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Abstract Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non‐disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site‐specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate‐based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology‐mimicking bacterial adhesins. 

Abstract Here we report the design ofN²‐carboxy‐4‐aryl‐1,2,3‐triazole‐lysines (CATKs) and their site‐specific incorporation into proteins via genetic code expansion. When introduced into the protein dimer interface, CATKs permitted spontaneous, proximity‐driven, site‐selective crosslinking to generate covalent protein dimers in living cells, with phenyl‐bearing CATK‐1exhibiting high reactivity toward the proximal Lys and Tyr. Furthermore, when introduced into theN‐terminal β‐strand of either a single‐chain VHH antibody or a supercharged monobody, CATK‐1enabled site‐specific, inter‐strand, orthogonal crosslinking with a proximal Tyr located on the opposing β‐strand. Compared with a non‐crosslinked monobody, the orthogonally crosslinked monobody displayed improved cellular uptake and enhanced proteolytic stability against an endosomal enzyme. The robust crosslinking reactivity of CATKs should facilitate the design of novel protein topologies with improved physicochemical properties.