Transition-path times of molecular shuttles under mechanical equilibrium show symmetry

Determining microscopic reversibility during the operation of synthetic devices is crucial for a fundamental understanding of the thermodynamic and kinetic aspects that rule their operation. Here, we used optical tweezers to measure transition paths of individual molecular shuttles oscillating between two different equilibrating co-conformations under the application of mechanical force. We experimentally confirm that the transition-path times of individual molecular shuttles under mechanical equilibrium show symmetry, as derived from the principle of microscopic reversibility. Furthermore, we show that the relation proposed by Bier, Astumian, and colleagues (Bier-Astumian relation), which is a corollary of microscopic reversibility, can be used to extract thermodynamic variables from the analysis of the transition-path times. These measurements provide a first look at the principle of microscopic reversibility in molecular shuttles at the single-molecule level and pave the way for a detailed and quantitative understanding of the dynamics of synthetic molecular machines.