Study reveals how liquid protein droplets age into rubber ball-like elastic solids

BUFFALO, N.Y. — Droplets of proteins, tied to an increasing number of cellular processes and human diseases, are known for their liquid-like ability to flow, exchange material and dissolve as needed.

Yet these droplets, known as biomolecular condensates, are something akin to childhood staple Silly Putty in that they can also transform into more solid-like structures.

Now, a collaborative effort between the University at Buffalo, St. Jude Children’s Research Hospital and Washington University in St. Louis has found that condensates’ unique viscoelastic properties are determined by the amino acid sequence of the proteins that form them. 

The team’s study, published July 2 in Nature Physics, found that whether the condensates behave more like a viscous liquid or an elastic solid depends on the strength and duration of the amino acids’ interactions. This can explain why condensates act like molecular putty and how they can even age into a solid similar to a rubber ball.

"Shedding light on the intricate behavior of condensates at the molecular level is crucial for advancing our knowledge of cellular biology and understanding their association with many neurodegenerative diseases,” says the study’s lead corresponding author, Priya R. Banerjee, PhD, associate professor of physics in the UB College of Arts and Sciences. “This work reveals the mechanisms behind cellular processes that have long been observed but never fully understood.”  

Biomolecular condensates are membraneless hubs that organize biomolecules in cells spatially and temporally. They’re abundant throughout cellular function and have been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia.

To better understand their role in disease, scientists need to interrogate their fundamental material properties. However, developing appropriate high-resolution technologies to probe these micron-scale droplets has been a key challenge, which Banerjee’s research group has successfully addressed recently.

The study’s co-corresponding author, Tanja Mittag, PhD, a faculty member in the St. Jude Department of Structural Biology, and senior author Rohit Pappu, PhD, of Washington University in St. Louis, previously helped establish a “stickers-and-spacers” model for predicting how proteins phase separate into droplets.

Stickers are amino acids that form contacts with other stickers, while spacers are amino acids necessary for patterning and arranging stickers and interactions with water.

In this new study, Banerjee, Mittag, Pappu and the rest of the team found that the strength of these sticker-sticker interactions determines whether the condensates behave as an elastic or viscous material. Plus, the material properties of these tiny droplets are robustly programmable by the identity of stickers and spacers.

“If we make stronger interactions, we can push their behavior more toward elastic properties,” Mittag says.

Throw putty at a wall, and it will bounce back. Hold it in your hand long enough, and it will eventually flow through your fingers. Viscoelastic material’s behavior depends on the timescale of the observer.

The making and breaking of the amino acids’ contacts happen on a timescale precisely encoded in the protein sequence, the study found.  

Prior work had focused on how proteins within aging condensates can arrange into fibrils, repeating patterns of proteins that have been linked to ALS and dementia, but the team identified an alternative path in condensate aging. 

“We found that if the spacer amino acids in the protein chain like water, we could get condensates to age into a solid state faster, but it was not bona fide fibrils. Instead, it was a viscoelastic solid that is semi-crystalline. This is like putty becoming a rubber ball,” says co-author Anurag Singh, PhD, a postdoctoral researcher in Banerjee’s lab. “The most intriguing part is that the millions of years of evolution likely shaped the protein sequence to contain a precise combination and patterning of stickers and spacers that control the solid-liquid behavior and the aging timescale of these droplets.”

Other UB contributors include the study’s co-first author Ibraheem Alshareedah, PhD, a former postdoctoral researcher in Banerjee’s lab who is currently a postdoctoral trainee at Harvard University.

Banerjee’s work was funded by the National Institutes of Health and St. Jude.