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

TL;DR

This study employs advanced single-molecule FRET techniques with DCO-ALEX to monitor elastic energy storage and rotational dynamics in the rotary motors of FoF1-ATP synthase, revealing reversible elastic deformations during enzyme operation.

Contribution

It introduces a novel application of DCO-ALEX FRET to observe transient elastic energy storage in the rotary components of ATP synthase at the single-molecule level.

Findings

Detected reversible elastic deformations between rotor parts.

Estimated maximum angular displacement during load-free rotation.

Validated the use of DCO-ALEX FRET for real-time monitoring of enzyme mechanics.

Abstract

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