How proteins breathe – and what makes them freeze | New insights from ISTA research

Advances in structural biology have allowed scientists to determine molecular structures with atomic‑level detail, sometimes yielding static snapshots that do not reflect the dynamism of proteins. However, these motions are often crucial for biological function. Researchers from the Institute of Science and Technology Austria (ISTA) together with international collaborators have now combined several methods to shed light on how proteins ‘breathe’ and how some experimental techniques freeze their motion. The findings—which could boost protein design approaches and improve AI-based structural prediction tools—were published in Nature Chemistry.

Despite serving as structural biology’s central pillar for over half a century, protein crystallography has yielded static molecular structures—like still frames from a video—far from the buzzing life inside cells.

“How much can these ‘frozen snapshots’ of protein structures really tell us about their true biological functions and bustling molecular environments?” asks Lea Becker, the study’s first author and a PhD student in Professor Paul Schanda’s group at the Institute of Science and Technology Austria (ISTA).

To address this fundamental question, Becker and Schanda have teamed up with international researchers, including Christophe Chipot from the Laboratoire International Associé CNRS in France and the University of Illinois at Urbana-Champaign in the United States, and Sylvain Engilberge from the European Synchrotron Radiation Facility in Grenoble, France. By combining insights from X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and molecular modeling, they overcame technical limitations and gained a more complete picture of proteins’ natural behavior.

Fleeting structures with a real function

Proteins have varying shapes and sizes, and their binding sites are often buried inside their cores. Therefore, they must change their shape considerably to allow other molecules to bind.

“Scientists have used the words ‘breathing motion’ to refer to this idea of a protein that transiently ‘opens’,” says Schanda. “What hides behind this oversimplified term is a complex molecular choreography, which is permanently ongoing in every protein. But we often lack a detailed understanding of this phenomenon.”

Several structural biology techniques cannot reveal all structural conformations that may be essential for biological function. In particular, crystallization can lock molecules into a limited set of rigid structures within the crystal lattice, while the same protein can breathe much more freely in solution.

“With our study, we aim to uncover the true dynamics of proteins as a function of time,” says Schanda.

Overcoming technical limitations

To explore this highly dynamic microscopic world, the team has investigated synergies between different experimental methods developed in recent years.

“We often think of experiments as objective windows into nature. However, each experimental method has its limitations and often only sheds light on part of the truth,” says Becker, whose research focuses on method development. “By developing methods and combining techniques, we aim to overcome limitations and broaden our insights.”

As a model system, Becker and the team used the protein GB1 to study its conformational flexibility with its binding partner, the antibody IgG. They did so both in the so-called solid phase, using X-ray crystallography and solid-state NMR, and in solution, by combining advanced labeling methods with quantitative NMR techniques. They also obtained molecular ‘movies’ of GB1 using enhanced-sampling molecular dynamics simulations.

Flipping aromatic rings

To gain detailed insights into how GB1 and IgG bind and breathe, the team examined the rotation of specific chemical groups found in some of their building blocks—the amino acids.

Amino acids link through common backbone components to form a protein chain. However, the distinct chemical compositions of the amino acids’ “side chains” ultimately determine how the entire protein folds on itself. These side chains also govern the possible shapes each part of the molecule can adopt after folding.

Some amino acid side chains include aromatic rings—chemical groups that have low affinity for water. As such, they are much more likely to be buried inside the protein’s core and in active sites, hiding away from the surrounding water molecules. This, together with the fact that aromatic rings can flip, makes them excellent indicators of protein motion under the conditions used in structural studies.

“For aromatic rings to flip, the entire protein needs to move considerably. This is what makes them reliable reporters of dynamics. So, by looking at how fast aromatic rings flip inside a protein’s core and in an enzyme’s active site during binding, we can read out how freely it can breathe,” explains Becker. “We already knew that crystallization can limit the proteins’ free movement. Our interdisciplinary approach now helps us understand some of these molecular details.”

Towards dynamic structures on demand?

Ultimately, understanding how substrates reach protein binding sites can reveal how proteins evolved to perform specific functions. This is because only a limited set of conformational dynamics permits certain molecular functions to arise.

On the other hand, the emerging field of de novo protein design—the computational creation of proteins with novel structures and functions from scratch—has had limited success in generating dynamic proteins, highlighting the need for further experimental data on protein dynamics in nature.

“Machine‑designed proteins have been optimized to reproduce static structures. But these frozen structures likely don’t provide access to the full array of functional conformations in nature,” says Schanda. “By uncovering protein dynamics experimentally, we may be able to model and design proteins with better functional relevance in the future.”

In turn, this experimental knowledge will help improve machine learning-based structural prediction tools such as AlphaFold that have transformed research in drug discovery and contributed to disease understanding.

“Understanding the dynamics of proteins and how they are linked to their biological functions makes structural biology truly exciting,” Schanda concludes.

Publication:

Lea M. Becker, Haohao Fu, Ben P. Tatman, Matthias Dreydoppel, Anna Kapitonova, Ulrich Weininger, Sylvain Engilberge, Christophe Chipot, and Paul Schanda. 2026. Aromatic Ring Flips Reveal Reshaping of Protein Dynamics in Crystals and Complexes. Nature Chemistry. DOI: 10.1038/s41557-026-02155-0

Funding information

This project was supported by funding from a DOC fellowship of the Austrian Academy of Sciences at the Institute of Science and Technology Austria (grant no. PR10660EAW01), the European Research Council (ERC) grant project 101097272, and the Métropole du Grand Nancy grant project “ARC”. This research was also supported by the Scientific Service Units (SSU) of the Institute of Science and Technology Austria (ISTA) through resources provided by the Nuclear Magnetic Resonance and the Lab Support Facilities.