Monitoring the rotary motors of single FoF1-ATP synthase by synchronized multi channel TCSPC

  title={Monitoring the rotary motors of single FoF1-ATP synthase by synchronized multi channel TCSPC},
  author={Nawid Zarrabi and Monika G. D{\"u}ser and Stefan Ernst and Rolf Reuter and Gary D Glick and Stanley D Dunn and J. Wrachtrup and Michael B{\"o}rsch},
  booktitle={SPIE Optics East},
Confocal time resolved single-molecule spectroscopy using pulsed laser excitation and synchronized multi channel time correlated single photon counting (TCSPC) provides detailed information about the conformational changes of a biological motor in real time. We studied the formation of adenosine triphosphate, ATP, from ADP and phosphate by FoF1-ATP synthase. The reaction is performed by a stepwise internal rotation of subunits of the lipid membrane-embedded enzyme. Using Förster-type… 

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

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.

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.

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.

Improving FRET-based monitoring of single chemomechanical rotary motors at work.

  • M. BörschJ. Wrachtrup
  • Chemistry
    Chemphyschem : a European journal of chemical physics and physical chemistry
  • 2011
This work summarizes the knowledge gathered from single-molecule FRET studies of the membrane-embedded rotary nanomotor F(o)F(1)-ATP synthase and new ideas and concepts to shift and extend the current limitations of the confocal FRET detection approach are discussed.

Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

A version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA is presented, which could hold single fluorescent nanobeads for more than 100 seconds and increase the observation times of a single particle more than 1000-fold.

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.

Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET

The study aims at the observation of conformational fluctuations within a single P-type ATPase functionally reconstituted into liposomes by single-molecule FRET and analysis by Hidden-Markov-Models.

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.

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

  • M. Börsch
  • Chemistry, Physics
    Biological chemistry
  • 2011
The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET and the results are summarized and perspectives for other transport ATPases are discussed.



3D-localization of the a-subunit in F0F1-ATP synthase by time resolved single-molecule FRET

Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps and the stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes and the position of the FRET donors at the asubunit was calculated.

3 D-localization of the a-subunit of FoF 1-ATP synthase by time resolved single-molecule FRET

Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps and the stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes.

ATP-driven stepwise rotation of FoF1-ATP synthase.

The rotational behavior of FoF1 resembles that of F1, and this finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation.

Direct observation of the rotation of F1-ATPase

It is shown that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion by attaching a fluorescent actin filament to the γ-subunit as a marker, which enabled us to observe this motion directly.

Analysis of single-molecule FRET trajectories using hidden Markov modeling.

An analysis scheme is developed that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the mostlikely time sequence of underlying states for each trajectory.

Movements of the ε‐subunit during catalysis and activation in single membrane‐bound H+‐ATP synthase

It is concluded that the active–inactive transition was associated with a conformational change of ε within the central stalk within F0F1‐ATP synthases, which catalyze proton transport‐coupled ATP synthesis in bacteria, chloroplasts, and mitochondria.

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

This work incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis.