Local Water Ordering Directs Early-Stage Nucleation in the Humidity-Responsive Self-Assembly of Phenylalanine Dipeptide at the Air Surface

The interaction between peptides and water is crucial for their assembly into higher-order architectures; however, the underlying mechanisms under trace water conditions remain poorly understood. Here, using the amyloid core sequence phenylalanine dipeptide (FF) as a model system, we investigate the kinetics of humidity-driven self-assembly at the FF/air interface by integrating surface-sensitive sum-frequency generation vibrational spectroscopy (SFG-VS) with Fourier-transform infrared spectroscopy (FTIR). Our results reveal that FF self-assembly follows a three-stage kinetic pathway─comprising lag, rapid growth, and plateau phases─which is effectively modulated by humidity variation. This process is accompanied by a conformational transition from disordered monomers to an ordered chiral supramolecular structure with C1 symmetry. Water initiates assembly by promoting key intermolecular C=O···H–N hydrogen bonds between FF units, which subsequently drive long-range molecular ordering. Notably, tracking the water bend–libration combination band via FTIR directly reveals enhanced ordering of hydration water within the peptide network during assembly. These findings demonstrate that the interfacial assembly is predominantly enthalpy-driven, with water acting as an active molecular switch that regulates kinetics through reorganization of the hydrogen-bond network. This work advances the molecular-scale understanding of amyloid fibrillation and provides a foundation for designing responsive functional nanomaterials.