Proteins systematically lose their protective hydration shell when their environment becomes more acidic. Until recently this was just a theory. State-of-the-art imaging techniques have helped researchers at Martin Luther University Halle-Wittenberg (MLU) directly observe this process for the first time at the level of the individual water molecule. This has answered a question in biochemistry that had remained unanswered for 50 years. In an article published in Proceedings of the National Academy of Sciences ( PNAS ), the team writes that this knowledge could help to develop more stable proteins.
Proteins are the machines of a cell, controlling all of the vital processes in an organism – from growth to metabolism. Their function depends largely on their spatial structure. Proteins are enveloped by a shell of water molecules which, in turn, helps to determine their shape and function. However, up until now, little had been known about the precise interaction between proteins, water molecules and their environment. “As early as 1974, Irwin Kuntz und Walter Kauzmann had posited that decreasing pH levels – in other words, increasing acidification – alter a protein’s hydration shell. However, there had been no direct evidence for this hypothesis,” explains biochemist Professor Panagiotis Kastritis from MLU.
In this new study, Kastritis’s team used high-resolution cryogenic electron microscopy (cryo-EM) to study this process in detail. During cryo-EM, proteins are flash-frozen and bombarded with an electron beam. The resulting signal can be used to create extremely detailed images that can be combined to produce 3D models. This revealed the protein’s structure at the level of just a few atoms. The researchers investigated the protein apoferritin which acts as an iron storage unit in cells. They observed it at seven different pH levels ranging from 9.0 (slightly alkaline) to 3.5 (acidic). Using cryo-EM, they individually mapped thousands of water molecules and tracked their behaviour as acidity increased. The experiments were accompanied by computer simulations which corroborated the observed behaviour.
“Our data allowed us to chart the movement of every single water molecule and observe how it moved in response to changes in pH levels. We were surprised to find that there were clear rules: certain amino acids bind water, while others release it. We hadn’t expected to see this,” says Dr Ioannis Skalidis, who completed his PhD at MLU and now works as a researcher at Utrecht University. Dr Farzad Hamdi from MLU adds: “We are the first group to succeed in providing this evidence at this level of mechanistic detail. Yes, it is an old theory but there has been no direct evidence for that.”
Other findings are also surprisingly precise: a protein loses around 100 water molecules for every unit by which the pH decreases. The researchers were even able to describe the behaviour of the individual amino acids making up the proteins. Glutamate and aspartate are the first to release their water molecules when acidified. Others retain the molecules regardless of pH value, so that a stable inner core consisting of around 40 per cent of the total number of water molecules is maintained regardless of the pH level.
Another unexpected finding: As pH levels decrease, not only does the hydration shell of the apoferritin change, but so does the position of the bound iron ions. These shift gradually away from their binding site. This is evidence of a structural mechanism by which rising acidity can trigger the release of metal ions from proteins – a process that plays a role, for example, in cellular iron regulation.
Other studies are needed to determine whether the rules that have been identified in the study also apply to other proteins. “We assume that this process is, at least, similar for other proteins. Such knowledge can be used to develop proteins that are more stable or more pH-tolerant,” says Kastritis. This could help improve industrial enzymes or protein-based drug delivery systems.
The study was funded by the European Union, the German Research Foundation (DFG), the Federal Ministry of Research, Technology and Space (BMFTR), and the State of Saxony-Anhalt.
Study: Hamdi F. et al. Direct evidence of acid-driven protein desolvation. Proceedings of the National Academy of Sciences (2026). DOI: 10.1073/pnas.2525949123
Proceedings of the National Academy of Sciences
Experimental study
Cells
Direct evidence of acid-driven protein desolvation
5-Mar-2026
he authors declare no competing interest.