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Proceedings Paper

Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor
Author(s): Thomas Heitkamp; Gabriele Deckers-Hebestreit; Michael Börsch
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Paper Abstract

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

Paper Details

Date Published: 1 March 2016
PDF: 14 pages
Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 97140B (1 March 2016); doi: 10.1117/12.2209123
Show Author Affiliations
Thomas Heitkamp, Jena Univ. Hospital, Friedrich Schiller Univ. Jena (Germany)
Gabriele Deckers-Hebestreit, Univ. of Osnabrück (Germany)
Michael Börsch, Jena Univ. Hospital, Friedrich Schiller Univ. Jena (Germany)


Published in SPIE Proceedings Vol. 9714:
Single Molecule Spectroscopy and Superresolution Imaging IX
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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