A series of studies from the University of Campinas, culminating in a prestigious journal cover, reveals the intricate dance between proteins and ionic liquids, paving the way for innovations in medicine, biotechnology, and sustainable materials.
In the world of biochemistry, proteins are the tireless workers. They build, repair, and regulate our bodies, acting as enzymes, antibodies, and structural components. To keep them working correctly, they need to be in the right environment, typically water. But what if we could design better environments? What if we could create custom solvents to protect fragile protein-based medicines, make industrial processes greener and more efficient, or even turn waste into valuable new materials?
This is the promise of ionic liquids (ILs). Through a series of insightful papers, Ph.D. researcher Vinicius Piccoli and his supervisor, Prof. Leandro Martínez of the Institute of Chemistry, have provided an atom-level view of how these remarkable solvents interact with proteins. Their work, supported by the Center for Computing in Engineering and Sciences (CCES), funded by the São Paulo Research Foundation (FAPESP), has unraveled a complex molecular ballet that was previously poorly understood.
The culmination of this research was recently featured as the cover article for The Journal of Physical Chemistry B, one of the world’s leading journals in the field, highlighting the global significance of their findings.
The Promise of Designed Salts
So, what are ionic liquids? Imagine salt, but instead of being a solid crystal at room temperature, it’s a liquid. These “designed salts” are made of large, complex, and often asymmetric positive ions (cations) and negative ions (anions). The beauty of ILs is their customizability; by swapping out the cation or the anion, scientists can fine-tune their properties—like viscosity, polarity, and solubility—for a specific task. This has made them a hot topic in green chemistry, with potential applications ranging from better batteries to safer industrial catalysts.
One of their most exciting applications is in biotechnology. Could ILs be used to stabilize protein-based drugs like insulin so they no longer require refrigeration? Could they make enzymes more robust for producing biofuels? Before these questions can be answered, a fundamental challenge must be overcome: we need to understand exactly how ILs and proteins interact in their native aqueous environment.
A Virtual Microscope into the Molecular World
To tackle this challenge, Piccoli and Martínez turned to one of the most powerful tools in modern science: high-performance computer simulations. Using the computational resources of the CCES, they built a “virtual microscope” capable of watching the movement of every single atom in their systems. This technique, called molecular dynamics, allowed them to simulate the protein Ubiquitin—a small, stable, and well-understood “model” protein—immersed in water and various ionic liquids.
Their first publication, in the Journal of Molecular Liquids in 2020, revealed that the interactions were far from simple. It wasn’t just a matter of a positive ion being attracted to a negative spot on the protein. Instead, they discovered a “cooperative” or “correlated” effect. The cation and anion in the IL, while having very different chemical personalities, are tethered by the fundamental need to keep the solution electrically neutral. They observed that an anion might form specific hydrogen bonds with the protein, but in doing so, it would drag its cation partner along to balance the charge nearby. This means that the effect of an ionic liquid cannot be predicted by looking at its ions in isolation; their combined, correlated dance is what truly matters.
Stabilizer or Denaturant? It’s All About Shape
Building on this, the team asked another crucial question: do these ionic liquids protect a protein’s delicate folded shape, or do they cause it to unravel (denature)?
In a 2022 follow-up paper, they simulated the protein not just in its active, folded “native” state, but also in various stages of unfolding. They found that, overwhelmingly, the studied ILs act as denaturants.
The reason lies in the protein’s changing surface. A folded protein typically tucks its greasy, water-repelling (hydrophobic) parts into its core, away from the surrounding water. When it denatures, this core is exposed. The team’s simulations showed that the IL cations, which often have hydrophobic parts themselves, are strongly attracted to this newly exposed greasy surface. They cluster around these regions, pushing water away and stabilizing the unfolded state. This creates a feedback loop: the more the protein unfolds, the more the IL is attracted to it, which in turn encourages even more unfolding.
This was a critical insight: even if an IL appears to be a “protector” for a perfectly folded protein, its overwhelming preference for the unfolded state makes it a potent denaturant in the bigger picture.
The Art of the Mix: Competing Ions
Having established the fundamentals, the researchers delved deeper into the tunability of these systems. What if you don’t use just one ionic liquid, but a mixture?
In a 2024 paper, they explored what happens when two different anions are present in the solution, competing for the protein’s attention. They found that the effects are not additive—you can’t just average the behaviors of the individual anions.
For example, the dicyanamide (DCA) anion is a very strong binder because it forms hydrogen bonds with the protein. Logic might suggest that mixing it with another anion that also forms hydrogen bonds would yield the strongest effect. However, the simulations revealed that mixing DCA with a weaker-binding anion like chloride (Cl⁻) could actually result in stronger overall protein solvation. This demonstrated that the process is a complex competition for specific binding sites on the protein, influenced by how the ions interact with each other and with water in the bulk solution. It is a powerful illustration of how a multi-component solvent can be designed for highly specific effects.
The Cover Story: A Surprising Reversal
The team’s most recent paper, which earned the journal cover, focused on the cation’s hydrophobicity. They compared two very similar cations: [EMIM]⁺ and [BMIM]⁺. The only difference is that [BMIM]⁺ has a slightly longer hydrocarbon tail, making it more hydrophobic .
Common wisdom suggested that the more hydrophobic cation would always interact more strongly with the protein. And at low concentrations, this was true. The simulations showed that [BMIM]⁺ was more effective at accumulating at the protein surface. But, in a fascinating twist, as the IL concentration increased, the effect reversed.
At higher concentrations, the prime hydrophobic spots on the protein surface become saturated . At this point, another factor takes over: the larger size of the [BMIM]⁺ ion means that, for the same molar concentration, it displaces more water from the solution. This subtle change in the bulk water concentration is enough to flip the script, making the less hydrophobic [EMIM]⁺ the preferentially interacting cation at high concentrations . This discovery of a concentration-dependent reversal of affinity is a testament to the intricate, and often counter-intuitive, physics at play.
From Virtual Labs to Real-World Impact
While this research provides deep fundamental knowledge, its impact is not confined to the theoretical realm. In a parallel study, Piccoli and Martínez collaborated on a project that showcases the direct application of this knowledge: creating a sustainable biorefinery process for squid pen waste.
Every year, the fishing industry generates enormous amounts of waste, including squid pens—the flexible, internal shell of a squid. These pens are rich in protein and β-chitin, a valuable biopolymer with applications in medicine, cosmetics, and agriculture. Traditionally, separating these components requires harsh chemicals and generates significant wastewater.
Applying their expertise, the research team used a biocompatible ionic liquid, choline acetate, to efficiently and cleanly fractionate the squid pens. Their molecular simulations explained why it worked so well: the acetate anion showed a particularly strong affinity for the proteins, helping to dissolve them away from the solid chitin. The project successfully recovered high-purity protein and chitin, which were then used to create new biocomposite films, demonstrating a complete “waste-to-value” pipeline.
This practical application is a powerful demonstration of how the fundamental insights gained from Piccoli and Martínez’s Ph.D. research can directly translate into innovative, sustainable technologies.
The journey from understanding the correlated dance of ions to designing a process for recycling seafood waste exemplifies the power of curiosity-driven research. The work of Vinicius Piccoli and Leandro Martínez at Unicamp has not only peeled back a layer on one of nature’s fundamental interactions but has also provided a detailed molecular roadmap for designing the next generation of green solvents and advanced biomaterials.