Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

  title={Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET},
  author={Eva Hammann and Andrea Zappe and Stefanie Keis and Stefan Ernst and Doreen Matthies and Thomas Meier and Gregory M. Cook and Michael Boersch},
Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a… 

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.

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.

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.

Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

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.

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.

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.

Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

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.



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.

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.

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.

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

A combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution and evaluates two different methods to trap the enzyme inside the confocal volume in order to extend the observation times.

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.

A Specific Adaptation in the a Subunit of Thermoalkaliphilic F1FO-ATP Synthase Enables ATP Synthesis at High pH but Not at Neutral pH Values*

Data suggest that amino acid residue Lys180 is a specific adaptation within the a subunit of TA2F1Fo to facilitate proton capture at high pH, which strongly imply that ATP synthesis under these conditions was sensitive to DCCD.

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.

Movements of the epsilon-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 epsilon within the central stalk of F0F1-ATP synthases, and the three states of the inactive enzyme were unequally populated.

Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ε Subunit

Reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1 exhibited ATP hydrolysis activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA 2F(o)F( 1).