title: Measurement of the conformational state of F(1)-ATPase by single-molecule rotation.
abstract: F(1)-ATPase (F(1)) is the water-soluble portion of ATP synthase and a rotary molecular motor in which the rotary shaft, the gamma subunit, rotates with 120 degrees steps against the alpha(3)beta(3) stator ring upon ATP hydrolysis. While the crystal structures of F(1) exhibit essentially one stable conformational state of F(1), single-molecule rotation studies revealed that there are two stable conformations of F(1) in each 120 degrees step: the ATP-binding dwell state and the catalytic dwell state. This chapter provides the experimental procedure for the determination of which catalytic state the crystal structures of F(1) represent, by the use of a cross-linking technique in the single-molecule rotation assay. The beta and gamma subunits are cross-linked through a disulfide bond between two cysteine residues genetically introduced at the positions where the beta and gamma subunits have a specific contact in the crystal structures of the ADP-bound form. In the single-molecule rotation assay, the cross-linked F(1) shows a pause at the catalytic dwell state that corresponds to the dwell angle in one turn where the beta subunit undergoes ATP hydrolysis. Thus, this experiment reveals not only that the crystal structure represents the catalytic dwell state but also that the ADP-bound beta subunit represents the catalytically active state. A protocol for inhibition of the wild-type F(1) with chemical inhibitors such as adenosine-5'-(beta,gamma-imino)-triphosphate (AMP-PNP) or/and N(3)(-) under crystallization conditions is also provided.
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Okuno
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Ikeguchi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008968:Molecular Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics
chemical_list: D006180:Proton-Translocating ATPases