Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
11015567 | Biochemical and Biophysical Research Communications | 2018 | 6 Pages |
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.
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Authors
Mitsuhiro Sugawa, Tomoko Masaike, Nagisa Mikami, Shin Yamaguchi, Keitaro Shibata, Kei Saito, Fumihiko Fujii, Yoko Y. Toyoshima, Takayuki Nishizaka, Junichiro Yajima,