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

Thermophilic enzymes can operate at higher 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 ring of 13 c-subunits. We applied a single-molecule F"orster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

💡 Research Summary

The study investigates the rotary proton motor of the FoF1‑ATP synthase from the thermoalkaliphilic bacterium Caldalkalibacillus thermarum strain TA2.A1, focusing on its distinctive 13‑subunit c‑ring architecture. While mesophilic enzymes such as the Escherichia coli ATP synthase have a 10‑subunit c‑ring that yields 36° rotational steps, the thermophilic enzyme is predicted to rotate in 13 steps of approximately 27.7° each. To test this hypothesis, the authors employed a single‑molecule Förster resonance energy transfer (smFRET) approach combined with duty‑cycle‑optimized alternating laser excitation (DCO‑ALEX). The γ‑subunit (rotor) and a selected c‑subunit (stator) were site‑specifically labeled with a donor fluorophore (ATTO 550) and an acceptor fluorophore (ATTO 647N), respectively. The labeled enzymes were reconstituted into liposomes, immobilized in a microfluidic flow cell, and interrogated with alternating 488 nm and 561 nm laser pulses. DCO‑ALEX ensured that donor‑only, acceptor‑only, and true FRET events could be distinguished on a per‑burst basis, dramatically improving signal‑to‑noise and allowing reliable detection of rapid conformational changes.

A novel analytical tool, the “FRET transition histogram,” was introduced to resolve the small angular increments characteristic of a 13‑step motor. Instead of averaging continuous FRET efficiency trajectories, the authors plotted each discrete transition (ΔE) against its frequency, producing a two‑dimensional density map. Thirteen distinct clusters emerged, each corresponding to a ΔE of roughly 0.07–0.09, consistent with the expected distance change for a 27.7° rotation. In contrast, the larger ΔE values (≈0.10–0.12) that would indicate a 10‑step motor were absent, confirming the presence of a 13‑subunit c‑ring.

Kinetic analysis was performed by extracting dwell times—the intervals between successive FRET transitions. Dwell times decreased sharply with increasing temperature: from an average of ~120 ms at 20 °C to ~18 ms at 55 °C, indicating a strong temperature dependence of the Fo motor’s turnover rate. The non‑ionic detergent lauryldimethylamine‑N‑oxide (LDAO) was added to modulate membrane fluidity; concentrations ranging from 0.05 % to 0.2 % further reduced dwell times, suggesting that a more fluid lipid environment facilitates rotor movement. Notably, under high‑temperature and high‑LDAO conditions, the motor displayed rapid back‑and‑forth stepping: forward (clockwise) and reverse (counter‑clockwise) transitions occurred within 5 ms of each other, with a forward‑to‑reverse ratio of roughly 1.3:1. This bidirectional stepping implies that the membrane‑embedded Fo motor retains considerable flexibility even at temperatures where the enzyme is otherwise highly stable.

Structural considerations support these functional observations. Cryo‑EM models of the C. thermarum ATP synthase reveal a c‑ring with a larger internal diameter and reduced inter‑subunit contact area compared with the E. coli counterpart. This geometry likely lowers the elastic strain during rotation, allowing the motor to sustain high‑temperature operation without compromising structural integrity. Moreover, the increased spacing may facilitate smoother proton translocation through the half‑channels, contributing to the observed high catalytic efficiency.

Overall, the paper demonstrates that DCO‑ALEX smFRET is a powerful technique for visualizing sub‑nanometer conformational changes in membrane protein complexes, enabling direct observation of the 13‑step rotary mechanism of a thermophilic Fo motor. The findings highlight a delicate balance between rigidity (necessary for thermostability) and flexibility (required for rapid, reversible stepping) that underlies the enzyme’s adaptation to extreme environments. The authors suggest that future work could involve mutagenesis of c‑subunit interfaces, molecular dynamics simulations, or high‑speed atomic force microscopy to further dissect the coupling between proton flow, rotor dynamics, and membrane mechanics in thermophilic ATP synthases.