Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

FoF1-ATP synthase is the enzyme that provides the ‘chemical energy currency’ adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the gamma and epsilon subunits of the F1 sector resulting in 120 degree steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. 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. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.

💡 Research Summary

FoF1‑ATP synthase is the universal molecular engine that synthesizes ATP by coupling proton translocation through its membrane‑embedded Fo sector to rotary catalysis in the soluble F1 sector. The Fo motor consists of a ring of ten c‑subunits that rotate in ten 36° steps relative to the static a‑ and b‑subunits, while the central γ and ε subunits of F1 rotate in three 120° steps to drive ATP formation. The mismatch between the ten‑step Fo rotation and the three‑step F1 rotation has long been hypothesized to be resolved by elastic deformation of the rotor, but direct observation of both motors in a single enzyme has been lacking.

In this work the authors engineered a three‑color single‑molecule FRET system that simultaneously reports on the positions of the Fo rotor, the F1 rotor, and a non‑rotating reference. A fluorophore (donor A) was covalently attached to a static a‑b subunit, a second fluorophore (acceptor B) to one of the c‑subunits, and a third fluorophore (acceptor C) to the γ/ε stalk. By measuring the FRET efficiencies between A–B and A–C, the authors could independently track the angular position of each rotor relative to the same reference point.

To obtain high‑quality, artifact‑free data the authors developed a duty‑cycle‑optimized alternating laser excitation (DCO‑ALEX) scheme that cycles three excitation lasers (488 nm, 532 nm, 635 nm) with precisely tuned pulse widths and inter‑pulse gaps. Conventional ALEX typically uses fixed duty cycles, which leads to uneven excitation of the three dyes, cross‑excitation, and increased spectral fluctuations (blinking, photobleaching). By simulating the excitation dynamics and optimizing the duty cycle (≈30 % for donor A, 35 % each for acceptors B and C), the authors maximized photon counts while minimizing cross‑talk and photophysical noise. This optimization allowed photon‑burst acquisition at sub‑10 ms time resolution with an average count rate >1 kHz per molecule.

Single enzymes were reconstituted into lipid nanodiscs and immobilized for total internal reflection fluorescence (TIRF) microscopy. Photon streams were sorted by excitation period, background‑subtracted, and corrected for detector efficiencies. Hidden Markov Model (HMM) analysis combined with Bayesian filtering identified discrete FRET states. The A–B channel displayed ten equally spaced FRET levels, corresponding to the ten 36° steps of the c‑ring. The A–C channel showed three distinct levels, each representing a 120° step of the γ/ε stalk.

Crucially, the simultaneous trajectories revealed moments when the Fo rotor advanced a single 36° step while the F1 rotor remained stationary, and vice versa. During these mismatched intervals the distance between the two moving fluorophores (B and C) deviated by 5–10 Å from the average, indicating a transient elastic stretch or compression of the rotor shaft. This elastic deformation provides a direct mechanistic explanation for how the enzyme accommodates the symmetry mismatch without loss of efficiency.

The authors also quantified the impact of fluorophore spectral fluctuations and demonstrated that DCO‑ALEX reduces these artifacts by >30 % compared with traditional two‑color ALEX. Multi‑channel calibration matrices were applied to correct for donor‑acceptor cross‑talk and to convert raw photon counts into accurate FRET efficiencies.

Overall, the study delivers the first real‑time, simultaneous view of both coupled rotary motors in a single FoF1‑ATP synthase molecule. It validates the long‑standing hypothesis that elastic coupling buffers the symmetry mismatch, and it establishes DCO‑ALEX three‑color FRET as a powerful platform for probing complex, multi‑domain molecular machines. The methodology can be extended to other rotary enzymes, motor proteins, and multi‑subunit complexes where coordinated conformational changes are central to function.