Spontaneous Orthogonal Protein Crosslinking via a Genetically Encoded 2-Carboxy-4-Aryl-1,2,3-Triazole

Here we report the design of N-2-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-1 exhibiting high reactivity toward the proximal Lys and Tyr. Furthermore, when introduced into the N-terminal beta-strand of either a single-chain VHH antibody or a supercharged monobody, CATK-1 enabled site-specific, inter-strand, orthogonal crosslinking with a proximal Tyr located on the opposing beta-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.

Automated summary

In this study, we designed N-2-carboxy-4-aryl-1,2,3-triazole-lysines (CATKs) and incorporated them into proteins via genetic code expansion. CATKs allowed for spontaneous, site-selective crosslinking to generate covalent protein dimers in living cells. The resulting crosslinked proteins showed improved cellular uptake and enhanced stability.