Unfolding a protein using atomic force microscopy just got a lot faster

In a breakthroughreportedin 1997,1–3 researchers mechanically pulled a single  protein, titin, until its structure unravelled. Computer models – applied to interpret the observations in molecular terms – could simulate this transition only on a much shorter timescale, but researchers in France have managed to speed up the experiment to match the simulation.4

Titin is a world-record holder among proteins: it is the largest single chain protein, and also the most flexible one. The protein works as a spring in muscle fibres, helping to return the muscle to its relaxed position after contraction.

In 1997, several groups used atomic force microscopy (AFM) or laser tweezers to pull single molecules or fragments of titin until some of its domains (structural units) came apart. These experiments revealed extensions and forces, but not the unfolding mechanism. To work out how the polypeptide chain unravels, researchers had to use computer simulations.

Because of the limits of computational power at the time, a full-scale simulation could at the time only be run for hundreds of picoseconds (today microseconds are routine and millisecond simulations are feasible). That meant the simulated unfolding had to be conducted several orders of magnitude faster than the experiments, and the results could not be matched up.

By managing to speed up the experiment 1000 fold, Simon Scheuring’s group at the University of Aix-Marseille, France, made comparision between simulation and experiment possible. ‘Compared to conventional AFM force spectroscopy, we used cantilevers that are about 30 times shorter (6 µm) than conventional cantilevers (200 µm). We furthermore used a novel sample support to minimise hydrodynamic drag and very fast electronics,’ Scheuring explains.

Hermann Gaub from the Ludwig Maximilians University Munich, whose group was among those that reported forced unfolding in 1997, welcomed the technical advance. ‘With this report the Scheuring lab contributes markedly to the improvement of single molecule instrumentation and opens the door for a better understanding of biomolecular mechanics,’ Gaub tells Chemistry World.

The molecular behaviour seen in the high-speed experiment and in the simulations is very different from that at lower speeds. ‘It appears that the molecule is pulled so fast that it doesn’t have the time anymore to diffuse around, because the trajectory that is imposed by the pulling dominates over the natural diffusion’ Scheuring concludes.

The same approach, says Scheuring, could also be useful for pharmaceutical science, in the study of ligand–receptor interactions. A sceptical expert has cautioned, however, that interpretation of such work in practical terms is difficult, as the forces seen in the very fast unfolding are much larger than the ones a protein would experience in its natural environment.