Self‐Assembling Synthetic Congener Collagen Mini‐Fibrils With Genetically Encoded Hydroxyproline and 3,4‐Dihydroxyphenylalanine for Wound Healing

ABSTRACT Collagen‐like proteins (CLPs) and their congeners can form stable triple helices but show limited fibril‐forming ability, restricting their application as biomaterials. To more closely replicate the structural features of natural collagen, we engineered an extended CLP‐CLP double domain (CLPdd) genetically encoded with Hydroxyproline (CLPdd‐Hyp) and 3,4‐dihydroxyphenylalanine (CLPdd‐DOPA) using a genetic code expansion strategy. This study presents the first report of the dual incorporation of Hydroxyproline (Hyp) and 3,4‐dihydroxyphenylalanine (DOPA) into the CLP double domain (CLPdd), yielding the variant CLPdd‐HD, which exhibited significantly enhanced fibrillation, thermal stability, and biomaterial potential. Among the engineered variants, CLPdd‐Hyp showed the most pronounced improvements in triple‐helical structure, fibrillation behavior, wound healing efficacy, and cell adhesion highlighting its promise as a biomaterial. Biocompatibility assessments further confirmed the suitability of CLPdd variants for biomedical applications. Notably, CLPdd‐HD demonstrated exceptional thermal stability and cell‐adhesive properties, underscoring its potential for further optimization. This work lays a foundation for tailoring bacterial CLPs through strategic NCAA incorporation, opening new avenues for developing advanced collagen‐mimetic biomaterials.