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BIOPHYSICS
The ATP-waiting conformation of rotating F₁-ATPase revealed by single-pair fluorescence resonance energy transfer

Ryohei Yasuda ^* , Tomoko Masaike  , Kengo Adachi ^¶, Hiroyuki Noji ^||, Hiroyasu Itoh ^** and Kazuhiko Kinosita, Jr. ^¶

^*Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; ATP System Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, 5800-3 Nagatsuta, Yokohama 226-0026, Japan; Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8503, Japan; ^¶Center for Integrative Bioscience, Okazaki National Research Institutes, Higashiyama 5-1, Myodaiji, Okazaki 444-8585, Japan; ^||Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Tokyo 153-8505, Japan; and ^**Hamamatsu Photonics KK, Tokodai, Tsukuba 300-2635, Japan

Edited by Paul D. Boyer, University of California, Los Angeles, CA, and approved June 16, 2003 (received for review December 23, 2002)

F₁-ATPase is an ATP-driven rotary motor in which a rod-shaped subunit rotates inside a cylinder made of ₃₃ subunits. To elucidate the conformations of rotating F₁, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three s and an acceptor on in single F₁ molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of . In the ATP-waiting state, the FRET yields indicated a position 40° counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F₁, suggesting that the crystal structures mimic a metastable state before product release.

Abbreviations: FRET, fluorescence resonance energy transfer; MF₁, mitochondrial F₁; DCCD, dicyclohexylcarbodiimide.

To whom correspondence should be addressed. E-mail: yasuda@cshl.org.