Single-site glycine-specific labeling of proteins

Abstract

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, ¹⁹F 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.

Fig. 1

Residue-specificity in labeling of proteins. a Nucleophilic addition of protein residues to an electrophile. b A latent electrophile, imine, renders the N-terminus labeling of proteins (path a–path c). c Latent nucleophile enables single-site N-terminus Gly labeling

Fig. 2

Stable aminoalcohol formation with glycine. a Aldehyde with H-bond acceptors forms a stable aminoalcohol with 1a (also see Supplementary Fig. 72). b Selective formation of aminoalcohol with 1a. c Stereo-stability of substituted amino acid (1n) under the reaction conditions. d Plausible mechanism for glycine specific formation of aminoalcohol

Fig. 3

N-terminus Gly labeling of proteins. a A user-friendly protocol delivers single-site labeling of proteins. Deconvoluted ESI-MS confirms the %conversion and rules out the possibility of side reactions (also see Supplementary Figs. 70 and 71). R indicates the fragment from reagent 2e attached to the protein. b Extension of methodology for labeling single protein (insulin 6d) in a mixture of proteins (6d–6j)

Fig. 4

Late-stage tagging and isolation of analytically pure tagged proteins. a Design elements of a symmetric bis-aldehyde. b Insulin is labeled through aminoalcohol formation with reagent 2g. The labeled insulin 9 can be treated with derivatives of O-hydroxylamine 10a–10c for tagging them with ¹⁹F-NMR probe, biotin, and coumarin. Alternatively, it can be immobilized on hydrazide functionalized resin through single-site to deliver 13. The unreacted insulin 6d is recovered and recycled. Subsequent treatment of 13 with 10a delivers analytically pure 11a after removing excess 10a through spin concentration. The hydrazide functionalized resin 12 is recovered and recycled multiple (5–8) times without loss of activity. Similar protocol renders pure 11b and 11c. For single-step late-stage installation of a probe, please see Supplementary Fig. 74

Fig. 5

Insulin bioactivity assay. a, Bioactivity measurement for native and modified insulin in a dose-dependent pAkt cell-based assay. The error bar represents standard error of mean (SEM). The assays were repeated at least three times (n = 3). b EC50 value determination from (a), absolute values obtained by extrapolation of graphs in a are represented by a simple bar graph. c Uptake of tagged insulin (green) and mixture of untagged and tagged insulin in cells. Chromatin (blue). Also see, Supplementary Fig. 77. d Activation of IR signaling and pAkt (red) accumulation in HEK293T cells after insulin treatment (scale bar:10 µm). Source data of Fig. 5 panel a provided as Source Data file

Fig. 6

Residue-specific labeling of a protein in the cell lysate. a Selective labeling of N-Gly SUMO1 with coumarin tag in cell lysate. b 12% SDS-PAGE of cell lysate (overexpressed with SUMO1) and coumarin-tagged SUMO1 7l in cell lysate followed by coomassie staining and fluorescence imaging. MW—molecular weight, Lanes 1 and 2: cell lysate before and after reaction (Coomassie); lanes 3 and 4: cell lysate before and after reaction (fluorescence). The band at ~17 kDa (m/z 11.6 kDa) in lanes corresponds to the SUMO1. The band at ~17 kDa (m/z 11.6 kDa) shown by fluorescence imaging in lane 4 confirms the selective tagging of SUMO1 (7l). Source data of Fig. 6 panel b provided as Source Data file