50-year-old mystery solved: For the first time, chemists have directly observed how the water shell of proteins changes under the influence of acid – thereby clarifying a long-standing question in biochemistry. The observations confirm that the acidic environment gradually removes the protective water molecules from the protein. Surprisingly, however, some amino acids lose their water earlier than others. In addition, iron atoms in the protein rearrange themselves – this was also unexpected, as the researchers explain.
Proteins are the most important tools of our cells. They control cell metabolism, control biochemical reactions, serve as messenger substances and form the basis for vital functions of our body. Whether a protein can carry out its function also depends on its three-dimensional structure. This in turn is shaped and stabilized by a dense shell of water molecules. “Life depends on proteins and proteins need water,” explain Farzad Hamdi from the Martin Luther University Halle-Wittenberg (MLU) and his colleagues.
Looking at the water molecules in a protein
It is all the more astonishing that a crucial question about the water shell of proteins has remained unanswered for 50 years: What happens to the watery protective shell of proteins when they are exposed to an acidic environment? “As early as 1974, Irwin Kuntz and Walter Kauzmann had theoretically predicted that a falling pH value, i.e. increasing acidification, would change the water shell of proteins. However, direct evidence for this hypothesis was missing until now,” explains Hamdi’s colleague Panagiotis Kastritis.
Using high-resolution cryo-electron microscopy, the team has now succeeded for the first time in tracking this acid-related water loss in a protein down to the level of individual water molecules. The protein apoferritin served as the test object. This resembles a hollow ball made of protein chains and serves as an iron store in our cells. Individual iron atoms can attach to different parts of the protein and be stored via complex compounds.
For their experiment, Hamdi and his colleagues exposed these proteins to seven different pH values, from slightly basic to strongly acidic (pH 3.5), frozen them using shock freezing and examined the molecular configuration in the electron microscope. The experiments were supplemented by computer simulations that explain the observed behavior.
Around 100 fewer water molecules per pH level
The images showed that up to an acidity level of around pH 5, the structure of apoferritin hardly changed and the basic structure of the protein also remained stable. But the first signs of dissolution appeared in the water shell: little by little, more and more water molecules were lost. “We see a clear trend in which the protein loses around 100 water molecules per pH unit,” report the researchers. “If the pH value is reduced from 9 to 3.5, the number of water molecules within a distance of up to five angstroms from the protein surface is reduced by half.”
From around pH 4, this water loss has clear consequences for apoferritin: the complex spatial structure begins to change. The water shell is now very irregular and limited to a thin layer of hydrate on the protein chains. “We saw hundreds of water molecules leaving the protein structure until only around 40 percent of them remained,” the team writes. These observations confirm the theory of acid-induced water loss from proteins that has existed for 50 years. “We are the first group to have succeeded in proving this,” says Hamdi.
Some amino acids are more stubborn than others
What was surprising, however, was the pattern of this water loss. “We were surprised that there seem to be clear rules: certain amino acids hold water, others let it go. That wasn’t expected,” says co-author Ioannis Skalidis from Utrecht University in the Netherlands. The side chains of the amino acids glutamate and aspartate bind most of the water molecules under normal conditions, but are the first to release them when acidified. The amino acids lysine and arginine as well as – to a lesser extent – amino acids with polar molecular parts also proved to be relatively susceptible.
Other protein building blocks, however, continue to hold on to their water molecules: regardless of the pH value, the water shell remains largely intact for amino acids with non-polar side chains such as alanine, leucine, phenylalanine or tryptophan. Although these protein components initially bind fewer water molecules, they hold on to them more stubbornly, as the cryo-electron microscopic images revealed.
Stored iron atoms also migrate
Another unexpected finding: as the pH value decreased, not only the water shell of the apoferritin changed, but also the position of the iron ions bound in the protein. These gradually moved from their original binding sites further into the interior of the protein complex. “This behavior is highly specific for iron and does not occur, for example, with magnesium built into the structure,” report Hamdi and his colleagues. This provides evidence of how acidity can also influence metal release from proteins.
“Our results clarify a long-standing question in biochemistry and reveal simple rules for how acidity influences the solvation of proteins,” write the researchers. They have only observed these processes on apoferritin. “We assume that this process is at least similar for other proteins. This knowledge can therefore help to make proteins more stable or pH-tolerant,” says Kastritis.
Source: Farzad Hamdi (Martin Luther University Halle-Wittenberg) et al., Proceedings of the National Academy of Sciences, 2026; doi: 10.1073/pnas.2525949123