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28 February 2014 Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase
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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 F1 portion of the Escherichia coli enzyme. 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 synthase in 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.
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Thomas M. Duncan, Monika G. Düser, Thomas Heitkamp, Duncan G. G. McMillan, and Michael Börsch "Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase", Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 89481J (28 February 2014);

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