title: Dissection of the angle of single fluorophore attached to the nucleotide in corkscrewing microtubules.
abstract: Direct dissection of the angles of single fluorophores under an optical microscope has been a challenging approach to study the dynamics of proteins in an aqueous solution. For angle quantifications of single substrates, however, there was only one report (Nishizaka et al., 2014) because of difficulties of construction of experimental systems with active proteins working at the single-molecule level. We here show precise estimation of orientation of single fluorescent nucleotides bound to single tubulins that comprise microtubule. When single-headed kinesins immobilized on a glass surface drive the sliding of microtubules, microtubules show corkscrewing with regular pitches (Yajima et al., 2005 & 2008). We found, by using a three-dimensional tracking microscope, that S8A mutant kinesin also showed precise corkscrewing with a 330-nm pitch, which is 13% longer than that of the wild type. The assay with the mutant was combined with a defocused imaging technique to visualize the rotational behavior of fluorescent nucleotide bound to corkscrewing microtubule. Notably, the defocused pattern of single TAMRA-GTP periodically changed, precisely correlating to its precession movement. The time course of the change in the fluorophore angle projected to the xy-plane enabled to estimate both the fluorophore orientation against microtubule axis and the precision of angle-determination of analyses system. The orientation showed main distribution with peaks at∼40°, 50° and 60°. To identify their molecular conformations, the rigorous docking simulations were performed using an atomic-level structure modeled by fitting x-ray crystal structures to the cryo-electron microscopy map. Among isomers, 2'-O-EDA-GDP labeled with 5- or 6-TAMRA were mainly specified as possible candidates as a substrate, which suggested the hydrolysis of TAMRA-GTP by tubulins.
authors.affiliation: Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Fujimura
authors.affiliation: Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Ikeguchi
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Adachi
authors.affiliation: Department of Life Sciences, Graduate School of Arts & Sciences, The University of Tokyo, Tokyo 153-8902, Japan.
authors.forename: Junichiro
authors.initials: J
authors.lastname: Yajima
authors.affiliation: Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan. Electronic address: takayuki.nishizaka@gakushuin.ac.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nishizaka
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020285:Cryoelectron Microscopy; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018715:Microscopy, Video / Q000379:methods; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D034421:Sus scrofa; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009711:Nucleotides; D014404:Tubulin; D016547:Kinesins
keywords: Defocus imaging; Docking simulation; Microtubule corkscrewing; Single-headed kinesin; TAMRA-EDA-GTP; TIRFM