title: Regulatory conformational changes of the ε subunit in single FRET-labeled FoF1-ATP synthase.
abstract: Subunit ε is an intrinsic regulator of the bacterial FoF1-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and β subunits, as revealed by the recent X-ray structure of the F1portion of theEscherichia colienzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthasein vitro. Labeling the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET) allowed monitoring of the conformation of ε in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in ε.
authors: Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
authors: Thomas M
authors: TM
authors: Duncan
authors: 3 Institute of Physics, Stuttgart University, Stuttgart, Germany.
authors: Monika G
authors: MG
authors: Düser
authors: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors: Thomas
authors: T
authors: Heitkamp
authors: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors: Duncan G G
authors: DG
authors: McMillan
authors: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors: Michael
authors: M
authors: Börsch
keywords: FoF1-ATP synthase; conformational change; single-molecule FRET; ε subunit