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

Author:
Sugawa, Mitsuhiro, Masaike, Tomoko, Mikami, Nagisa, Yamaguchi, Shin, Shibata, Keitaro, Saito, Kei, Fujii, Fumihiko, Toyoshima, Yoko Y., Nishizaka, Takayuki, Yajima, Junichiro
Source:
Biochemical and biophysical research communications 2018 v.504 no.4 pp. 709-714
ISSN:
0006-291X
Subject:
H-transporting ATP synthase, adenosine triphosphate, chemical interactions, dissociation, fluorescence, fluorescent dyes, image analysis, microtubules, molecular motor proteins, nanorods, protein subunits
Abstract:
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.
Agid:
6159185