PMID:15110252
| Citation |
Steigmiller, S, Zimmermann, B, Diez, M, Börsch, M and Gräber, P (2004) Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer. Bioelectrochemistry 63:79-85 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1016/j.bioelechem.2003.08.008 |
| Keywords |
Adenosine Triphosphate/chemistry; Bacterial Proton-Translocating ATPases/analysis; Bacterial Proton-Translocating ATPases/chemistry; Bacterial Proton-Translocating ATPases/classification; Binding Sites; DNA-Binding Proteins/analysis; DNA-Binding Proteins/chemistry; Escherichia coli/enzymology; Fluorescence Resonance Energy Transfer/methods; Nucleotides/chemistry; Photons; Protein Binding |
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