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

@inproceedings{Su2015ObservingCO,
  title={Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap},
  author={Bertram Su and Monika G. D{\"u}ser and Nawid Zarrabi and Thomas Heitkamp and Ilka Starke and Michael B{\"o}rsch},
  booktitle={Photonics West - Biomedical Optics},
  year={2015}
}
To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Förster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic… 
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TLDR
This work measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy and confirmed the binding of Cy5 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.
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References

SHOWING 1-10 OF 85 REFERENCES
Manipulating freely diffusing single 20-nm particles in an Anti-Brownian Electrokinetic Trap (ABELtrap)
TLDR
An ABELtrap based on a laser focus pattern generated by a pair of acousto-optical beam deflectors and controlled by a programmable FPGA chip is presented, which increased observation times of a single particle by a factor of 1000.
Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap
TLDR
A combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution and evaluates two different methods to trap the enzyme inside the confocal volume in order to extend the observation times.
Subunit rotation in single FRET-labeled F1-ATPase hold in solution by an anti-Brownian electrokinetic trap
TLDR
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.
Improving FRET-based monitoring of single chemomechanical rotary motors at work.
  • M. Börsch, J. Wrachtrup
  • Chemistry
    Chemphyschem : a European journal of chemical physics and physical chemistry
  • 2011
TLDR
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.
The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap
TLDR
This work labels F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer and compares FRET changes in single F1 and FRET histograms for different biochemical conditions to evaluate the proposed regulatory mechanism.
Monitoring the rotary motors of single FoF1-ATP synthase by synchronized multi channel TCSPC
TLDR
The action mode of bactericidal drugs, i.e. inhibitors of FoF1-ATP synthase like aurovertin, could be investigated by the time resolved single-molecule FRET approach.
Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer.
TLDR
An Anti-Brownian Electrokinetic trap capable of trapping individual fluorescently labeled protein molecules in aqueous buffer is presented, and confinement of single fluorophores of the dye Cy3 in water is shown.
Spectrally resolved anti-Brownian electrokinetic (ABEL) trapping of single peridinin-chlorophyll-proteins in solution
We use an Anti-Brownian ELectrokinetic (ABEL) trap to probe spectral emission shifts in solution-phase single Peridinin-Chlorophyll-Proteins (PCPs). The ABEL trap allows localization of single
Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET
TLDR
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.
Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX
TLDR
To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed and simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.
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