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

  title={Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap},
  author={Maria Dienerowitz and M. O. Ilchenko and Bertram Su and Gabriele Deckers-Hebestreit and G{\"u}nter Mayer and Thomas Henkel and Thomas Heitkamp and Michael B{\"o}rsch},
  booktitle={SPIE BiOS},
Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap — a microfluidic device to actively counteract Brownian motion of single nanoparticles with… 
9 Citations

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

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.

Measuring nanoparticle diffusion in an ABELtrap

Monitoring the Brownian motion of individual nanoscopic objects is key to investigate their transport properties and interactions with their close environment. Most techniques rely on transient

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).

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.

Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

Their mitochondrial nano-environment was investigated by FLIM and superresolution microscopy in living human cells and different lifetimes and anisotropy values were found.

The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.

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.



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.

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 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.

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.

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.

Watching conformational- and photo-dynamics of single fluorescent proteins in solution

A complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models is observed and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant.

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 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.

Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1.

TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range.