Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap

@inproceedings{Rendler2011MonitoringSM,
  title={Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap},
  author={Torsten Rendler and Marc Renz and Eva Hammann and Stefan Ernst and Nawid Zarrabi and Michael Boersch},
  booktitle={BiOS},
  year={2011}
}
FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We… 
Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap
TLDR
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.
Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap
TLDR
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.
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.
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.
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.
Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution
TLDR
It is reported that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap), and broad distributions of ATP-dependent catalytic rates were revealed.
Fast ATP-Dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution.
TLDR
It is reported that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an anti-Brownian electrokinetic trap (ABEL trap), and broad distributions of ATP-dependent catalytic rates were revealed.
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.
Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET
TLDR
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 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.
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