NMR Innovation Offers Insights into Protein Interaction

The findings were published the week of June 24, 2013, in the Proceedings of the National Academy of Sciences of the United States of America (PNAS). The interaction the researchers became the first ones to describe is nearly universal across all of life. A protein unit called a chaperone takes hold of a disordered smaller protein to help it find its correct folded conformation. The scientists, in this instance, initiated test-tube experiments where they hoped to visualize the capsule-shaped bacterial chaperone GroEL capture a disordered amyloid beta (A-beta) protein, a molecule that in humans is central in Alzheimer's disease.

The two proteins are well researched, but the motions they go through when they first meet, i.e., when the open GroEL capsule captures its target, have been invisible to scientists. Electron microscopy and X-ray crystallography are only good for taking snapshots of easily frozen moments in time. NMR is capable of sensing the interactions and kinetics of protein interactions as they occur, but in some cases, any single technique can provide only clues into what is actually going on.

Brown University (Providence, RI, USA) biologist Dr. Nicolas Fawzi, who was a post-doc in the group of Marius Clore’s at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the US National Institutes of Health (NIH; Bethesda, MD, USA), worked with coauthors and NIDDK researchers Drs. David Libich, Jinfa Yang, and Marius Clore assembling the interactions of the proteins by combining four different NMR techniques. They determined what each one could inform them about the interaction and built the case presented in PNAS.

“None of the four techniques alone gave us sufficient information,” said Dr. Fawzi, now an assistant professor in Brown’s department of molecular pharmacology, physiology, and biotechnology. “Only by using them all together would we be able to figure out the structure and motions of A-beta; when it was bound to GroEL. By having four indirect measurements together, that was able to give us a complete picture.”

The NMR techniques they used were lifetime line broadening, Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion spectroscopy, and exchange-induced chemical shifts. “The fourth technique we employed was dark-state exchange saturation transfer [DEST] spectroscopy, which we had developed in my lab at the NIH in 2011,” said Dr. Clore, also the article’s corresponding author. “We were able to more effectively conduct our research by using that tool to corroborate and extend the information afforded by the other three measurements.”

The elusive process debated among molecular biologists was about what the GroEL chaperone requires of its captives at the moment they engage. Does it force them into a specific conformation? Does it hold on tightly while it closes its capsule lid around the smaller protein, or does the captive stay in motion at all?

What the investigators observed is that the GroEL is a permissive captor. It bound A-beta; at just two hydrophobic sites, leaving the smaller protein to otherwise swing in a range of conformations. It also did not keep it bound the entire time, letting it instead detach and re-bind. Basically, A-beta would jump off and on within GroEL’s binding cavity.

“By using these four techniques together we were able to extract information about the structure of the protein while it binds as well as how fast it comes on and off and what it's doing at each position,” Dr. Fawzi said. “Instead of forming more particular structure upon binding it appears to retain great conformational heterogeneity.”

The lifetime line-broadening technique, for example, informed them that the A-beta; was interacting with something big (GroEL), while the CPMG and chemical shift observations combined to show the length of time A-beta; spent on GroEL before unbinding, as well as the structural characteristics of A-beta; when it was bound to GroEL. DEST provided data that could validate much of the story of the other techniques.

Dr. Fawzi noted that GroEL’s relaxed strategy could be a matter of being able to bind many different proteins in disordered conformations, but also of saving energy. Forcing proteins into a specific conformation just to make and sustain the initial capture would require more energy than it is worth. Eventually, in moments after those the team resolved in this study, GroEL closes its lid and encapsulates its target proteins fully, according to Dr. Fawzi said. Thatis when it capitalizes on in compelling them to fold in the correct manner.

For molecular and structural biologists, the newly proven combination of NMR techniques could create a number of other cold cases of elusive interactions. “We can now look at how these big machines can do their job while they are working,” Dr. Fawzi said. “This is not just limited to this GroEL machine.”

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