Mussel foot protein with genetically incorporated hydroxyproline and DOPA exhibits enhanced phase separation and underwater adhesion

Mussel foot protein-5 (mfp-5) is a key biological macromolecule responsible for the remarkable ability of mussels to adhere to wet and diverse surfaces. While the function of L-3,4-dihydroxyphenylalanine (Dopa) in underwater adhesion is well-characterised, the structural and functional role of hydroxyproline (Hyp) in mfp-5 remains underexplored, largely due to challenges in achieving site-specific dual post-translational modifications (PTMs). In this study, we employed residue-specific genetic code expansion strategies to incorporate either Dopa or Hyp into mfp-5 and systematically analyzed their effects on liquid–liquid phase separation (LLPS), redox stability, and interfacial interactions. Spectroscopic and microscopic analyses revealed that Hyp promotes coacervate formation, preserves Dopa in its reduced state, and enhances substrate binding by facilitating π–π, cation-π, and CH/π interactions. These results demonstrate that mfp-5 functions as a dynamic phase-separating macromolecule whose adhesion efficiency is modulated by Hyp-mediated regulation. This work offers new molecular insight into mfp-5's dual-modification chemistry and highlights its potential in the design of biomimetic, water-resistant adhesives for biomedical applications, including tissue sealing, drug delivery, and regenerative materials.