# Mesostructured Water Enhances Stability of ProteinMPNN-Designed Ubiquitin-Fold Proteins

## TL;DR

This study shows that a special water structure around AI-designed proteins helps them stay stable even under extreme conditions.

## Contribution

The discovery of mesostructured hydration as a sequence-encoded mechanism for protein stability is novel.

## Key findings

- ProteinMPNN-designed variants R4/R10 and ICVs remain stable above 120 °C and in extreme denaturing conditions.
- Mesostructured hydration strengthens hydrogen bonding between water and proteins, preventing unfolding.
- Charge enrichment on protein surfaces drives the formation of this stabilizing hydration shell.

## Abstract

AI-designed protein variants have demonstrated remarkable
resistance
to heat and chemical stress, yet the molecular mechanisms underlying
this stability remain unclear. Here, we present a comprehensive biophysical
and nuclear magnetic resonance (NMR) analysis of thermally stable
ubiquitin and its ProteinMPNN-designed variants, R4 and R10, together
with a second system based on the less stable ISG15 C-terminal domain
(ISG15-CTD). Both R4/R10 and ProteinMPNN-designed ISG15-CTD variants
(ICVs) exhibit extraordinary thermostability beyond 120 °C, and
resist extreme denaturation at pH 3.0 in 8 M urea. NMR relaxation
and hydrogen–deuterium exchange, and molecular-dynamics simulations
reveal a protective mesostructured hydration shell that strengthens
the hydrogen bonding network between protein-bound and bulk water,
thereby suppressing unfolding. Sequence and electrostatic analyses
indicate that this hydration arises from charge enrichment and clustering
on the protein surface. These findings identify mesostructured hydration
as a general, sequence-encoded mechanism of ProteinMPNN-driven stability
and provide a physical framework for designing highly resilient biomolecules.