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

TL;DR

This study employs a three-color FRET technique with duty cycle-optimized triple-ALEX to simultaneously monitor the coupled motors of FoF1-ATP synthase at the single-molecule level, revealing elastic deformations during rotation.

Contribution

It introduces a novel three-color FRET method with DCO-ALEX for real-time, simultaneous observation of both motors in ATP synthase, enhancing understanding of their coupled dynamics.

Findings

Simultaneous monitoring of Fo and F1 motors during ATP synthesis.

Detection of reversible elastic deformations between rotor parts.

Improved fluorescence measurement accuracy with DCO-ALEX.

Abstract

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.