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Journal of Biochemistry Advance Access originally published online on October 17, 2007
Journal of Biochemistry 2007 142(6):691-698; doi:10.1093/jb/mvm183
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© 2007 The Japanese Biochemical Society.

Photocontrol of Kinesin ATPase Activity Using an Azobenzene Derivative

Masafumi D. Yamada, Yuki Nakajima, Hidekatsu Maeda and Shinsaku Maruta*

Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan

*To whom correspondence should be addressed. Tel: +81 (426) 91-9443, Fax: +81 (426) 91-9312, E-mail: shinsaku{at}t.soka.ac.jp

Received September 3, 2007; Accepted September 14, 2007


  Abstract

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis–trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV–VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.

Key Words: ATPase, kinesin, motor protein, photochromic molecule, photocontrol

Abbreviations: DTT, dithiothreitol; EGTA, ethylene glycol bis (b-aminoethyl ether)-N, N, N', N'-tetraacetic acid; KIF5a, mouse brain kinesin; LDH, lactate dehydrogenase; L5, loop 5; L11, loop 11; L12, loop 12; MT, microtubule; NADH, nicotinamide adenine dinucleotide; PK, pyruvate kinase; PEP, phosphoenolpyruvic acid; SDS, sodium dodecyl sulfate; SH, sulfhydryl; WT, wild-type


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