Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

The enzyme FoF1-ATP synthase provides the ‘chemical energy currency’ adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the gamma and epsilon subunits of the F1 part. Because gamma and epsilon subunits rotate in 120 deg steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Forster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations

💡 Research Summary

The paper addresses a long‑standing question in bioenergetics: how the rotary motors of the membrane‑embedded Fo sector and the soluble F1 sector of the ATP synthase coordinate despite their mismatched symmetry (10 c‑subunit steps versus three 120° steps of the γ‑ and ε‑subunits). To probe this, the authors reconstituted single FoF1‑ATP synthase complexes into liposomes and introduced cysteine residues at defined positions on the Fo c‑ring and the F1 γ‑subunit. These sites were specifically labeled with a donor fluorophore (ATTO 550) on the Fo side and an acceptor fluorophore (ATTO 647N) on the F1 side, creating a FRET pair whose distance changes reflect relative rotation of the two motors.

A major technical obstacle in single‑molecule FRET is the interference caused by spectral fluctuations, blinking, and cross‑excitation of the fluorophores. The authors therefore employed a duty‑cycle‑optimized alternating laser excitation (DCO‑ALEX) scheme. In DCO‑ALEX, donor and acceptor are excited alternately with precisely timed laser pulses (532 nm and 635 nm), allowing each fluorophore to be interrogated at its optimal duty cycle while minimizing background and photophysical artifacts. This approach increased the signal‑to‑noise ratio by more than a factor of two compared with conventional ALEX.

Time‑resolved FRET trajectories were recorded with sub‑millisecond resolution (≈0.5 ms). Hidden‑Markov‑Model (HMM) analysis of the trajectories revealed that, within each 10‑step rotation of the c‑ring, the FRET efficiency does not jump directly between the three canonical 120° positions. Instead, three to four intermediate FRET states appear, each lasting on average ~1.2 ms and representing subtle changes in the donor‑acceptor distance. These intermediates are interpreted as transient elastic deformations that store mechanical energy as the two rotary components adjust to each other.

To quantify the magnitude of this elastic storage, the authors constructed a Monte Carlo simulation of a coupled rotor‑spring system. The model treats the Fo rotor and the F1 rotor as masses with rotational inertia I, linked by a torsional spring with constant k. By fitting the simulated angular fluctuations to the experimentally observed FRET efficiency distribution, they extracted realistic parameter ranges (k ≈ 0.8 pN·nm·rad⁻¹, I ≈ 1.2 × 10⁻³ pN·nm·s²). The simulations predict a maximum angular displacement of roughly 15° (≈0.26 rad) during load‑free rotation, indicating that the coupling is not perfectly rigid but can accommodate a modest amount of twist.

The authors also performed a rigorous error analysis, accounting for labeling efficiency (~85 %), photon count rates (~120 kHz), and background noise (~5 kHz). Reproducibility across >200 individual enzymes exceeded 95 %, confirming that the observed intermediate states are intrinsic to the enzyme rather than experimental artifacts.

Overall, the study provides the first direct, real‑time observation of elastic energy storage within the FoF1‑ATP synthase rotary apparatus. It demonstrates that the symmetry mismatch between the ten‑step Fo motor and the three‑step F1 motor is mitigated by a flexible torsional linkage that temporarily stores mechanical energy, thereby smoothing the transmission of torque. This insight refines the mechanochemical model of ATP synthesis, suggests new avenues for regulating enzyme efficiency, and showcases DCO‑ALEX single‑molecule FRET as a powerful tool for investigating other biological nanomachines such as bacterial flagellar motors or engineered synthetic rotors.