A breakthrough by researchers at the University of Notre Dame has uncovered a crucial step in the life of a protein that may determine whether it works properly or instead ends up misfolded and non-functional. Their findings reveal a surprising race at the heart of protein folding, and offer new hope for understanding diseases like Alzheimer’s, Parkinson’s, and others linked to protein malfunction.

The research, led by Qing Luan and Patricia Clark, the Rev. John Cardinal O’Hara C.S.C. Professor of Biochemistry, focuses on how a large protein called pertactin, produced by bacteria, manages to fold into its correct shape. Proteins are long linear polymers that have different folding properties depending on their chemical structure and folding environment. Pertactin is much larger than most proteins typically used by scientists as models to study protein folding, but it is similar to the average size of all proteins in our cells. In the test tube, pertactin folds so slowly that scientists have long suspected it might be getting stuck along the way, and that the environment in the cell might help it avoid getting stuck in misfolded shapes.

Now, thanks to a clever new approach, the Notre Dame team has discovered a short-lived, “in-between” structure — named PFS* — that lies along the pathway for folding and acts like a fork in the road. At this crucial point, pertactin can either continue toward its proper folded form or fall into a misfolded trap that it may never escape.

The Tortoise and the Hare, Reimagined

To explain their findings, Luan and Clark use a familiar story: Aesop’s fable The Tortoise and the Hare. In their version, the “hare” follows the correct folding path: fast and efficient. The “tortoise” takes the fork in the road that leads to the misfolded shape that is quite stable. It’s a slow process but difficult to undo.

“If the protein hesitates too long in this intermediate state, the tortoise wins,” said Professor Clark. “It ends up misfolded. But if it keeps moving quickly, it can get to the correct shape. The hare wins — and the protein functions as it should.”

This balance between speed and stability helps explain why protein folding can go wrong, and why some conditions — like those inside cells — can help proteins fold correctly while others increase the risk of failure.

Finding What’s Been Hiding in Plain Sight

The newly discovered folding intermediate, PFS*, is tricky to spot. It looks almost identical to the stable misfolded form of the protein (called simply PFS) when viewed using traditional methods to study protein folding. The difference? PFS* is unstable and temporary, while PFS is stable and stuck.

To catch this fleeting moment at the junction between folding and misfolding, Luan and Clark designed a special experiment known as a “double-jump denaturant challenge.” They briefly allowed pertactin to fold, waiting just long enough for PFS* to form, then quickly added just enough of a chemical called a denaturant to rapidly unfold PFS* but leave the stable misfolded PFS state unaffected. Then, they watched how the protein reacted. If pertactin unfolded quickly when the denaturant was added, they knew they had caught PFS* before it turned into the misfolded PFS.

“This gave us a time-lapse snapshot of the decision point between folding correctly and misfolding,” said Luan. “And for the first time, we could see that PFS* is a separate state that determines the fate of the folding process.”

A Folding Process with Direction

Another key insight from the study is that pertactin folds in a specific direction, from one end to the other: specifically, from its C-terminal end to its N-terminal end. This was a surprising discovery because it matches how pertactin folds in living bacteria, where it folds while it is “pushed” out of the cell, starting with its C-terminal end. Luan and Clark found that progressive folding from the C-terminal end helps prevent parts of the protein from getting tangled with each other, which can slow down folding and lead to misfolding.

Why This Matters for Human Health

Misfolded proteins are a known factor in many serious illnesses. Misfolded proteins can clump together and form harmful aggregates that damage cells. By identifying the exact moment when a protein chooses between proper folding and misfolding, this study opens the door to ultimately developing new treatments that could tip the balance in favor of healthy, properly folded proteins.

“If we can find ways to help proteins move through PFS* to the correctly folded structure more quickly, we might be able to prevent them from ever misfolding,” said Clark.

A Path Forward

The research offers not just a scientific advance, but a shift in how we think about folding: it’s not just the final shape that matters, but the journey to get there — and how fast it happens.

The study, “Identification of an On-Pathway Protein Folding Intermediate Illuminates the Kinetic Competition Between Folding and Misfolding”, was published in Proceedings of the National Academy of Sciences. Research in Clark’s lab is funded by the National Institutes of Health.

Originally published by Samantha Keller at science.nd.edu on August 07, 2025.