Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

Thomas Heitkamp; Gabriele Deckers-Hebestreit; Michael Börsch

doi:10.1117/12.2209123

1 March 2016 Observing single F_oF₁-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

Thomas Heitkamp, Gabriele Deckers-Hebestreit, Michael Börsch

Proceedings Volume 9714, Single Molecule Spectroscopy and Superresolution Imaging IX; 97140B (2016) https://doi.org/10.1117/12.2209123
Event: SPIE BiOS, 2016, San Francisco, California, United States

Abstract

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme F_oF₁-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 F_o 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 F_oF₁-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.

Conference Presentation

Citation Download Citation

Thomas Heitkamp, Gabriele Deckers-Hebestreit, and Michael Börsch "Observing single F_oF₁-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor", Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 97140B (1 March 2016); https://doi.org/10.1117/12.2209123

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