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Updated: Aug 18, 2026

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
Published on: July 27, 2016
Spontaneous Orthogonal Protein Crosslinking via a Genetically Encoded 2-Carboxy-4-Aryl-1,2,3-Triazole
Yali Xu1, Abdur Rahim1, Qing Lin1
1Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260-3000, USA.
Abstract:
Here we report the design of N2 -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 β-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 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.
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