Stepwise rotation of the γ‐subunit of EF0F1‐ATP synthase observed by intramolecular single‐molecule fluorescence resonance energy transfer 1

The EF0F1‐ATP synthase mutants bQ64C and γT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b‐subunit and with a cyanine (Cy5) at the γ‐subunit. After reconstitution into liposomes, these double‐labelled enzymes catalyzed ATP synthesis at a rate of 33 s−1. Fluorescence of TMR and Cy5 was measured with a confocal set‐up for single‐molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET‐states were identified, indicating stepwise rotation of the γ‐subunit of EF0F1.

[1]  U. Hahn,et al.  A general method for rapid site-directed mutagenesis using the polymerase chain reaction. , 1990, Gene.

[2]  R. H. Fillingame,et al.  Arrangement of the Multicopy H+-translocating Subunit c in the Membrane Sector of the Escherichia coliF1F0 ATP Synthase* , 1998, The Journal of Biological Chemistry.

[3]  Helmut Grubmüller,et al.  Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase , 2002, Nature Structural Biology.

[4]  M Dahan,et al.  Ratiometric single-molecule studies of freely diffusing biomolecules. , 2001, Annual review of physical chemistry.

[5]  M. Müller,et al.  Inter‐subunit rotation and elastic power transmission in F0F1‐ATPase , 2001, FEBS letters.

[6]  P. Boyer.  ATP synthase--past and future. , 1998, Biochimica et biophysica acta.

[7]  U. Lücken,et al.  Molecular architecture of Escherichia coli F1 adenosinetriphosphatase. , 1989, Biochemistry.

[8]  M Dahan,et al.  Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Robert Aggeler,et al.  Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor. , 2002, Trends in biochemical sciences.

[10]  R. Aggeler,et al.  Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex. The gamma-epsilon subunits are essentially randomly distributed relative to the alpha3beta3delta domain in the intact complex. , 1997, The Journal of biological chemistry.

[11]  Jan Pieter Abrahams,et al.  Structure at 2.8 Â resolution of F1-ATPase from bovine heart mitochondria , 1994, Nature.

[12]  Stefan Fischer,et al.  The Activity of the ATP Synthase from Escherichia coli Is Regulated by the Transmembrane Proton Motive Force* , 2000, The Journal of Biological Chemistry.

[13]  A. E. Senior,et al.  Integration of F1 and the membrane sector of the proton-ATPase of Escherichia coli. Role of subunit "b" (uncF protein). , 1983, The Journal of biological chemistry.

[14]  Masasuke Yoshida,et al.  ATP synthase — a marvellous rotary engine of the cell , 2001, Nature Reviews Molecular Cell Biology.

[15]  R. Aggeler,et al.  Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis. , 1992, The Journal of biological chemistry.

[16]  Peter Gräber,et al.  Comparison of ΔpH‐ and Δφ‐driven ATP synthesis catalyzed by the H+‐ATPases from Escherichia coli or chloroplasts reconstituted into liposomes , 1999 .

[17]  V. V. Bulygin,et al.  Rotation of subunits during catalysis by Escherichia coli F1-ATPase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Capaldi,et al.  Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function. , 1984, Biochemistry.

[19]  T. Yanagida,et al.  Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation. , 1999, Science.

[20]  R. Aggeler,et al.  Disulfide Bond Formation between the COOH-terminal Domain of the Subunits and the and Subunits of the Escherichia coli F-ATPase , 1995, The Journal of Biological Chemistry.

[21]  Kazuhiko Kinosita,et al.  Direct observation of the rotation of F1-ATPase , 1997, Nature.

[22]  Kazuhiko Kinosita,et al.  F1-ATPase Is a Highly Efficient Molecular Motor that Rotates with Discrete 120° Steps , 1998, Cell.

[23]  C. Seidel,et al.  Conformational changes of the H+‐ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence , 1998, FEBS letters.

[24]  R. Capaldi,et al.  ATP hydrolysis-driven structural changes in the gamma-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues. , 1994, The Journal of biological chemistry.

[25]  Hiroyasu Itoh,et al.  Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase , 2001, Nature.

[26]  J. Weber,et al.  The molecular mechanism of ATP synthesis by F1F0-ATP synthase. , 2002, Biochimica et biophysica acta.

[27]  M. Wilce,et al.  Structure of the γ–ɛ complex of ATP synthase , 2000, Nature Structural Biology.

[28]  W. Junge,et al.  Intersubunit rotation in active F-ATPase , 1996, Nature.