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Journal of Biochemistry Advance Access published online on October 30, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp173
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Kinesin-Calmodulin Fusion Protein as a Molecular Shuttle

Hideki Shishido1, Kiyoshi Nakazato1, Eisaku Katayama2, Shigeru Chaen3 and Shinsaku Maruta1

1Department of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan; 2Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan; 3Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, Setagaya-ku, Tokyo 156-8550, Japan

Author for correspondence: Prof. Shinsaku Maruta, Department of Bioinformatics, Graduate School of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, JAPAN Phone: +81-426-91-9443, Fax: +81-426-91-9312, E-mail: maruta{at}soka.ac.jp

Received September 22, 2009; Accepted September 28, 2009


  Abstract

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca2+-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca2+, while both dimeric and tetrameric configurations in the presence of Ca2+. Further, Ca2+-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca2+.

Key Words: calmodulin, fusion protein, kinesin, molecular shuttle, motor protein


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