title: Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer.
abstract: F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The enzyme has three catalytic nucleotide binding sites, one on each beta-subunit; three non-catalytic binding sites are located mainly on each alpha-subunit. In order to observe substrate binding to the enzyme, the H(+)-ATP synthase from Escherichia coli was labelled selectively with the fluorescence donor tetramethylrhodamine (TMR) at position T106C of the gamma-subunit. The labelled enzymes were incorporated into liposomes and catalysed proton-driven ATP synthesis. The substrate ATP-Alexa Fluor 647 was used as the fluorescence acceptor to perform intermolecular fluorescence resonance energy transfer (FRET). Single molecules are detected with a confocal set-up. When one ATP-Alexa Fluor 647 binds to the enzyme, FRET can be observed. Five stable states with different intermolecular FRET efficiencies were distinguished for enzyme-bound ATP-Alexa Fluor 647 indicating binding to different binding sites. Consecutive hydrolysis of excess ATP resulted in stepwise changes of the FRET efficiency. Thereby, gamma-subunit movement during catalysis was directly monitored with respect to the binding site with bound ATP-Alexa Fluor 647.
authors.affiliation: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 23a, D-79104 Freiburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Steigmiller
authors.forename: B
authors.initials: B
authors.lastname: Zimmermann
authors.forename: M
authors.initials: M
authors.lastname: Diez
authors.forename: M
authors.initials: M
authors.lastname: Börsch
authors.forename: P
authors.initials: P
authors.lastname: Gräber
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D025243:Bacterial Proton-Translocating ATPases / Q000032:analysis / Q000737:chemistry* / Q000145:classification; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry*; D017785:Photons; D011485:Protein Binding
chemical_list: D004268:DNA-Binding Proteins; D009711:Nucleotides; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases