The Picosecond Dance of a Single Water Molecule at the Protein's Surface

The dynamics of water molecules directly hydrogen-bonded to the peptide backbone are crucial for protein structure and function. However, experimentally capturing the behavior of weakly-bound, mobile biological water has been a long-standing challenge. Using N-ethylpropionamide as a model β-peptide, we combined ultrafast two-dimensional infrared (2D IR) spectroscopy, FTIR, and molecular dynamics simulations to probe the hydration shell of the amide carbonyl group. Our results reveal two distinct solvation states: a strongly hydrogen-bonded (SHB) state and a weakly hydrogen-bonded (WHB) state, characterized by a 13 cm⁻¹ frequency difference in the amide-I band. 2D IR cross-peaks unambiguously demonstrate chemical exchange between these states. The kinetics show that the SHB-to-WHB transition occurs in ~0.5 ps at room temperature, with an activation energy of 13 kJ/mol. This work provides direct experimental evidence of a transient, weakly-bound water molecule in the immediate hydration layer of the peptide backbone, offering new insights into the picosecond dynamics of biological water.