Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

  title={Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET},
  author={Stefan Ernst and M. G. Dueser and Nawid Zarrabi and Michael Boersch},
  booktitle={Other Conferences},
The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ε subunits of the F1 part. Because γ and ε subunits rotate in 120… 
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.
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.
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.
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.
The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria
The one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells is obtained and the limited size of the observation area in the membrane with its significant membrane curvature has to be considered.
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.
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.


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.
Three-color Förster resonance energy transfer within single F₀F₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.
This work presents a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work, and labels this enzyme with three fluorophores, specifically at the stator part and at the two rotors.
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.
Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET
Progress is reported of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.
Stepwise rotation of the γ-subunit of EFoF1-ATP synthase during ATP synthesis: a single-molecule FRET approach
A stepwise rotation of the γ-subunit is detected under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition).
3D-localization of the a-subunit in F0F1-ATP synthase by time resolved single-molecule FRET
Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps and the stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes and the position of the FRET donors at the asubunit was calculated.
Asymmetry of rotational catalysis of single membrane-bound F0F1-ATP synthase
Computer simulations of the FRET signals and non-equally distributed orientations of ε strongly corroborate asymmetry of catalysis in F0F1-ATP synthase.
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.
Energy transduction in the F1 motor of ATP synthase
It is concluded that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.