High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.

Journal article

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: Bilyard, T; Nakanishi-Matsui, M; Steel, B; Pilizota, T; Nord, A

• Publication status: Published
• Journal: Philosophical transactions of the Royal Society of London. Series B, Biological sciences
• Volume: 368
• Issue: 1611
• Pages: 20120023
• Publication date: 2013-02-01
• DOI: 10.1098/rstb.2012.0023
• EISSN: 1471-2970
• ISSN: 0962-8436
• Language: English
• Keywords: Optical Imaging; Time Factors; Hydrolysis; Escherichia coli Proteins; Magnesium; Bacterial Proton-Translocating ATPases; Binding Sites; Actin Cytoskeleton; Temperature; Protein Conformation; Protein Binding; Adenosine Triphosphate; Hydrodynamics; Escherichia coli; Adenosine Diphosphate; Enzyme Activation; Enzyme Assays