Synthesis of Fluorescent-NTA and its Application to the Labeling of Photo-Controlled Kinesin Eg5

All members of the kinesin superfamily contain a structurally conserved loop L5 near the ATP-binding site. The length and the amino acids composition of L5 vary among the kinesin superfamily members. It is believed that L5 may be related to the enzymatic and motor function of kinesin. Interestingly, L5 of Eg5 is uniquely longer than that of other kinesins. Moreover, it was demonstrated that this loop undergoes conformational changes that are related to the nucleotide binding and neck linker docking. Previously we attempted to control Eg5 function photo-reversibly by incorporating photochromic molecules into the L5. We prepared Eg5 mutants, E116C, E118C, T125C, W127C, D130C, which have a single cysteine residue in L5 in order to incorporate photochromic molecules specifically. The Eg5 mutants modified with azobenzene and spiropyran derivatives showed photo-reversible alteration of microtubules dependent ATPase activities.In this study, we synthesized a novel thiol reactive photochromic molecule, monoiodoacetyl-flugide (IAFG). Fulgimide performs photo-reversible isomerization between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light, respectively. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Although the modified Eg5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photo-reversible alteration upon UV and visible light irradiations. We utilized fluorescent probe bound NTA-Ni to label the His-tagged Eg5 modified with photochromic molecules. According to the methods of Soh et al., Dansyl-NTA and other fluorescent-NTA were synthesized and conjugated with Ni2+. The Dansyl-NTA-Ni2+ bound to His tagged Eg5. Using the fluorescent NTA-Ni2+, interaction of photo-regulated Eg5 with microtubules was monitored.