From 4-arm star proteins to diverse stimuli-responsive molecular networks enabled by orthogonal genetically encoded click chemistries

Abstract

Protein topology engineering has opened the door to various nonlinear biomacromolecular architectures, holding promise for designing some unprecedented biomaterials. Here, we report the synthesis of 4-arm star protein molecules both in vitro and in vivo, which further served as versatile building blocks to create protein networks suitable for 3D cell culturing. To achieve these, we leveraged two orthogonal genetically encoded click chemistries (GECCs), corresponding to the two peptide/protein pairs, SnoopTag/SnoopCatcher and SpyTag/SpyCatcher, that can spontaneously form covalent isopeptide bonds under physiological conditions with great efficiency and selectivity. These molecular networks were also designed to contain stimuli-responsive motifs such as the AdoB₁₂-dependent photoreceptor protein (CarH_C) and the Ni²⁺-cleavable peptide (SNAC tag), leading to photo- and Ni²⁺-responsive hydrogels, respectively. This study illustrates the integrated use of protein topology engineering and stimuli-responsive peptide/protein motifs as a powerful approach for designing smart materials.