Regulatory conformational changes of the epsilon subunit in single FRET-labeled FoF1-ATP synthase

Thomas M. Duncan; Monika G. Düser; Thomas Heitkamp; Duncan G. G. McMillan; Michael Börsch

doi:10.1117/12.2040463

28 February 2014 Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F₀F₁-ATP synthase

Thomas M. Duncan, Monika G. Düser, Thomas Heitkamp, Duncan G. G. McMillan, Michael Börsch

Author Affiliations +

Proceedings Volume 8948, Multiphoton Microscopy in the Biomedical Sciences XIV; 89481J (2014) https://doi.org/10.1117/12.2040463
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Subunit ε is an intrinsic regulator of the bacterial F_oF₁-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 F_oF₁-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, Thomas M. Duncan, Monika G. Düser, Monika G. Düser, Thomas Heitkamp, Thomas Heitkamp, Duncan G. G. McMillan, Duncan G. G. McMillan, Michael Börsch, 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); doi: 10.1117/12.2040463; https://doi.org/10.1117/12.2040463

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