Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer.

Content Provider	Semantic Scholar
Author	Steigmiller, Stefan Zimmermann, Birgit Díez, Manuel Börsch, Michael Gräber, Peter
Copyright Year	2004
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.
Starting Page	79
Ending Page	85
Page Count	7
File Format	PDF HTM / HTML
DOI	10.1016/j.bioelechem.2003.08.008
PubMed reference number	15110252
Journal	Medline
Volume Number	63
Issue Number	1-2
Alternate Webpage(s)	http://www.atpase.uni-freiburg.de/dokumente/publikationen/2004_steigmiller_bioelectrochemistry.pdf
Alternate Webpage(s)	https://doi.org/10.1016/j.bioelechem.2003.08.008
Journal	Bioelectrochemistry
Language	English
Access Restriction	Open
Content Type	Text
Resource Type	Article