Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

@inproceedings{Renz2012DiffusionPO,
  title={Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy},
  author={Marc Renz and Torsten Rendler and Michael B{\"o}rsch},
  booktitle={Other Conferences},
  year={2012}
}
FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related… 
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References

SHOWING 1-10 OF 38 REFERENCES
Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET
TLDR
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.
3D-localization of the a-subunit in F0F1-ATP synthase by time resolved single-molecule FRET
TLDR
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.
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.
Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX
TLDR
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.
Clustering and dynamics of cytochrome bd‐I complexes in the Escherichia coli plasma membrane in vivo
The cytochrome bd‐I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and
Diffusion of Green Fluorescent Protein in Three Cell Environments in Escherichia Coli
TLDR
Fluorescence recovery after photobleaching is used to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin and shows that both cell compartments are relatively fluid environments.
Three-color Förster resonance energy transfer within single F₀F₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.
TLDR
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.
Macromolecular organization of ATP synthase and complex I in whole mitochondria
TLDR
It is proposed that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as propton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.
Structural organization of the V-ATPase and its implications for regulatory assembly and disassembly.
TLDR
Reversible assembly/disassembly of V-ATPases has certain conformational constraints, which are best fulfilled by adopting a unique conformation before disassembly, which leads to an efficient shutdown of ATP consumption.
Are Escherichia coli OXPHOS complexes concentrated in specialized zones within the plasma membrane?
TLDR
It is observed that a fluorescently labelled terminal oxidase, the cytochrome bd complex, is heterogeneously distributed in the Escherichia coli plasma membrane, and this observation forms the basis of a working hypothesis that patches of the E. coli plasma membranes are dedicated to respiratory function by the high concentration of OXPHOS components in these zones relative to the adjacent membrane.
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