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High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F-1-ATPase

Research output: Contribution to journalArticle

  • Thomas Bilyard
  • Mayumi Nakanishi-Matsui
  • Bradley C. Steel
  • Teuta Pilizota
  • Ashley L. Nord
  • Hiroyuki Hosokawa
  • Masamitsu Futai
  • Richard M. Berry

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Original languageEnglish
Article number20120023
JournalPhilosophical Transactions of the Royal Society B: Biological Sciences
Volume368
Issue number1611
DOIs
Publication statusPublished - 5 Feb 2013

Abstract

The rotary motor F-1-ATPase from the thermophilic Bacillus PS3 (TF1) is one of the best-studied of all molecular machines. F-1-ATPase is the part of the enzyme F1FO-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 (EF1) is governed by the same mechanism as TF1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the gamma-subunit of single surface-bound EF1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF1. We also show that the ATP-binding and catalytic states are separated by 35 +/- 3 degrees. At room temperature, chemical processes occur faster in EF1 than in TF1, 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.

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