Researchers have long wanted to better understand the way an unfolded protein chain reaches its final folded structure. But experimental tools needed to probe this complex process have been limited.
An apparatus developed by Philip Anfinrud, Ad Bax, and coworkers at the National Institutes of Health now opens a new window on the process by allowing nuclear magnetic resonance spectroscopy (NMR) to follow protein folding at an atomic level of detail in real time (Proc. Natl. Acad. Sci. USA 2018, DOI: 10.1073/pnas.1803642115).
Proteins unfold when subjected to high pressure and then refold when normal pressure is restored, a handy trick that researchers pair with spectroscopy to track folding. Fluorescence spectroscopy has been used in this way, but it provides only low-resolution information.
NMR can obtain highly precise data at every atom position that naturally contains or can be endowed with a magnetic isotope, like hydrogen-1, carbon-13, or nitrogen-15. But NMR analysis of protein folding has typically involved one-shot methods that can’t easily achieve high time resolution. The challenge was finding a way to rapidly and repeatedly impose and release very high pressure on NMR samples without destroying the equipment.
The apparatus from the NIH group safely changes the pressure as much as 2.7 kilobars in 1 to 3 milliseconds. The energy of each pressure pulse is “equivalent to setting off a firecracker” in the NMR sample cell, Bax says. And the process is reversible. “We typically switch back and forth between high and low pressure more than 100,000 times on any given sample.” Nevertheless, the NMR’s sample cell and its cryogenic probe, an expensive and delicate component that boosts NMR sensitivity, survive the dramatic changes.
The approach “overcomes the not inconsiderable difficulties and dangers of changing pressure rapidly inside a million-dollar piece of apparatus,” says protein folder Peter G. Wolynes of Rice University. “That is impressive.”
With their special setup, the team learned that about half of unfolded molecules of the small regulatory protein ubiquitin fold in two steps, first to a partially folded intermediate and then to a fully folded form. And the other half fold in a single step with no long-lived intermediate. The work also shows that ubiquitin makes numerous failed attempts to cross the transition-state barrier to folding before it succeeds. Similar findings on ubiquitin have been proposed theoretically but have had scarce experimental support.
The study “provides precise insights into folding events that were previously more or less just assumed,” says biomolecular NMR expert Stephan Grzesiek of the University of Basel. “The sheer detail is amazing.”
The methodology makes it possible to determine atomic-resolution structures of folding intermediates and to study disease-related events such as protein aggregation and fibril formation. “The importance of the work is clear when one considers the significance of protein misfolding diseases to human health and therefore the importance of developing techniques capable of dissecting folding and misfolding pathways and characterizing the states that populate them,” says protein NMR specialist Lewis E. Kay of the University of Toronto.
More generally, the researchers say, the system could probe any chemical reaction or process whose equilibrium is affected significantly by pressure changes of a few kilobars.