The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap

@inproceedings{Bockenhauer2014TheRS,
  title={The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap},
  author={Samuel D Bockenhauer and Thomas M. Duncan and W. E. Moerner and Michael B{\"o}rsch},
  booktitle={Photonics West - Biomedical Optics},
  year={2014}
}
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary… 
Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase
TLDR
An experimental system is developed that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthase in vitro and labels the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET).
Regulatory conformational changes of the epsilon subunit in single FRET-labeled FoF1-ATP synthase
TLDR
Labeling the C-terminal domain of epsilon and the gamma subunit specifically with two different fluorophores for single-molecule Foerster resonance energy transfer (smFRET) allowed monitoring of the conformation of ePSilon in the reconstituted enzyme in real time.
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 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.
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.
The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
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.
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.
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References

SHOWING 1-10 OF 73 REFERENCES
Stepwise rotation of the γ-subunit of EFoF1-ATP synthase during ATP synthesis: a single-molecule FRET approach
TLDR
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).
Activation and Stiffness of the Inhibited States of F1-ATPase Probed by Single-molecule Manipulation
F1-ATPase (F1), a soluble portion of FoF1-ATP synthase (FoF1), is an ATP-driven motor in which γϵ subunits rotate in the α3β3 cylinder. Activity of F1 and FoF1 from Bacillus PS3 is attenuated by the
Intersubunit rotation in active F-ATPase
TLDR
An intersubunit rotation in real time in the functional enzyme F-ATPase is recorded by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled γ in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilization F1.
F1-ATPase of Escherichia coli
TLDR
The rapid effects of catalytic site ligands on conformational changes of F1-bound ϵ suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position, and ϵ inhibition may provide a new target for antimicrobial discovery.
Direct observation of the rotation of F1-ATPase
TLDR
It is shown that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion by attaching a fluorescent actin filament to the γ-subunit as a marker, which enabled us to observe this motion directly.
Rotation of subunits during catalysis by Escherichia coli F1-ATPase.
TLDR
The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis, and similar reactivities of unlabeled and radiolabeled beta sub units with gamma C87 upon reoxidation.
The Proton-translocating a Subunit of F0F1-ATP Synthase Is Allocated Asymmetrically to the Peripheral Stalk*
TLDR
The position of the a subunit of the membrane-integral F0 sector of Escherichia coli ATP synthase was investigated, finding that this relationship provides stability to the membrane interface between a and b2, allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.
Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase.
TLDR
It is concluded that the active-inactive transition was associated with a conformational change of epsilon within the central stalk of F0F1-ATP synthases, and the three states of the inactive enzyme were unequally populated.
The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer
TLDR
F1-ATPase is an ATP-driven rotary motor in which a rod-shaped γ subunit rotates inside a cylinder made of α3β3 subunits, suggesting that the crystal structures mimic a metastable state before product release.
Asymmetry of rotational catalysis of single membrane-bound F0F1-ATP synthase
TLDR
Computer simulations of the FRET signals and non-equally distributed orientations of ε strongly corroborate asymmetry of catalysis in F0F1-ATP synthase.
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