Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

@inproceedings{Zarrabi2009SimultaneousMO,
  title={Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX},
  author={Nawid Zarrabi and Stefan Ernst and Monika G. D{\"u}ser and Anastasiya Golovina-Leiker and Wolfgang Becker and Rainer Erdmann and Stanley D Dunn and Michael B{\"o}rsch},
  booktitle={BiOS},
  year={2009}
}
FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ε subunits of the F1 sector resulting in 120… 

Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

The detection of reversible elastic deformations between the rotor parts of Fo and F1 is reported and the maximum angular displacement during the load-free rotation is estimated using Monte Carlo simulations.

Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap

FoF1-ATP synthase is the membrane protein catalyzing the synthesis of the 'biological energy currency' adenosine triphosphate (ATP) and single-molecule Förster resonance energy transfer (FRET) is applied to monitor subunit rotation in the two coupled motors F1 and Fo.

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

The novel FRET donor mNeonGreen is evaluated as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP to evaluate the biochemical purification procedures and activity measurements of the fully functional mutant enzyme.

Subunit rotation in single FRET-labeled F1-ATPase hold in solution by an anti-Brownian electrokinetic trap

Monte Carlo simulations are used to reveal that stepwise FRET efficiency changes can be analyzed by Hidden Markov Models even at the limit of a low signal-to-background ratio that was expected due to high background count rates caused by the microfluidics of the ABELtrap.

Unraveling the Rotary Motors in FoF1-ATP Synthase by Time-Resolved Single-Molecule FRET

Detection of single fluorophore molecules was reported 25 years ago, at first in a crystalline matrix at cryogenic temperatures but quickly followed by single-molecule studies of biological machines

Three-color Förster resonance energy transfer within single F₀F₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.

This work presents a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work, and labels this enzyme with three fluorophores, specifically at the stator part and at the two rotors.

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

An experimental system is developed that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthase in vitro and labels the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET).

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

Labeling the C-terminal domain of epsilon and the gamma subunit specifically with two different fluorophores for single-molecule Foerster resonance energy transfer (smFRET) allowed monitoring of the conformation of ePSilon in the reconstituted enzyme in real time.

Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

A single-molecule FRET approach using duty cycle-optimized alternating laser excitation and new FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme, indicating a high flexibility in the membrane part of this thermophilic enzyme.

Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP).

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