University of Massachusetts Medical School Faculty Publications

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Department of Biochemistry and Molecular Pharmacology; Thompson Lab

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Amino Acids, Peptides, and Proteins | Biochemistry | Enzymes and Coenzymes | Medicinal and Pharmaceutical Chemistry | Medicinal Chemistry and Pharmaceutics | Nucleic Acids, Nucleotides, and Nucleosides


Citrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an engineered E. coli-derived leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterize the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and show that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This technology possesses the potential to decipher the biology of citrullination.


Synthetic biology, Chemical genetics, Proteomics, Hydrolases

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Copyright © The Author(s) 2021. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

DOI of Published Version



Mondal S, Wang S, Zheng Y, Sen S, Chatterjee A, Thompson PR. Site-specific incorporation of citrulline into proteins in mammalian cells. Nat Commun. 2021 Jan 4;12(1):45. doi: 10.1038/s41467-020-20279-w. PMID: 33398026; PMCID: PMC7782748. Link to article on publisher's site

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Nature communications

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Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.