Kinesin-Calmodulin fusion protein as a molecular shuttle.
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. Ca(2+)-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 Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-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 Ca(2+).
Shishido
Hideki
H
Department of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
Nakazato
Kiyoshi
K
Katayama
Eisaku
E
Chaen
Shigeru
S
Maruta
Shinsaku
S
D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014461:Ultracentrifugation
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D016547:Kinesins