Single-site glycine-specific labeling of proteins

• Published in: Nature communications 2019-06, Vol.10 (1), p.2539-2539, Article 2539
• Main Authors: Purushottam, Landa, Adusumalli, Srinivasa Rao, Singh, Usha, Unnikrishnan, V. B., Rawale, Dattatraya Gautam, Gujrati, Mansi, Mishra, Ram Kumar, Rai, Vishal
• Format: Article
• Language: English
• Subjects: 101/58; 14/35; 140/58; 631/92/2783; 631/92/96; 639/638/92/2783; 639/638/92/96; 82/29; 82/58; 82/80; 82/81; 82/83; Aldehydes; Aldehydes - chemistry; Amino acids; Cellular structure; Escherichia coli; Fluorescent Dyes - chemistry; Fluorine; Glycine; Glycine - chemistry; HEK293 Cells; Humanities and Social Sciences; Humans; Insulin; Insulin - chemistry; Labeling; multidisciplinary; N-Terminus; NMR; Nuclear magnetic resonance; Proteins; Proteins - chemistry; Receptor, Insulin - chemistry; Science; Science (multidisciplinary); Selectivity; Staining and Labeling - methods; Structure-function relationships
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10503-7

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19 F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed. Single-site labelling of proteins is desirable, e.g., for analytical purposes. Here, the authors developed a method in which they use an aldol-type reaction to modify proteins at N-terminal glycine residues in an efficient and selective manner, which is also applicable to cell lysates.