The cell is no longer viewed only as a biochemical entity. In an increasing manner, the focus of research is on trying to understand the physical processes that go on inside and around the cell as well. Cells use molecules to sense and exert forces that are important in biological function. Adhesion molecules on the cell surface sense tension in the surrounding tissue, motor proteins apply force to move cargo in the cell, and proteins in the muscle mechanically move the tissue and allow motion. Both large and small mechanical effects are ultimately mediated by tiny forces that take place within and between individual molecules.

Over the past decades, methods based on optical tweezers and atomic force microscopy (AFM) have allowed researchers to apply small forces to single molecules and measure the molecule's response, relating protein mechanics, structure and folding. Combinations of AFM experiments with molecular dynamic simulations have led to rich atomic-level descriptions of processes such as receptor-ligand binding or the detailed unfolding of muscle proteins and have pushed forward the emerging field of single-molecule mechanics.

But up until now, the two methodologies, experimental force spectroscopy and in silico simulations, have not been easy to compare side by side because the experimental methods could not attain the pulling speeds of simulations.

High-speed AFM methods have, however, existed for quite some time, explains Simon Scheuring, who works at the INSERM (the French National Institute of Health and Medical Research) at Aix-Marseille University. These methods are based on using small cantilevers with high resonance frequency as well as fast scanners, feedback controllers and electronics. In the past, high-speed AFM has been used for imaging experiments, not for measuring forces, says Scheuring. In a paper recently published by his group, Scheuring and colleagues adapted the methodology to perform high-speed force spectroscopy measurements.

High-speed force spectroscopy pulls molecules at high speeds to study their unfolding. Image courtesy of F. Rico.

Scheuring and collaborators used a short cantilever and a small piezoelectric actuator like those used in previous high-speed AFM setups, but they added a few optimizations of their own. For instance, they tilted the surface to which the molecule is attached to on one end, reducing hydrodynamic drag and laser interferences in the force measurements. They also improved the electronics and data-collection processes. This allowed them to exert pulling forces at a speed of 4 millimeters per second, more than two orders of magnitude faster than in conventional AFM.

The technique offers two main advantages, says Scheuring. “One can do very fast velocity force spectroscopy experiments, or one can pull relatively slowly and use the high sampling resolution to see intermediate folding steps.” Using this approach, the team studied how the muscle protein, Titin, unfolds in response to forces exerted at high and low speeds. Because of the high sampling resolution, they could decipher the detailed molecular dynamics that take place as Titin's domains unfold and refold. They also measured the dynamic force spectrum for this protein and found that it was not linear, something that had been previously predicted by simulations but that had not been experimentally confirmed.

“Our hope is that this type of work will stimulate new theory,” says Scheuring, “and that we will be able to go back to back between the experiments and simulations.” Scheuring is planning to apply the method to study ligand-receptor interactions now and envisions a future, not too far away, in which these types of measurements will be performed directly on molecules in cells and tissues.