Local Water Ordering Directs Early-Stage Nucleation in the Humidity-Responsive Self-Assembly of Phenylalanine...

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.