Photo Regulation of Kinesin Motor Activity Utilizing Photochromic Dronpa-Loop11 Fusion Protein

Abstract

Kinesin is an ATP driven motor protein that moves along microtubules. Kinesin plays physiologically important roles of intracellular transport. Structure and energy transducing mechanism of kinesin have been well studied. Previous crystallographic studies revealed that kinesin has several unique loops in its motor domain. Loop11 and Loop12 are located in the microtubule binding region of kinesin. They were considered to bind to microtubule alternately during motility cycle with ATP hydrolysis. Previously we have demonstrated that incorporation of photochromic molecules into the microtubules binding site of kinesin enabled photo-reversible regulation of kinesin ATPase activity. In this study, we designed the photochromic protein “Dronpa derivative 145K-145N” fused with kinesin microtubule binding site, “Loop11” and “Loop12” in order to control kinesin motor activity photo-reversibly. First, the Dronpa 145K-L11-145N fusion protein was prepared. 145K-L11-145N showed reversible absorption spectral changes upon 400 nm and 500 nm light irradiation in a manner consistent with original Dronpa tandem dimer 145K-145N. Ionic strength dependent microtubules pelleting assay suggested that 145K-L11-145N associate with microtubules electrostatic interaction. Bright state of 145K-L11-145N inhibited conventional kinesin driven microtubules gliding in the in vitro motility assay. Moreover, Bright state of 145K-L11-145N inhibited microtubule dependent kinesin ATPase activity with the IC50 of 2 μM. We examined also dark state of 145K-L11-145N.