Stepwise rotation of the gamma-subunit of EF(0)F(1)-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer.
The EF(0)F(1)-ATP synthase mutants bQ64C and gamma T106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the gamma-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 gamma-subunit of EF(0)F(1).
Börsch
Michael
M
Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 23 a, 79104, Freiburg, Germany. m.borsch@physik.uni-stuttgart.de
Diez
Manuel
M
Zimmermann
Boris
B
Reuter
Rolf
R
Gräber
Peter
P
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D012399:Rotation; D013050:Spectrometry, Fluorescence / Q000379:methods
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008081:Liposomes; D021122:Protein Subunits; D012235:Rhodamines; C085321:cyanine dye 5; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases