NMR spectroscopy learns new tricks under pressure


Researchers are rightly cautious about tinkering with the mechanics of expensive pieces of equipment like a nuclear magnetic resonance (NMR) spectrometer, but for a team at the National Institutes of Health in Bethesda, Maryland, the reward was well worth the risk. By transforming the machine’s sample cell into a high-speed pressure vessel that could achieve 2.7 kilobars in just a few milliseconds, they have opened up a new window on the fundamental process of protein folding.

The team responsible, led by Adriaan Bax and Philip Anfinrud, published their findings in the Proceedings of the National Academy of Sciences. The work was reviewed by Lewis Kay, a professor in the University of Toronto’s Department of Biochemistry and winner of this year’s Gerhard Herzberg Canada Gold Medal for Science and Engineering. Kay’s own research has dealt with improvements to NMR to improve protein imaging, so he welcomed this significant development in the field.


“It provides us with a tool kit to begin asking questions about how proteins fold and mis-fold,” he explains. “You’re starting out with a basic amino acid sequence, a code that dictates how a protein folds up. We don’t understand the rules of the game very well and hence the need to do experiments to try to understand how this primary amino acid sequence gives rise to a three-dimensional fold.”

In contrast to conventional fluorescence spectroscopy, which provides only low-resolution information about where all the parts of a complex molecule are, NMR can offer much more accurate data. However, it typically does so in a one-off fashion, simply capturing the current state of a sample molecule. However, by using pressure to fold and unfold a sample over and over again, the NIH array can track a sample through tens of thousands of iterations, capturing a data point for each one and then combining them to compose a complete picture.

Kay regards this strategy as an ingenious response to the limitations of the technology and a valuable asset for anyone interested in protein folding. He points to his own study of diseases related to misfolding, a phenomenon that remains baffling in many instances. “This is an area of research that we’re involved with as well,” he says. “We have looked at proteins that are implicated in diseases such as ALS.”

The PNAS paper showcases the new NMR method with the well-studied protein ubiquitin, whose folding characteristics had nevertheless remained elusive.

“Interestingly, our data shed light on the prior debate regarding the two- or three-state nature of ubiquitin’s folding process,” the authors state with a specific reference to the role of energy barriers in encouraging or discouraging the appearance of an intermediate. “For ubiquitin, about one-half of the protein molecules reach their final folded state through this intermediate state, whereas the other half clears a single free-energy barrier. Our data therefore indicate that, contrary to the common view that regards intermediates as kinetic traps, three-state folding can be as efficient as two-state.”