Mechanical Processes of a Single Synthetic Molecular Machine Studied by AFM-based Force Spectroscopy

Some biomolecules are able to generate directional forces by rectifying random thermal motions. This allows these molecular machines to perform mechanical tasks such as intracellular cargo transport or muscle contraction in plants and animals. Although some artificial molecular machines have been synthesized and used collectively to perform mechanical tasks, so far there have been no direct measurements of mechanical processes at the single-molecule level. Here we report measurements of the mechanical work performed by a synthetic molecule less than 5 nm long. We show that biased Brownian motion of the submolecular components in a hydrogen-bonded [2]rotaxane -a molecular ring threaded onto a molecular axle- can be harnessed to generate significant directional forces. We used the cantilever of an atomic force microscope to apply a mechanical load to the ring during single-molecule pulling-relaxing cycles. The ring was pulled along the axle, away from the thermodynamically favoured binding site, and was then found to travel back to this site against an external load of 30 pN. Using fluctuation theorems, we were able to relate the measurements of the work done at the level of individual molecules to the free energy change measured previously by ensemble measurements. Finally, we used dynamic single-molecule force spectroscopy to probe kinetic information of the interaction between the molecular ring and the preferred binding site. The results also demonstrate that AFM-based single-molecule force spectroscopy, which has been widely used to investigate the mechanochemical behaviour of (bio)macromolecules, can be applied to a molecule that is less than 5 nm in its extended form.