title: High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
abstract: The rotary motor F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) is one of the best-studied of all molecular machines. F(1)-ATPase is the part of the enzyme F(1)F(O)-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F(1)-ATPase from Escherichia coli (EF(1)) is governed by the same mechanism as TF(1) under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF(1) molecules, we characterized the ATP-binding, catalytic and inhibited states of EF(1). We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF(1) than in TF(1), and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.
authors.affiliation: Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bilyard
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakanishi-Matsui
authors.forename: Bradley C
authors.initials: BC
authors.lastname: Steel
authors.forename: Teuta
authors.initials: T
authors.lastname: Pilizota
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Nord
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hosokawa
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Futai
authors.forename: Richard M
authors.initials: RM
authors.lastname: Berry
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000737:chemistry; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry*; D001665:Binding Sites; D004789:Enzyme Activation; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D057446:Hydrodynamics; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D061848:Optical Imaging / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D008274:Magnesium