Imagine standing on a railway platform at rush hour: people jostle, cluster, and form little knots of conversation before dispersing again. Inside every cell, proteins can do much the same thing — crowding into tiny condensates for a moment before breaking apart again. However, in the early 2000s, biologists believed that only intrinsically disordered proteins (proteins with a region that does not form one stable structure) could create such liquid droplets. Fast forward two decades, and a team from IIT Bombay has upended that view: nearly every protein, regardless of shape or sequence, can condense into droplets — if you crowd them enough. This phenomenon, where biomolecules reside inside droplets (organelles lacking membranes), is called liquid-liquid phase separation. So, what does it mean when even highly structured proteins can suddenly behave like their sloppy, phase-separating relatives?

Samir Maji, Professor, Dept. of Biosciences and Bioengineering,  IIT Bombay, and his team selected 23 different proteins and peptides to explore this idea. These ranged from well-folded enzymes to simple 10-amino-acid peptides. Each sample was mixed with increasing amounts of PEG-8000, mimicking the cell’s crowded interior. Tiny, round droplets appeared when the mixture hit a threshold called the saturation concentration. These droplets fused on contact and allowed molecules inside to rearrange freely, proving they were liquid-like rather than solid clumps.

Manisha Poudyal, the first author of the paper (former PhD student from Maji’s lab and currently a Postdoctoral Research Associate at St. Jude Children’s Research Hospital, Memphis, USA), explained why this study was needed. “Several studies have reported that unstructured, intrinsically disordered domains and low complexity domains are the prerequisites for phase separation. We hypothesised that phase separation could be a generic property of proteins irrespective of their sequence and structure.” By testing proteins with drastically different shapes, charges, and sizes, the authors found something striking: every single one formed droplets when crowded enough. This showed that “It might be an intrinsic feature of a protein to undergo condensate formation, however, with diverse modes of intermolecular interactions and concentration,” says Poudyal. 

To discover what holds droplets together, the team treated them with different chemicals. Adding salt weakened electrostatic attractions between opposite charges on proteins. The compound 1,6-hexanediol disrupted hydrophobic interactions — the tendency of “water-fearing” parts to stick together. Urea helped in weakening the hydrogen bonds. They found that each protein depended on one or more of these forces to remain in a droplet — for instance, a protein with large hydrophobic patches formed droplets that dissolved only when hexanediol was added.

The researchers also measured how strongly each protein was bound to itself in solution. Proteins that self-associated more tightly needed lower concentrations to form droplets, while those with  weaker self-binding required much higher levels. This link between binding strength and droplet formation explains why any protein can condense once its local concentration crosses a critical threshold.

The study extended beyond full-length proteins. Maji’s team made separate chains of ten residues of glycine, valine, arginine, or aspartic acid. Even these minimal peptides formed droplets under suitable conditions. Valine chains clumped via hydrophobic attraction, while charged chains only condensed when their charges were balanced by salt or by mixing oppositely charged peptides. This confirmed that basic chemical principles, not just complex, folded structures, drive phase separation.

The work demonstrates that nearly every protein can form liquid droplets when crowded. This changes our understanding of cellular organisation. Inside a living cell, proteins and nucleic acids occupy over thirty per cent of the volume, creating a bustling environment. Small shifts, such as a protein moving closer to partners or being modified by enzymes, could push it past the threshold and into a droplet. While droplet formation is essential for normal processes like stress response, droplets that harden could lead to harmful aggregates and disease.

Looking ahead, these findings open exciting paths in both medicine and biotechnology. In disease contexts, Poudyal noted that one could “Develop some small molecules or chaperones that can inhibit phase separation and subsequent liquid-to-solid transition of the protein. Preventing the solidification and abnormal protein aggregation using such small molecules, chaperones, or chaperone homologues might offer a promising therapeutic strategy.” By targeting the forces driving droplets to age into solids, researchers may reduce or prevent the formation of harmful aggregates in conditions like Alzheimer’s or Parkinson’s. Understanding universal phase separation in biotechnology could help design artificial compartments that concentrate enzymes, speed up reactions, or deliver drugs with precision.

The journey to these insights was not simple. Poudyal recalled, “The main challenge was handling so many different proteins and polypeptides. Mapping the saturation concentration for all of them, each with its own solubility and behaviour, was difficult.”

This study highlights a fundamental aspect of molecular life: proteins’ ability to self-organise into dynamic, liquid compartments. By showing that this capacity is built into every protein’s chemistry, the work challenges textbook ideas and inspires fresh possibilities. When we next imagine a cell, we can picture millions of tiny droplets constantly forming and dissolving — a lively, ever-changing landscape where proteins dance together, driven by forces as simple as hydrophobicity and electrostatic interactions.