Direct evidence of acid-driven protein desolvation

Water and its ability to modulate the protonation states of biomolecules govern the physical chemistry of life, dictating their metabolic functions( 1 ). However, how amino acid protonation alters protein hydration and solubility( 2, 3 ) is an open question since Kuntz and Kauzmann proposed p H -driven protein desolvation in 1974( 4 ). Here, in a series of high-resolution cryo-electron microscopy structures of a protein complex at different p H values (from p H 9.0 to 3.5) we examined thousands of observable hydration sites. Cryo-EM data, in agreement with constant-p H molecular dynamics simulations, show that nearly half of protein-bound waters exchanged with the bulk solvent upon acidification, with ∼100 waters lost per p H unit per molecule. The loss of waters was most significant around the side chains of glutamate and aspartate residues while specific polar residues, mostly asparagine, anchored persistent waters. A positionally conserved hydration layer was observed across all p H conditions, accounting for 40% of resolved waters. Those waters displayed denser packing than less persistent waters, forming a p H -independent solvation shell. Acid-induced water exchange also displaced bound iron, providing a mechanistic link between solvation and metal release. Our findings demonstrate the core principles of acid-driven protein desolvation, resolving a 50-year-old biochemical hypothesis( 4 ).