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- W2164073471 abstract "In the present study, we have demonstrated that the novel 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. We successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca2+ by the total internal reflection fluorescence microscopy. Reversible Ca2+dependent cargo-loading system was achieved by changing the Ca2+ concentration in the flow cell. K560-CaM was adsorbed onto the fluorescently unlabeled microtubule adhered on the glass surface in flow cell using non-hydrolyzable ATP analogue, AMP-PNP which stabilize the microtubule binding state of kinesin. Subsequently, Qdot-M13 was added in the presence of Ca2+ to be loaded on K560-CaM adsorbed on the microtubule. The fluorescence of Qdot-M13 loaded onto K560-CaM along a microtubule was observed after washing excess unbound Qdot-M13. When the Ca2+ solution in the flow cell was replaced by the Ca2+ free solution, Qdot-M13 was unloaded. Even after the several times alternate exchange of the solution in the flow cell with Ca2+ and EGTA solutions, the K560-CaM adsorbed onto microtubule by AMP-PNP showed stable Ca2+ dependent cargo loading. When excess ATP was added into the flow cell, K560-CaM-Qdot started to move along the microtubule. Interestingly, 145 seconds later, K560-CaM-Qdot accumulated at plus end of microtubule and showed fluorescent clumps as marker for plus end of microtubule." @default.
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- W2164073471 date "2010-01-01" @default.
- W2164073471 modified "2023-10-01" @default.
- W2164073471 title "Kinesin-Calmodulin Fusion Protein as a Molecular Shuttle and Marker for Plus End of Microtubule" @default.
- W2164073471 doi "https://doi.org/10.1016/j.bpj.2009.12.1959" @default.
- W2164073471 hasPublicationYear "2010" @default.
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