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

TL;DR

This study introduces an improved fluorescent protein, mNeonGreen, as a FRET donor to observe single FoF1-ATP synthase enzyme activity, enhancing resolution over previous fluorophores like EGFP.

Contribution

The paper demonstrates the use of mNeonGreen as a novel FRET donor for single-molecule studies of ATP synthase, improving spatial and temporal resolution.

Findings

mNeonGreen successfully labels FoF1-ATP synthase without impairing activity

Compared to EGFP, mNeonGreen offers better photophysical properties for smFRET

The enzyme remains fully functional after fusion with mNeonGreen

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.