5 was much shorter than that of conventional kinesin. Therefore, the novel conformation of the L5 of K16 may determine the unique enzymatic properties. Therefore, it is expected that photo regulation of ATPase activity of K16 by incorporation of photochromic molecules in to L5. We prepared the K16 mutants that have elongated different length L5. The result of these mutants ATPace activity suggests that the length of L5 is important for rice kinesin k16 ATPase activity. Subsequently, we have prepared the K16 mutant Q101C C214S and L5 KIF5A H101C C214S that have a single reactive cysteine reside in the L5. Photochromic molecule of azobenzene derivative PAM or spiropyran derivative MA-SP was incorporated into the cysteine residue to induce conformational change of L5 by ultraviolet and visible light irradiation. The ATPase activities of kinesin mutants modified by PAM or MA-SP showed reversible alteration by ultra-violet and visible light irradiations.