Monika G. Düser

36° step size of proton-driven c-ring rotation in FoF1-ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by FoF1‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the Fo motor powers catalysis in the F1 motor. Although F1 uses 120° stepping during ATP synthesis, models of Fo predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.

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 by single molecule fluorescence resonance energy transfer studies utilizing a fusion of enhanced green fluorescent protein to the C terminus of the a subunit and fluorescent labels attached to specific positions of the ϵ or γ subunits. Three fluorescence resonance energy transfer levels were observed during rotation driven by ATP hydrolysis corresponding to the three resting positions of the rotor subunits, γ or ϵ, relative to the a subunit of the stator. Comparison of these positions of the rotor sites with those previously determined relative to the b subunit dimer indicates the position of a as adjacent to the b dimer on its counterclockwise side when the enzyme is viewed from the cytoplasm. 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.

Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary F(O) motor connected to a 3-stepped F(1) motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer.

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Monitoring the rotary motors of single F o F 1 -ATP synthase by synchronized multi channel TCSPC

Confocal time resolved single-molecule spectroscopy using pulsed laser excitation and synchronized multi channel time correlated single photon counting (TCSPC) provides detailed information about the conformational changes of a biological motor in real time. We studied the formation of adenosine triphosphate, ATP, from ADP and phosphate by FoF1-ATP synthase. The reaction is performed by a stepwise internal rotation of subunits of the lipid membrane-embedded enzyme. Using Förster-type fluorescence resonance energy transfer, FRET, we detected rotation of this biological motor by sequential changes of intramolecular distances within a single FoF1-ATP synthase. Prolonged observation times of single enzymes were achieved by functional immobilization to the glass surface. The stepwise rotary subunit movements were identified by Hidden Markov Models (HMM) which were trained with single-molecule FRET trajectories. To improve the accuracy of the HMM analysis we included the single-molecule fluorescence lifetime of the FRET donor and used alternating laser excitation to co-localize the FRET acceptor independently within a photon burst. The HMM analysis yielded the orientations and dwell times of rotary subunits during stepwise rotation. In addition, 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.

Regulatory conformational changes of the ε subunit in single FRET-labeled FoF1-ATP synthase.

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 hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthase in vitro. Labeling the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET) allowed monitoring of the conformation of ε in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in ε.

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

To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Förster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle more than 1000-fold. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.

Manipulating freely diffusing single 20-nm particles in an Anti-Brownian Electrokinetic Trap (ABELtrap)

Conformational changes of individual fluorescently labeled proteins can be followed in solution using a confocal microscope. Two fluorophores attached to selected domains of the protein report fluctuating conformations. Based on Förster resonance energy transfer (FRET) between these fluorophores on a single protein, sequential distance changes between the dyes provide the real time trajectories of protein conformations. However, observation times are limited for freely diffusing biomolecules by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices with 4 electrodes for an Anti-Brownian Electrokinetic Trap (ABELtrap). Here we present an ABELtrap based on a laser focus pattern generated by a pair of acousto-optical beam deflectors and controlled by a programmable FPGA chip. Fluorescent 20-nm beads in solution were used to mimic freely diffusing large proteins like solubilized FoF1-ATP synthase. The ABELtrap could hold these nanobeads for about 10 seconds at the given position. Thereby, observation times of a single particle were increased by a factor of 1000.