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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12 Citations
Background Citations
1
Methods Citations
3

12 Citations

Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap
TLDR
This ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip increases the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000, and monitors conformational changes of individual membrane proteins in real time.
Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor
TLDR
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.
Single-molecule FRET dynamics of molecular motors in an ABEL Trap
TLDR
The capabilities of the ABEL trap are exemplified in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication.
Confining Brownian motion of single nanoparticles in an ABELtrap
TLDR
The ABELtrap is an active feedback system cancelling the nano-object’s Brownian motion by applying an electric field and it is shown how the induced electrokinetic forces confine the motion of nanoparticles and proteoliposomes to the centre of the trap.
Tetherless, precise and extended observation of single-molecule FRET in an Anti-Brownian trap
A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Förster resonance energy transfer…
Analyzing conformational changes in single FRET-labeled A1 parts of archaeal A1AO-ATP synthase
TLDR
The lifetimes of fluorescence donor and acceptor dyes are analyzed to distinguish between smFRET signals of conformational changes and potential artefacts to prevent wasteful ATP hydrolysis.
Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases
TLDR
The enzyme’s rotary progression during ATP hydrolysis is monitored by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods, and it is found that one dwell in the three-steppedrotary progression lasting longer than the other two by a factor of up to 1.6.
Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap
TLDR
The oligomerization state of the human NTSR1 tagged with mRuby3 is reported by dissolving the plasma membranes of living HEK293T cells into 10 nm-sized soluble lipid nanoparticles by addition of styrene-maleic acid copolymers (SMALPs).
The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching
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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