Direct evidence of acid-driven protein desolvation

TL;DR

This paper shows how acidity affects water molecules around proteins, revealing a long-standing biochemical question about protein solvation.

Contribution

The study provides direct atomic-level evidence of acid-driven protein desolvation using cryoelectron microscopy and simulations.

Findings

Acidification causes nearly half of protein-bound waters to exchange with bulk solvent, losing ~100 waters per pH unit per molecule.

Persistent hydration shells remain around specific residues like asparagine, forming a pH-independent solvation layer.

Acid-induced water exchange displaces bound iron, linking solvation changes to metal release.

Abstract

Life depends on proteins, and proteins depend on water. Yet for 50 y, the open question around what happens to the water around proteins in acidic conditions has not been resolved. Here, we visualized biomolecular hydration at the atomic level as a function of increasing acidity. We saw hundreds of water molecules leave the structure, while a persistent shell of water remained, organized by ~40% of resolved waters. We also found that acidity shifts how specific metals, i.e., iron, are held in their binding sites. Our results resolve a long-standing question in biochemistry and reveal simple rules for how acidity affects protein solvation. Our findings may also aid the design of more stable or pH-tolerant proteins, critical for biotechnological applications.

Water and its ability to modulate the protonation states of biomolecules govern the physical chemistry of life, dictating their metabolic functions. However, how amino acid protonation alters protein hydration and solubility is an open question since Kuntz and Kauzmann proposed pH-driven protein desolvation in 1974. Here, in a series of high-resolution cryoelectron microscopy structures of a protein complex at different pH values (from pH 9.0 to 3.5), we examined thousands of observable hydration sites. Cryoelectron microscopy data, in agreement with constant-pH molecular dynamics simulations, show that nearly half of protein-bound waters exchanged with the bulk solvent upon acidification, with ~100 waters lost per pH 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 pH conditions, accounting for 40% of resolved waters. Those waters displayed denser packing than less persistent waters, forming a pH-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-y-old biochemical hypothesis.

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