First Hydration Shell Integrity: Key to Protein Stability.

Protein stabilizers and denaturants can be used to elucidate the fundamental principles of hydration, which is crucial for biological functions and biotechnology. Despite decades of work, existing molecular models of such stabilizers and denaturants have not yet been fully validated because few experimental methods can detect water structures within the hydration shell in situ under ambient conditions. Here, we devise a molecular model guided by the application of chiral-selective vibrational sum frequency generation spectroscopy to a solvated protein. We show that urea disrupts the first hydration shell, weakening the protein's hydrogen bonds. Conversely, trimethylamine N-oxide resides outside the shell and stabilizes the protein by strengthening water hydrogen bonds within the first hydration shell. In mixtures, trimethylamine N-oxide drives urea out of the shell, neutralizing urea's destabilizing effect. We conclude that protein stability directly correlates with first hydration shell integrity. These insights have broad implications for understanding solvent effects on biocatalysis and heterogeneous cellular environments.