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Targeting the N terminus for site-selective protein modification

Abstract

The formation of well-defined protein bioconjugates is critical for many studies and technologies in chemical biology. Tried-and-true methods for accomplishing this typically involve the targeting of cysteine residues, but the rapid growth of contemporary bioconjugate applications has required an expanded repertoire of modification techniques. One very powerful set of strategies involves the modification of proteins at their N termini, as these positions are typically solvent exposed and provide chemically distinct sites for many protein targets. Several chemical techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. A growing number of N-terminus-specific enzymatic ligation strategies have provided additional possibilities. This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each. Examples of specific N-terminal protein conjugates are provided, along with their uses in a number of diverse biological applications.

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Figure 1: Schematic representation of the average abundance of the amino acids commonly used for bioconjugation, including the corresponding pKa values of their side chains.
Figure 2: Selective modification of protein N termini using pH control.
Figure 3: Modification of specific amino acids at protein N termini.
Figure 4: Transamination of protein N termini, resulting in carbonyls that can be further reacted with alkoxyamines.
Figure 5: One-step modification of protein N termini.
Figure 6: Immobilization of proteins through the N terminus.
Figure 7: Enzyme-mediated N-terminal protein modification.
Figure 8: Subtiligase-mediated N-terminal labeling to identify proteolytic cleavage sites.

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Acknowledgements

This work was supported by the National Science Foundation (CHE-1413666). C.B.R. was supported by the Villum Kann Rasmussen Foundation. The authors thank J.I. MacDonald for helpful comments and suggestions for this manuscript.

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Rosen, C., Francis, M. Targeting the N terminus for site-selective protein modification. Nat Chem Biol 13, 697–705 (2017). https://doi.org/10.1038/nchembio.2416

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