Time-resolved NMR probes the dynamics of protein folding

In vivo proteins somehow navigate tortuous free-energy landscapes to transform from linear chains to three-dimensional structures in a few microseconds. Their final configurations must be just right to function properly; protein misfolding is thought to underlie diseases such as Parkinson’s and Alzheimer’s. Despite long-standing techniques for probing a protein’s structure, the rapidity of folding has stymied attempts to track the process.

Now Jaekyun Jeon, Robert Tycko , and coworkers at the National Institutes of Health in Maryland have introduced a new way to track protein folding. To achieve the necessary temporal resolution, the process relies on two novel features: a device that can start and stop protein folding on millisecond time scales and a signal-enhancing NMR technique.

For their proof-of-concept experiment, the researchers investigated melittin, a peptide found in bee venom. At low pH, the peptides are linear chains, but in neutral to high pH, each peptide forms two helices and the twisted chains assemble into tetramers. The entire transition happens in less than 10 ms, so whether those steps happen concurrently or sequentially has been hard to discern.

To trigger assembly, the researchers mixed a low-pH melittin solution with a high-pH buffer to produce a neutral solution. Their homemade device, shown in the picture, mixes a tiny volume of the two solutions in just 1.6 ms. Then it shoots the mixture onto a 100 K rotating copper plate that freezes the solution—and the protein’s configuration—in less than 0.5 ms. The speed of the fluid and the distance between the mixer and the plate determine how long the protein has been allowed to fold. In experiments, those were 2.2, 4.6, 9.4, and 29 ms.

Jeon, Tycko, and coworkers then used solid-state NMR to look at the time series of frozen proteins (see Physics Today, October 2016, page 19 ). At the low protein concentration needed for folding, the NMR signal would normally be too weak to analyze. To make the signal stronger, the researchers used dynamic nuclear polarization, a technique that enhances nuclear spin alignment by coupling it to the electron cloud through microwave radiation (see Physics Today, March 2016, page 16 ). They found that both the helical and tetrameric structures formed within 6–9 ms, which suggests the processes are concurrent rather than sequential. The time-resolved NMR technique can also be used to study the folding and assembly of larger proteins relevant to human pathology. (J. Jeon et al., Proc. Natl. Acad. Sci. USA 116, 16717, 2019 .)