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

  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},
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… 
Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap
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
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
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
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
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
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
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
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.


Manipulating freely diffusing single 20-nm particles in an Anti-Brownian Electrokinetic Trap (ABELtrap)
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
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
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
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
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
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.
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
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
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.