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Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins

Sugawa, Mitsuhiro, Masaike, Tomoko, Mikami, Nagisa, Yamaguchi, Shin, Shibata, Keitaro, Saito, Kei, Fujii, Fumihiko, Toyoshima, Yoko Y., Nishizaka, Takayuki, Yajima, Junichiro
Biochemical and biophysical research communications 2018 v.504 no.4 pp. 709-714
H-transporting ATP synthase, adenosine triphosphate, chemical interactions, dissociation, fluorescence, fluorescent dyes, image analysis, microtubules, molecular motor proteins, nanorods, protein subunits
Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.