pH ‐Dependent Assembly and Disassembly of β‐Sheet Peptide Nanofibers Tuned by Histidine Positioning

Histidine (His)‐containing β‐sheet assemblies are promising pH‐responsive nanomaterials that function in mildly acidic biological environments. Although these assemblies have been extensively studied, it remains unclear how the number and positioning of His residues in β‐sheet‐forming sequences define the pH range for β‐sheet formation and nanofiber assembly and how they regulate pH‐triggered structural transitions in pre‐formed nanofibers. Here, we designed five FVIFLD‐based β‐sheet‐forming peptide–oligo(ethylene glycol) (OEG) conjugates by systematically varying the number and positioning of His residues. Circular dichroism measurements over pH 3.9–9.0 showed that four conjugates exhibited a characteristic three‐region pH dependence, displaying random coil‐like signatures at high and low pH and β‐sheet‐like signatures within an intermediate pH range, whereas one conjugate remained random coil‐like across the entire pH range. Notably, even with the same total number of His residues, the pH region favorable for β‐sheet formation shifted depending on their positioning. Transmission electron microscopy confirmed nanofiber formation within the β‐sheet‐forming pH range. In addition, pre‐formed nanofibers underwent a decrease in β‐sheet order and shifted toward random coil‐like conformations upon acidification to mildly acidic pH. The midpoint pH for acid‐triggered destabilization of pre‐formed nanofibers (pH D50 ) was not determined solely by the total number of His residues but was also influenced by their positioning within the sequence, and it did not necessarily correlate with the midpoint pH for assembly (pH A50 ). Together, these results provide a practical reference for designing His‐mediated pH‐responsive β‐sheet assemblies.