Proteins – the building blocks of life – consist of a long chain of molecules called amino acids folded into a 3D shape. An atomic force microscope (AFM) can be used to study this folding by attaching one end of a protein to a substrate and the other end to the AFM’s cantilever. As the protein is stretched, the cantilever is oscillated and the force restoring the protein and cantilever back into equilibrium is measured.

In theory, monitoring this non-equilibrium force should provide information about the many intermediate equilibrium energy states that the protein goes through on its way to being fully extended. In practice, however, interpreting the data has proved controversial, and until now researchers have only had a clear understanding of the equilibrium states at the beginning and end of the folding process.

Now Ching-Hwa Kiang and colleagues from Rice University have improved the AFM technique to determine the intermediate states. To do this, they built a computer program based on an equation formulated by University of Maryland physicist Chris Jarzynski a decade ago.

Although scientists had believed “Jarzynski’s equality” could be used to obtain equilibrium information from non-equilibrium measurements, none had been able to apply it successfully. “Through numerous discussions with Jarzynski, we had a thorough understanding of where and how the theory applies,” Kiang explained.

The Rice group proved their technique works using section of “titin”, the largest known protein and the one that constitutes elastic muscle in the heart, and managed to map eight individual energy states as they used an AFM to unfold it. This, they say, paves the way for investigating how environmental changes such as temperature affect protein folding.