Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase

  title={Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase},
  author={Thomas M. Duncan and Monika G. D{\"u}ser and Thomas Heitkamp and Duncan G. G. McMillan and Michael B{\"o}rsch},
  booktitle={Photonics West - Biomedical Optics},
Subunit ε is an intrinsic regulator of the bacterial FoF1-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and β subunits, as revealed by the recent X-ray structure of the F1 portion of the Escherichia coli enzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP… 
The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
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.
Conformational dynamics of the rotary subunit F in the A3B3DF complex of Methanosarcina mazei Gö1 A‐ATP synthase monitored by single‐molecule FRET
This work investigated the nucleotide‐dependent conformational changes of subunit F relative to subunit D during ATP hydrolysis in the A3B3DF complex of the Methanosarcina mazei Gö1 A‐ATP synthase using single‐molecule Förster resonance energy transfer and found two conformations.
Fast ATP-Dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution.
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.
Insights into the regulatory function of the ɛ subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data
It is concluded that the ɛ subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.
The NMR solution structure of Mycobacterium tuberculosis F‐ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine
The Mtε structure provides a novel mechanistic model of coupling c‐ring‐ and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α3β3‐headpiece.
Three‐color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells
3‐color FRET assays further support in vitro results about the role of IQGAP1, Rac1, and CDC42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N‐WASP with IQG AP1.


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.
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).
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.
Movements of the ε‐subunit during catalysis and activation in single membrane‐bound H+‐ATP synthase
It is concluded that the active–inactive transition was associated with a conformational change of ε within the central stalk within F0F1‐ATP synthases, which catalyze proton transport‐coupled ATP synthesis in bacteria, chloroplasts, and mitochondria.
Rotation of subunits during catalysis by Escherichia coli F1-ATPase.
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*
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.
F1-ATPase of Escherichia coli
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.
Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation*
Results suggest that the ϵ subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.
Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coli FoF1-ATP Synthase*
The results suggest that the C-terminal domain of the ϵ subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.