Hidden protein-folding states unveiled

For 30 years, atomic force microscopy (AFM) has been imaging surfaces with subnanometer resolution. As a thin, flexible cantilever with an atomically sharp tip is scanned back and forth across the surface, the tip’s vertical position is monitored by reflecting a laser beam off it. The cantilever’s minute deflections reveal the surface topography.

But AFM is not limited to determining where atoms and molecules are: It can also manipulate them. In single-molecule force spectroscopy, for example, an AFM tip attached to one end of a protein is gently raised to unravel the folded chain of amino acids. From the points where the cantilever stalls on its upward trajectory, researchers infer the presence of stable intermediate states of the partially unfolded molecule. Those states can offer clues about the energetics of how the molecule folds into its correct structure to begin with.

In contrast to the atomic-resolution surface images, AFM measurements have offered only a coarse view of protein-folding dynamics. In the case of a standard 0.2-mm-long cantilever, the drag exerted by the aqueous environment can slow the response time to 0.1 ms. But simulations predict that many intermediate states are occupied for just a few microseconds, so they’re invisible to such a setup.

For the past decade, Thomas Perkins and colleagues of JILA in Boulder, Colorado, have been working to develop the perfect cantilever for protein-folding measurements. Using a focused ion beam, they modify commercially available ultrashort cantilevers—just 9 µm long—to make them flexible enough to be both sensitive to subpiconewton forces and responsive to microsecond time scales.

Now the JILA researchers have put their cantilevers to work to probe the folding dynamics of bacteriorhodopsin, a protein that harvests photon energy to pump protons across the cell membrane. Molecular dynamics simulations find that the pair of helices shown in blue in the figure should have 10–12 folded intermediate states. Prior experiments have detected just two; the new JILA experiments find 14, some of them separated by the folding of just two amino acids. As shown in the short data segment in the figure, the molecule rapidly hops between adjacent intermediates in a process more intricate than folding experiments had ever detected before. (H. Yu et al., Science 355, 945, 2017 .)