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

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.