An improved version of single-molecule force spectroscopy (SMFS) has enabled researchers to study the unfolding of a membrane protein with much higher time resolution and force precision than ever before, revealing a multitude of new details.

Proteins must adopt specific three-dimensional shapes to work properly. If they don’t, they can malfunction or cause protein-misfolding conditions such as Alzheimer’s disease. Scientists would therefore like to better understand how proteins fold and unfold, including all the intermediate states they adopt along the way.

One type of SMFS technique probes protein-folding intermediates by using an atomic force microscopy (AFM) cantilever to pull on a single protein molecule, unraveling it a little at a time. Most unfolding steps are reversible, so studying unfolding reveals a protein’s folding behavior as well.

In the past, SMFS studies found two intermediate states in the unfolding of two helices in the membrane protein bacteriorhodopsin. But molecular dynamics simulations predicted that this process is much more complex, with 10 unfolding intermediates.

Thomas T. Perkins of JILA in Boulder, Colo., and coworkers shortened, reshaped, and surface-modified AFM cantilevers to develop an SMFS system that analyzes proteins 100 times as fast as conventional AFM-based SMFS and with 10 times better force precision. The researchers have now used it to reveal experimentally that two of bacteriorhodopsin’s helices unfold with 14 intermediates (Science 2017, DOI: 10.1126/science.aah7124).

The findings show that the molecular dynamics results were not far off. “Without sufficiently high time resolution, many intermediate states are masked by instrumental limitations,” Perkins says.

The modified system can observe the unfolding of as few as two amino acids over microseconds, whereas conventional AFM-based SMFS typically observes the unfolding of six to 60 amino acids over milliseconds. Another technique called optical trapping can analyze protein unfolding with microsecond time resolution and higher stability than AFM-based SMFS, but it isn’t well suited for studying membrane proteins.

The new system’s time-resolution and force-precision improvements “represent a tour de force, and I use the pun intentionally,” comments Steven Block of Stanford University, an expert on nanoscale biomolecular motions. “The study shows that, with the right instrumentation, you can begin to resolve all the intermediates in protein unfolding,” bringing experiment and theory closer together. “The work is a true breakthrough that will in the future lead to all kinds of new insights,” Block says.