A Macrocycle-Assisted Platform Approach to Protein Cross-Linking Via Chemically Inactive Residues

Cross-linking proteins using cross-linkers that chemically target primary amine and/or carboxyl residues has been a technically mature and robust method in protein engineering. However, depletion of chemically active residues over cross-linking presents a significant challenge to the ability of the resulting bioassemblies to be further engineered and/or maintain specific biological functions. Here, we report a platform approach to cross-link natural proteins via the otherwise chemically inactive residues. This method exploits noncovalent and selective binding of molecularly engineered cucurbit[7]uril macrocycle to aromatic residues that endows the parent protein with additional unique handles for cross-linking. Various proteins are amenable to this approach, yielding bioassemblies with mechanical strength and thermal and enzymatic stability comparable to or exceeding counterparts prepared by some “gold-standard” chemical cross-linkers. This macrocycle-assisted platform approach offers a new paradigm for fabricating valuable bioassemblies that overcome the intrinsic limitations of existing methodologies.